Nucleic Acids Res, 2001 Dec 1, 29(23), 4834 - 42 Nearest neighbour analysis of MCM protein complexes in Drosophila melanogaster; Crevel G et al.; The MCM proteins are a group of six proteins whose action is vital for DNA replication in eukaryotes . It has been suggested that they constitute the replicative helicase, with a subset of the proteins forming the catalytic helicase (MCM4,6,7) while the others have a loading or control function . In this paper we show that all six MCM proteins are present in equivalent amounts in soluble extracts and on chromatin . We have also analysed soluble and chromatin-associated MCM protein complexes under different conditions . This suggests that all six MCM proteins are always found in a complex with each other, although the interaction between the individual MCM proteins is not equivalent as stringent salt conditions are able to break the intact complex into a number of stable subcomplexes . These data contribute to the ongoing debate about the nature of MCM complexes, supporting the hypothesis that they act as a heterohexamer rather than as a number of different subcomplexes . Finally, using protein-protein cross-linking we have shown that MCM2 interacts directly with MCM5 and MCM6; MCM5 with MCM3 and MCM2; and MCM6 with MCM2 and MCM4 . This provides the first direct information about specific subunit contacts in the MCM complex. Nucleic Acids Res, 2001 Dec 1, 29(23), 4826 - 33 Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells; Manivasakam P et al.; Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination . We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells . A plasmid containing the neo(R) expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells . Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells . In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells . Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neo(R) gene flanked by exon 3 of HPRT: The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells . However, all three enzymes decreased the absolute frequency of homologous integration approximately 2-fold, and the percentage of homologous integration decreased >10-fold . This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search. Nucleic Acids Res, 2001 Dec 1, 29(23), 4808 - 17 Replication protein A is sequentially phosphorylated during meiosis; Brush GS et al.; Phosphorylation of the cellular single-stranded DNA-binding protein, replication protein A (RPA), occurs during normal mitotic cell cycle progression and also in response to genotoxic stress . In budding yeast, these reactions require the ATM homolog Mec1, a central regulator of the DNA replication and DNA damage checkpoint responses . We now demonstrate that the middle subunit of yeast RPA (Rfa2) becomes phosphorylated in two discrete steps during meiosis . Primary Rfa2 phosphorylation occurs early in meiotic progression and is independent of DNA replication, recombination and Mec1 . In contrast, secondary Rfa2 phosphorylation is activated upon initiation of recombination and requires Mec1 . While the primary Rfa2 phosphoisomer is detectable throughout most of meiosis, the secondary Rfa2 phosphoisomer is only transiently generated and begins to disappear soon after recombination is complete . Extensive secondary Rfa2 phosphorylation is observed in a recombination mutant defective for the pachytene checkpoint, indicating that Mec1-dependent Rfa2 phosphorylation does not function to maintain meiotic delay in response to DNA double-strand breaks . Our results suggest that Mec1-dependent RPA phosphorylation could be involved in regulating recombination rather than cell cycle or meiotic progression. J Biol Chem, 2002 Feb 22, 277(8), 6530 - 5 Epub 2001 Nov 28. Identification of a region of RyR1 that participates in allosteric coupling with the alpha(1S) (Ca(V)1.1) II-III loop; Proenza C et al.; In skeletal muscle, excitation-contraction (EC) coupling and retrograde signaling are thought to result from direct interactions between the ryanodine receptor (RyR1) and the alpha(1) subunit of the dihydropyridine receptor (alpha(1S)) . Previous work has shown that the s53 region of alpha(1S) (residues 720-765 in the II-III loop) and regions R10 (1635-2636) and R9 (2659-3720) of RyR1 are involved in this signaling . Using the yeast two-hybrid system, we here report an interaction between s53 and the sR16 region of RyR1 (1837-2168, within R10), whereas no interaction was seen using upstream residues of the alpha(1S) II-III loop (s31, 666-709) . The specificity of the s53-sR16 interaction was tested by using fragments of the cardiac RyR (RyR2) and DHPR (alpha(1C)) that correspond to sR16 and s53, respectively . No interaction was observed for sR16 x c53 (alpha(1C) 850-897), but weak interaction was occasionally observed for s53 x cR16 (RyR2 1817-2142) . To test the functional significance of the s53 x sR16 interaction, we expressed in dyspedic myotubes a chimeric RyR (chimeraR16) in which sR16 was substituted for the corresponding region of RyR2 . ChimeraR16 was found to mediate weak skeletal-type EC coupling . To test the necessity of sR16 sequence for coupling, we used "chimeraR16-rev," in which sR16 and a small upstream region of RyR1 were replaced by RyR2 sequence . ChimeraR16-rev did not differ from RyR1 in its ability to mediate EC coupling . Thus, interaction between residues 720-765 of alpha(1S) and residues 1837-2168 of RyR1 appears to contribute to but is not essential for EC coupling in skeletal muscle. Hum Mol Genet, 2001 Nov 1, 10(23), 2651 - 60 Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex; Otsuki T et al.; Fanconi anemia (FA) is a genetic disorder that predisposes to hematopoietic failure, birth defects and cancer . We identified an interaction between the FA protein, FANCA and brm-related gene 1 (BRG1) product . BRG1 is a subunit of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity . FANCA was demonstrated to associate with the endogenous SWI/SNF complex . We also found a significant increase in the molecular chaperone, glucose-regulated protein 94 (GRP94) among BRG1-associated factors isolated from a FANCA-mutant cell line, which was not seen in either a normal control cell line or the mutant line complemented by wild-type FANCA . Despite this specific difference, FANCA did not appear to be absolutely required for in vitro chromatin remodeling . Finally, we demonstrated co-localization in the nucleus between transfected FANCA and BRG1 . The physiological action of FANCA on the SWI/SNF complex remains to be clarified, but our work suggests that FANCA may recruit the SWI/SNF complex to target genes, thereby enabling coupled nuclear functions such as transcription and DNA repair. EMBO J, 2001 Dec 3, 20(23), 6889 - 98 The intracellular location of two aminoacyl-tRNA synthetases depends on complex formation with Arc1p; Galani K et al.; In yeast, two aminoacyl-tRNA synthetases, MetRS and GluRS, are associated with Arc1p . We have studied the mechanism of this complex formation and found that the non-catalytic N-terminally appended domains of MetRS and GluRS are necessary and sufficient for binding to Arc1p . Similarly, it is the N-terminal domain of Arc1p that contains distinct but overlapping binding sites for MetRS and GluRS . Localization of Arc1p, MetRS and GluRS in living cells using green fluorescent protein showed that these three proteins are cytoplasmic and largely excluded from the nucleus . However, when their assembly into a complex is inhibited, significant amounts of MetRS, GluRS and Arc1p can enter the nucleus . We suggest that the organization of aminoacyl-tRNA synthetases into a multimeric complex not only affects catalysis, but is also a means of segregating the tRNA- aminoacylation machinery mainly to the cytoplasmic compartment. EMBO J, 2001 Dec 3, 20(23), 6856 - 65 Release of U18 snoRNA from its host intron requires interaction of Nop1p with the Rnt1p endonuclease; Giorgi C et al.; An external stem, essential for the release of small nucleolar RNAs (snoRNAs) from their pre-mRNAs, flanks the majority of yeast intron-encoded snoRNAs . Even if this stem is not a canonical Rnt1p substrate, several experiments have indicated that the Rnt1p endonuclease is required for snoRNA processing . To identify the factors necessary for processing of intron-encoded snoRNAs, we have raised in vitro extracts able to reproduce such activity . We found that snoRNP factors are associated with the snoRNA- coding region throughout all the processing steps, and that mutants unable to assemble snoRNPs have a processing-deficient phenotype . Specific depletion of Nop1p completely prevents U18 snoRNA synthesis, but does not affect processing of a dicistronic snoRNA-coding unit that has a canonical Rnt1p site . Correct cleavage of intron-encoded U18 and snR38 snoRNAs can be reproduced in vitro by incubating together purified Nop1p and Rnt1p . Pull-down experiments showed that the two proteins interact physically . These data indicate that cleavage of U18, snR38 and possibly other intron-encoded snoRNAs is a regulated process, since the stem is cleaved by the Rnt1p endonuclease only when snoRNP assembly has occurred. EMBO J, 2001 Dec 3, 20(23), 6816 - 25 CBP/p300 and muscle differentiation: no HAT, no muscle; Polesskaya A et al.; Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both muscle-specific and ubiquitous genes . Two distinct families of transcriptional co-activators, GCN5/PCAF and CREB-binding protein (CBP)/p300, are crucial to this process . While both possess histone acetyl-transferase (HAT) activity, previous studies have failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation . We have addressed this issue directly using a chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant . Our results clearly demonstrate that CBP/p300 HAT activity is critical for myogenic terminal differentiation . Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression . These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program. EMBO J, 2001 Dec 3, 20(23), 6648 - 59 Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores; Fraschini R et al.; The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3, Bub1, Bub3 and Mps1 proteins, prevents anaphase entry and mitotic exit by inhibiting the anaphase promoting complex activator Cdc20 in response to monopolar attachment of sister kinetochores to spindle fibres . We show here that Cdc20, which had previously been shown to interact physically with Mad2 and Mad3, associates also with Bub3 and association is up-regulated upon checkpoint activation . Moreover, co-fractionation experiments suggest that Mad2, Mad3 and Bub3 may be concomitantly present in protein complexes with Cdc20 . Formation of the Bub3-Cdc20 complex requires all kinetochore checkpoint proteins but, surprisingly, not intact kinetochores . Conversely, point mutations altering the conserved WD40 motifs of Bub3, which might be involved in the formation of a beta-propeller fold devoted to protein-protein interactions, disrupt its association with Mad2, Mad3 and Cdc20, as well as proper checkpoint response . We suggest that Bub3 could serve as a platform for interactions between kinetochore checkpoint proteins, and its association with Mad2, Mad3 and Cdc20 might be instrumental for checkpoint activation. Plant Mol Biol, 2001 Nov, 47(5), 663 - 75 Identification of arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein; Huang Z et al.; Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is required for virus movement . The CaMV movement protein (MP) was used in a yeast 2-hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-interacting (MPI) proteins . Three different clones were found encoding proteins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV MP . The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants . A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the 2-hybrid system . MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis . MPI7 is related in sequence, size and hydropathy profile to mammalian proteins (such as rat PRA1) described as a rab acceptor . The gene encoding MPI7 is expressed widely is Arabidopsis plants . and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentrated in punctate spots . In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed . At these sites, fluorescence resonance energy transfer (FRET) between fluorophores was observed indicating an interaction in planta between the CaMV MP and MPI7. Yi Chuan Xue Bao, 2001 Nov, 28(11), 1077 - 84 {Bioinformatics analysis of autophagy 5 gene structure}; Chen Y et al.; In order to study the relationship between autophagy and apoptosis, APG5 gene structure was revealed by bioinformatics analysis and meantime a new isoform resulted from alternative splicing of the hAPG5 gene was confirmed, which was hereby designated as human autophagy 5 beta (hAPG5 beta; LOCUS AF293841, GenBank) . We cloned and sequenced the cDNAs from fetal brain and B cell cDNA libraries using the known hAPG5 cDNA open reading frame sequences as primers . The cDNA obtained from the human fetal brain cDNA library was identical to the known hAPG5 cDNA . However, the cDNA from adult brain cDNA library was 129 bp shorter in length, lacking the sequence corresponding to those from positions 434 to 563 of the hAPG5 cDNA . Through search in public database and sequence comparisons and assembly 4 related sequences, APG5 genomic sequence was obtained . We found that the hAPG5 gene had 8 exons, and those sequences missing in hAPG5 beta cDNA exactly corresponded to exon 3 . By bioinformatics software, the structure of introns, exons, splicing sites, promoter and polyA were demonstrated . Moreover, we were able to express both hAGP5 and 5 beta cDNA clones in human hepatocytes and HeLa cells using pEGFP-C1 vector . In conclusion, our data indicate that a systematic bioinformatics method of finding protein diversity from alternative splicing is a good approach in post-genome biology. Mol Biotechnol, 2001 Oct, 19(2), 169 - 77 Virus-like particles as vaccine adjuvants; Gilbert SC; Virus-like particles (VLPs) consist of one or more viral coat proteins that assemble into particles . They can be taken up by antigen presenting cells (APC), peptides derived from them are presented on MHC class I molecules at the cell surface, and thereby prime a CD8+ T cell response, either against the particle-forming protein itself (such as Hepatitis B surface antigen) or additional peptide sequences that are produced as fusions with the particle-forming protein . This article describes the preparation of Ty-VLPs in Saccharomyces cerevisiae, a system that can easily be handled in the laboratory or scaled up for manufacture, and is safe in use. Proc Natl Acad Sci U S A, 2001 Dec 4, 98(25), 14304 - 9 Epub 2001 Nov 27. A vertebrate fatty acid desaturase with Delta 5 and Delta 6 activities; Hastings N et al.; Delta5 and Delta6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids arachidonic, eicosapentaenoic, and docosahexaenoic acids . They are encoded by distinct genes in mammals and Caenorhabditis elegans . This paper describes a cDNA isolated from zebrafish (Danio rerio) with high similarity to mammalian Delta6 desaturase genes . The 1,590-bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b(5) domain and three histidine boxes, believed to be involved in catalysis . When the zebrafish cDNA was expressed in Saccharomyces cerevisiae it conferred the ability to convert linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) to their corresponding Delta6 desaturated products, 18:3n-6 and 18:4n-3 . However, in addition it conferred on the yeast the ability to convert di-homo-gamma-linoleic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), respectively, indicating that the zebrafish gene encodes an enzyme having both Delta5 and Delta6 desaturase activity . The zebrafish Delta5/Delta6 desaturase may represent a component of a prototypic vertebrate polyunsaturated fatty acids biosynthesis pathway. J Cell Biol, 2001 Nov 26, 155(5), 797 - 808 Epub 2001 Nov 26. A Gip1p-Glc7p phosphatase complex regulates septin organization and spore wall formation; Tachikawa H et al.; Sporulation of Saccharomyces cerevisiae is a developmental process in which a single cell is converted into four haploid spores . GIP1, encoding a developmentally regulated protein phosphatase 1 interacting protein, is required for spore formation . Here we show that GIP1 and the protein phosphatase 1 encoded by GLC7 play essential roles in spore development . The gip1Delta mutant undergoes meiosis and prospore membrane formation normally, but is specifically defective in spore wall synthesis . We demonstrate that in wild-type cells, distinct layers of the spore wall are deposited in a specific temporal order, and that gip1Delta cells display a discrete arrest at the onset of spore wall deposition . Localization studies revealed that Gip1p and Glc7p colocalize with the septins in structures underlying the growing prospore membranes . Interestingly, in the gip1Delta mutant, not only is Glc7p localization altered, but septins are also delocalized . Similar phenotypes were observed in a glc7-136 mutant, which expresses a Glc7p defective in interacting with Gip1p . These results indicate that a Gip1p-Glc7p phosphatase complex is required for proper septin organization and initiation of spore wall formation during sporulation. J Cell Biol, 2001 Nov 26, 155(5), 763 - 74 Epub 2001 Nov 26. Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15 during chromosome segregation; Kang J et al.; We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae . Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle . We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle . The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP) . Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function . Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants . Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1-Sli15 are functionally important in vivo . Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo . Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules. J Biol Chem, 2002 Feb 1, 277(5), 3357 - 63 Epub 2001 Nov 27. Cysteine-directed cross-linking to subunit B suggests that subunit E forms part of the peripheral stalk of the vacuolar H+-ATPase; Arata Y et al.; We have employed a combination of site-directed mutagenesis and covalent cross-linking to identify subunits in close proximity to subunit B in the vacuolar H(+)-ATPase (V-ATPase) complex . Unique cysteine residues were introduced into a Cys-less form of subunit B, and the V-ATPase complex in isolated vacuolar membranes from each mutant strain was reacted with the bifunctional, photoactivable maleimide reagent 4-(N-maleimido)benzophenone . Photoactivation resulted in cross-linking of the unique sulfhydryl groups on subunit B with other subunits in the complex . Four of the eight mutants constructed containing a unique cysteine residue at Ala(15), Lys(45), Glu(494), or Thr(501) resulted in the formation of cross-linked products, which were recognized by Western blot analysis using antibodies against both subunits B and E . These products had a molecular mass of 84 kDa, consistent with a cross-linked product of subunits B and E . Molecular modeling of subunit B places Ala(15) and Lys(45) near the top of the V(1) structure (i.e . farthest from the membrane), whereas Glu(494) and Thr(501) are predicted to reside near the bottom of V(1), with all four residues predicted to be oriented toward the external surface of the complex . A model incorporating these and previous data is presented in which subunit E exists in an extended conformation on the outer surface of the A(3)B(3) hexamer that forms the core of the V(1) domain . This location for subunit E suggests that this subunit forms part of the peripheral stalk of the V-ATPase that links the V(1) and V(0) domains. J Biol Chem, 2002 Feb 1, 277(5), 3767 - 75 Epub 2001 Nov 27. Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein (APP); Scheinfeld MH et al.; The familial Alzheimer's disease gene product amyloid beta precursor protein (APP) is sequentially processed by beta- and gamma-secretases to generate the Abeta peptide . The biochemical pathway leading to Abeta formation has been extensively studied since extracellular aggregates of Abeta peptides are considered the culprit of Alzheimer's disease . Aside from its pathological relevance, the biological role of APP processing is unknown . Cleavage of APP by gamma-secretase releases, together with Abeta, a COOH-terminal APP intracellular domain, termed AID . This peptide has recently been identified in brain tissue of normal control and patients with sporadic Alzheimer's disease . We have previously shown that AID acts as a positive regulator of apoptosis . Nevertheless, the molecular mechanism by which AID regulates this process remains unknown . Hoping to gain clues about the function of APP, we used the yeast two-hybrid system to identify interaction between the AID region of APP and JNK-interacting protein-1 (JIP1) . This molecular interaction is confirmed in vitro, in vivo by fluorescence resonance energy transfer (FRET), and in mouse brain lysates . These data provide a link between APP and its processing by gamma-secretase, and stress kinase signaling pathways . These pathways are known regulators of apoptosis and may be involved in the pathogenesis of Alzheimer's disease. J Biol Chem, 2002 Mar 1, 277(9), 6846 - 51 Epub 2001 Nov 27. Insulin receptor substrate-3 functions as transcriptional activator in the nucleus; Kabuta T et al.; The family of insulin receptor substrates (IRSs) has been reported to play important roles for signal transduction of various hormones . Four members of the IRS family have been described . Each IRS is believed to have different functions; however, the distinct physiological roles of each IRS are unclear . This study was undertaken to determine the intracellular localization of IRS-3 . IRS-3 was expressed in COS-7 cells as fusion with a green fluorescent protein (GFP), and subcellular localization of the chimera protein was analyzed by fluorescent microscopy . Surprisingly, GFP-IRS-3 was localized not only adjacent to the plasma membrane but also in the nucleus . We confirmed by immunostaining with anti-IRS-3 antibody that non-fused IRS-3 protein is also localized in the nucleus of COS-7 cells that were transfected with IRS-3 cDNA . In addition, we detected endogenous IRS-3 in the nucleus of isolated rat adipocytes . We then studied subcellular localization of deletion mutants and fragments of IRS-3 fused with GFP . We found that the region corresponding to amino acid residues 192-223 in the phosphotyrosine binding domain played an important role in nuclear localization . This region includes sequences that are unique to IRS-3 . We then investigated intracellular localization of other IRSs fused with GFP . GFP-IRS-1, GFP-IRS-2, and GFP-IRS-4 were mainly localized in the cytosol or plasma membranes . Chimeric protein, Gal4 DNA binding domain fused with IRS-3 C-terminal region, increased transcription of the reporter gene containing Gal4 binding site in human embryonic kidney 293 cells . These results suggest that intracellular localization of IRS-3 is determined by a different mechanism from other IRS proteins, and that IRS-3 possesses a transcription-regulating activity. Bioinformatics, 2001 Nov, 17(11), 1065 - 6 Segmentation of long genomic sequences into domains with homogeneous composition with BASIO software; Ramensky VE et al.; We present a software system BASIO that allows one to segment a sequence into regions with homogeneous nucleotide composition at a desired length scale . The system can work with arbitrary alphabet and therefore can be applied to various (e.g . protein) sequences . Several sequences of complete genomes of eukaryotes are used to demonstrate the efficiency of the software . AVAILABILITY: The BASIO suite is available for non-commercial users free of charge as a set of executables and accompanying segmentation scenarios from To obtain the source code, contact the authors. Bioinformatics, 2001 Nov, 17(11), 1019 - 26 Combining frequency and positional information to predict transcription factor binding sites; Kielbasa SM et al.; MOTIVATION: Even though a number of genome projects have been finished on the sequence level, still only a small proportion of DNA regulatory elements have been identified . Growing amounts of gene expression data provide the possibility of finding coregulated genes by clustering methods . By analysis of the promoter regions of those genes, rather weak signals of transcription factor binding sites may be detected . RESULTS: We introduce the new algorithm ITB, an Integrated Tool for Box finding, which combines frequency and positional information to predict transcription factor binding sites in upstream regions of coregulated genes . Motifs are extracted by exhaustive analysis of regular expression-like patterns and by estimating probabilities of positional clusters of motifs . ITB detects consensus sequences of experimentally verified transcription factor binding sites of the yeast Saccharomyces cerevisiae . Moreover, a number of new binding site candidates with significant scores are predicted . Besides applying ITB on yeast upstream regions, the program is run on human promoter sequences . AVAILABILITY: ITB is available upon request. Z Naturforsch {C}, 2001 Sep-Oct, 56(9-10), 781 - 6 Very-long-chain fatty acid biosynthesis is inhibited by cafenstrole, N,N-diethyl-3-mesitylsulfonyl-1H-1,2,4-triazole-1-carboxamide and its analogs; Takahashi H et al.; The rice herbicide cafenstrole and its analogs inhibited the incorporation of {1-(14)C}-oleate and (2-(14)C}-malonate into very-long-chain fatty acids (VLCFAs), using Scenedesmus cells and leek microsomes from Allium porrum . Although the precise mode of interaction of cafenstrole at the molecular level is not completely clarified by the present study, it is concluded that cafenstrole acts as a specific inhibitor of the microsomal elongase enzyme involved in the biosynthesis of fatty acids with alkyl chains longer than C18 . For a strong VLCFA biosynthesis inhibition an -SO2- linkage of the 1,2,4-triazole-1-carboxamides was required . Furthermore, N,N-dialkyl substitution of the carbamoyl nitrogen and electron-donating groups such as methyl at the benzene ring of 1,2,4-triazole-1-carboxamides produced a strong inhibition of VLCFA formation . A correlation was found between the phytotoxic effect against barnyardgrass (Echinochloa oryzicola) and impaired VLCFA formation. Neurology, 2001 Nov 27, 57(10), 1849 - 57 Gating of myotonic Na channel mutants defines the response to mexiletine and a potent derivative; Desaphy JF et al.; BACKGROUND: Myotonia and periodic paralysis caused by sodium channel mutations show variable responses to the anti-myotonic drug mexiletine . OBJECTIVE: To investigate whether variability among sodium channel mutants results from differences in drug binding affinity or in channel gating . METHODS: Whole-cell sodium currents (I(Na)) were recorded in tsA201 cells expressing human wild-type (WT) and mutant skeletal muscle sodium channels (A1156T, hyperkalemic periodic paralysis; R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia) . RESULTS: At a holding potential (hp) of -120 mV, mexiletine produced a tonic (TB, 0.33 Hz) and a use-dependent (UDB, 10 Hz) block of peak I(Na) with a potency following the order rank R1448C > WT approximately equal A1156T > G1306E . Yet, when assayed from an hp of -180 mV, TB and UDB by mexiletine were similar for the four channels . The different midpoints of channel availability curves found for the four channels track the half-maximum inhibitory value (IC50) measured at -120 mV . Thus differences in the partitioning of channels between the closed and fast-inactivated states underlie the different IC50 measured at a given potential . The mexiletine-derivative, Me7 (alpha-{(2-methylphenoxy)methyl}-benzenemethanamine), behaved similarly but was approximately 5 times more potent than mexiletine . Interestingly, the higher drug concentrations ameliorated the abnormally slower decay rate of myotonic I(Na) . CONCLUSIONS: These results explain the basis of the apparent difference in block of mutant sodium channels by mexiletine and Me7, opening the way to a more rationale drug use and to design more potent drugs able to correct specifically the biophysical defect of the mutation in individual myotonic patients. Gene, 2001 Nov 14, 279(1), 49 - 62 Species-specific mechanisms control the activity of the Pit1/PIT1 phosphate transporter gene promoter in mouse and human; Palmer G et al.; The Pit1 phosphate transporter is involved in regulated phosphate handling in bone forming cells . In this study, we compared the structure of the murine and human Pit1/PIT1 promoters and characterized cis-acting elements controlling Pit1/PIT1 expression . The Pit1/PIT1 promoter sequence and its location relative to the first transcribed exon are conserved and similar transcription factor binding sites are found at identical positions in mouse and human . Luciferase reporter gene assays in transiently transfected mouse ATDC5 chondrocytes and human SaOS-2 osteoblasts indicated that the activity of the mouse Pit1 promoter depends on several cis-acting elements, including ATF/CREB, Sp1 and AP-1 sites, an E-box and a TATA box . In contrast, the activity of the human promoter essentially requires a TATA-like sequence and one single Sp1 site . This Sp1 site binds Sp1, Sp3, as well as unidentified proteins present in SaOS-2 nuclear extracts and co-transfection experiments in SL2 cells indicate that Sp1 and Sp3 activate transcription from the human PIT1 promoter . These data suggest that, despite similarities in promoter structure, changes in the relative importance of conserved transcription factor binding sites cause species-dependent differences in Pit1 promoter function, which allow Sp1-related proteins to play a particularly important role in human. Gene, 2001 Nov 14, 279(1), 1 - 16 The molecular biology of the SIR proteins; Gasser SM et al.; Silent or heritably repressed genes constitute the major fraction of genetic information in higher eukaryotic cells . Budding yeast has very little consecutively repressed DNA, but what exists has served as a paradigm for the molecular analysis of heterochromatin . The major structural constituents of repressed chromatin in yeast are the four core histones and three large chromatin factors called Silent information regulators 2, 3 and 4 . How these components assemble DNA into a state that is refractory to transcription remains a mystery . Nonetheless, there have been many recent insights into their molecular structures . This review examines the impact of these results on our understanding of silencing function in budding yeast. Br J Haematol, 2001 Oct, 115(1), 150 - 2 TAFI antigen and D-dimer levels during normal pregnancy and at delivery; Chabloz P et al.; We have investigated whether the levels of thrombin-activatable fibrinolysis inhibitor (TAFI) were correlated with D-dimer levels during pregnancy and at delivery . From the 10th week of pregnancy to delivery, 519 samples from 144 women (mean age 29.3 +/- 5, range 19-43) were obtained . We confirm the gradual increase of D-dimer levels, and provide reference intervals for D-dimer measurements throughout normal pregnancy . TAFI levels increased moderately during pregnancy but no inverse correlation with D-dimer levels was observed. J Hum Genet, 2001, 46(11), 626 - 32 A novel in vitro system for analyzing parental allele-specific histone acetylation in genomic imprinting; Yoshioka H et al.; One of the obstacles in studying human genomic imprinting is distinguishing the parental origin of alleles in diploid cells . To solve this problem, we have constructed a library of mouse A9 hybrids in which individual clones contain a single human chromosome of known parental origin . Here we extend this in vitro system to the analysis of the role of histone acetylation in the allelic expression of human imprinted genes . The levels of histone H4 acetylation of the imprinted human LIT1, H19, and SNRPN genes were examined by a chromatin immunoprecipitation (ChIP) assay in mouse A9 hybrids with a single human chromosome of known parental origin . We demonstrated that H4 histones associated with the actively expressed alleles of imprinted LIT1, H19, and SNRPN genes were highly acetylated, whereas they were hypoacetylated in the silent alleles . Furthermore, treatment of A9 hybrids with trichostatin A (TSA), an inhibitor of histone deacetylase, resulted in transcriptional reactivation of the silent alleles for LIT1 and SNRPN, suggesting that histone deacetylation is one of the key regulatory mechanisms in genomic imprinting . These results indicate that our monochromosomal hybrid system is a new technology for analyzing histone modifications between parental alleles in human imprinted genes. Neurosci Lett, 2001 Dec 4, 316(1), 50 - 4 Human Nck-associated protein 1 and its binding protein affect the metabolism of beta-amyloid precursor protein with Swedish mutation; Yamamoto A et al.; Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system, and beta-amyloid precursor protein (betaAPP) plays a pivotal role in AD pathology . We previously reported that the suppression of human Nck-associated protein 1 (Nap1) whose expression was down-regulated in sporadic AD led to apoptosis in human neuroblastoma cells, and also its binding protein, hNap1BP was identified . Here, we examined whether these molecules were involved in the regulation of betaAPP metabolism . Human Nap1 and hNap1BP were found not to effect the amount of intracellular betaAPP but induced sAPPalpha secretion . Interestingly, they didn't reduce but slightly increased the extracellular level of Abeta . Furthermore, neither human Nap1 nor hNap1BP influenced the ratio of Abeta42/43 to total Abeta . Taken together, human Nap1 and hNap1BP may play a role in regulation of beta-secretase activity in the processing of betaAPP. Curr Issues Mol Biol, 2001 Oct, 3(4), 71 - 8 The RNase III family: a conserved structure and expanding functions in eukaryotic dsRNA metabolism; Lamontagne B et al.; The last few years have witnessed the appreciation of dsRNA as a regulator of gene expression, a potential antiviral agent, and a tumor suppressor . However, in spite of these clear effects on the cell function, the mechanism that controls dsRNA maturation and stability remains unknown . Recently, the discovery of eukaryotic orthologues of the bacterial dsRNA specific ribonuclease III (RNase III) suggested a central role for these enzymes in the regulation of dsRNA and eukaryotic RNA metabolism in general . This article reviews the structure-function features of the eukaryotic RNase III family and their roles in dsRNA metabolism with an emphasis on the yeast RNase III . Yeast RNase III is involved in the maturation of the majority of snRNAs, snoRNAs, and rRNA . In addition, perturbation of the expression level of yeast RNase III alters meiosis and causes sterility . These basic functions of the yeast RNase III appear to be widely conserved which makes it a good model to understand the importance of eukaryotic dsRNA metabolism. Mol Biol Evol, 2001 Dec, 18(12), 2280 - 8 Study of intrachromosomal duplications among the eukaryote genomes; Achaz G et al.; Complete eukaryote chromosomes were investigated for intrachromosomal duplications of nucleotide sequences . The analysis was performed by looking for nonexact repeats on two complete genomes, Saccharomyces cerevisiae and Caenorhabditis elegans, and four partial ones, Drosophila melanogaster, Plasmodium falciparum, Arabidopsis thaliana, and Homo sapiens . Through this analysis, we show that all eukaryote chromosomes exhibit similar characteristics for their intrachromosomal repeats, suggesting similar dynamics: many direct repeats have their two copies physically close together, and these close direct repeats are more similar and shorter than the other repeats . On the contrary, there are almost no close inverted repeats . These results support a model for the dynamics of duplication . This model is based on a continuous genesis of tandem repeats and implies that most of the distant and inverted repeats originate from these tandem repeats by further chromosomal rearrangements (insertions, inversions, and deletions) . Remnants of these predicted rearrangements have been brought out through fine analysis of the chromosome sequence . Despite these dynamics, shared by all eukaryotes, each genome exhibits its own style of intrachromosomal duplication: the density of repeated elements is similar in all chromosomes issued from the same genome, but is different between species . This density was further related to the relative rates of duplication, deletion, and mutation proper to each species . One should notice that the density of repeats in the X chromosome of C . elegans is much lower than in the autosomes of that organism, suggesting that the exchange between homologous chromosomes is important in the duplication process. J Biol Chem, 2002 Feb 1, 277(5), 3274 - 9 Epub 2001 Nov 21. Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster; Chen Q et al.; Recent studies have shown that trehalose plays a protective role in yeast in a variety of stresses, including heat, freezing and thawing, dehydration, hyperosmotic shock, and oxidant injury . Because (a) heat shock and anoxia share mechanisms that allow organisms to survive, (b) Drosophila melanogaster is tolerant to anoxia, and (c) trehalose is present in flies and is metabolically active, we asked whether trehalose can protect against anoxic stress . Here we report on a new role of trehalose in anoxia resistance in Drosophila . We first cloned the gene trehalose-6-phosphate synthase (tps1), which synthesizes trehalose, and examined the effect of tps1 overexpression as well as mutation on the resistance of Drosophila to anoxia . Upon induction of tps1, trehalose increased, and this was associated with increased tolerance to anoxia . Furthermore, in vitro experiments showed that trehalose reduced protein aggregation caused by anoxia . Homozygous tps1 mutant (P-element insertion into the third intron of the gene) leads to lethality at an early larval stage, and excision of the P-element rescues totally the phenotype . We conclude that trehalose contributes to anoxia tolerance in flies; this protection is likely to be due to a reduction of protein aggregation. Curr Biol, 2001 Nov 13, 11(22), R932 - 4 Membrane transport: ubiquitylation in endosomal sorting; Dupre S et al.; In yeast, membrane proteins from the biosynthetic and endocytic pathways must be ubiquitylated for sorting to inward-budding vesicles in late endosomes, which give rise to multivesicular bodies . A conserved protein complex containing the yeast Vps23p or its mammalian counterpart Tsg101 may act as the ubiquitin receptor. Curr Biol, 2001 Nov 13, 11(22), R920 - 4 DNA repair: how Ku makes ends meet; Doherty AJ et al.; The recently determined crystal structure of the Ku heterodimer, in both DNA-bound and unbound forms, has shed new light on the mechanism by which this protein fulfills its key role in the repair of DNA double-strand breaks. Curr Biol, 2001 Nov 13, 11(22), 1788 - 93 Organized microtubule arrays in gamma-tubulin-depleted Drosophila spermatocytes; Sampaio P et al.; To assess the role of gamma-tubulin in spindle assembly in vivo, we have followed meiosis progression by immunofluorescence and time-lapse video microscopy in gammaTub23C(PI) mutant spermatocytes . We have found that centrosomes associate with large numbers of astral microtubules even though gamma-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells . Several lines of evidence suggest that these cones may be related to wild-type central spindles . First, they are assembled midway through meiosis and elongate during anaphase . Second, they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone . Third, Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones . Finally, ectopic cytokinesis furrows are often formed at the distal end of the cone . Our results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a gamma-tubulin-independent manner in Drosophila spermatocytes . However, gamma-tubulin seems to be essential for spindle assembly in these cells . Finally, our results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles. Curr Biol, 2001 Nov 13, 11(22), 1781 - 7 The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition; Hendrickson C et al.; Clb2 mitotic cyclin inhibits cell cycle progression by preventing mitotic exit and DNA synthesis . To allow cell cycle progression, Clb2 proteolysis is triggered by Cdc20 during the metaphase-to-anaphase (M-A) transition and by Hct1 during mitotic exit and G1 {1-6} . A cis element called the destruction box is required for this proteolysis {7-11} . Recently, an additional cis element called the "KEN box" was also shown to be required for proteolysis of human CDC20 and Securin {3,12} . Using a novel color assay, we show that a Clb2 KEN box is required to target a fusion protein containing the first 124 amino acids of Clb2 for proteolysis . We further show that full-length Clb2 bearing mutations in the KEN box is degraded efficiently during the M-A transition, but poorly during G1 . If the destruction box of Clb2 is mutated in combination with mutation of the KEN box, then this form of Clb2 is more stable than Clb2 bearing either mutation by itself during both M-A and G1 . Our results show that the KEN box and the destruction box act together during both M-A and G1 to regulate Clb2 proteolysis. Mech Ageing Dev, 2002 Jan, 123(2-3), 65 - 73; discussion 75-9 Keynote: mechanisms of senescence--complificationists versus simplificationists; Martin GM; It strikes me that among our relatively small community of gerontologists concerned with genetic approaches to our science, there is somewhat of a dichotomization . On the one hand, there are those of us, like myself, who tend to be dour 'complificationists' . Journalists talk to us, but are usually disappointed by the encounter . We are perhaps too impressed with the enormous diversity of genetic modulations of human senescence and with our interpretations of the implications of the evolutionary biological theory of senescence, namely that senescent phenotypes per se are non-adaptive, non-determinative, subject to stochastic events as well as highly polygenic modulations, with resulting wide variability in mechanisms of senescence among and within species . Quite happily, however, there are wonderful optimists among us . They seem to be convinced that there are likely to be a rather small number of major gene effects for a few major mechanisms . They include most Saccharomyces cerevisiae and Caenorhabditis elegans geneticists, some Drosophila melanogaster geneticists, and some mouse geneticists . They also include caloric restriction enthusiasts . Let's call these colleagues 'simplificationists' . Journalists and friends generally find them to be delightful companions . Where does the truth lie? Perhaps the truth lies somewhere between these two extremes and is largely dependent upon the organisms and the range of environments being investigated. Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13808 - 13 Impairment of MAD2B-PRCC interaction in mitotic checkpoint defective t(X;1)-positive renal cell carcinomas; Weterman MA et al.; The papillary renal cell carcinoma (RCC)-associated (X;1)(p11;q21) translocation fuses the genes PRCC and TFE3 and leads to cancer by an unknown molecular mechanism . We here demonstrate that the mitotic checkpoint protein MAD2B interacts with PRCC . The PRCCTFE3 fusion protein retains the MAD2B interaction domain, but this interaction is impaired . In addition, we show that two t(X;1)-positive RCC tumor cell lines are defective in their mitotic checkpoint . Transfection of PRCCTFE3, but not the reciprocal product TFE3PRCC, disrupts the mitotic checkpoint in human embryonic kidney cells . Our results suggest a dominant-negative effect of the PRCCTFE3 fusion gene leading to a mitotic checkpoint defect as an early event in papillary RCCs. Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13705 - 9 Surface structure of the COPII-coated vesicle; Matsuoka K et al.; The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated vesicle surfaces . The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/24p >> Sec13/31p . Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated vesicles . Sec23/24 resembles a bow tie, and Sec13/31p contains terminal bilobed globular structures bordering a central rod . The surface structure of COPII vesicles revealed a coat built with polygonal units . The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of Sec13/31 . Partially uncoated profiles revealed strands of Sec13/31p stripped from the vesicle surface . We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat. J Biol Chem, 2002 Jan 25, 277(4), 2716 - 24 Epub 2001 Nov 20. Novel vacuolar H+-ATPase complexes resulting from overproduction of Vma5p and Vma13p; Keenan Curtis K et al.; The vacuolar H(+)-ATPase (V-ATPase) is a multisubunit complex composed of two sectors: V(1), a peripheral membrane sector responsible for ATP hydrolysis, and V(0), an integral membrane sector that forms a proton pore . Vma5p and Vma13p are V(1) sector subunits that have been implicated in the structural and functional coupling of the V-ATPase . Cells overexpressing Vma5p and Vma13p demonstrate a classic Vma(-) growth phenotype . Closer biochemical examination of Vma13p-overproducing strains revealed a functionally uncoupled V-ATPase in vacuolar vesicles . The ATP hydrolysis rate was 72% of the wild-type rate; but there was no proton translocation, and two V(1) subunits (Vma4p and Vma8p) were present at lower levels . Vma5p overproduction moderately affected both V-ATPase activity and proton translocation without affecting enzyme assembly . High level overexpression of Vma5p and Vma13p was lethal even in wild-type cells . In the absence of an intact V(0) sector, overproduction of Vma5p and Vma13p had a more detrimental effect on growth than their deletion . Overproduced Vma5p associated with cytosolic V(1) complexes; this association may cause the lethality. J Am Chem Soc, 2001 Nov 28, 123(47), 11608 - 13 Design of allele-specific protein methyltransferase inhibitors; Lin Q et al.; Protein arginine methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to arginine side chains in target proteins, regulate transcription, RNA processing, and receptor-mediated signaling . To specifically address the functional role of the individual members of this family, we took a "bump-and-hole" approach and designed a series of N(6)-substituted S-adenosylhomocysteine (SAH) analogues that are targeted toward a yeast protein methyltransferase RMT1 . A point mutation was identified (E117G) in Rmt1 that renders the enzyme susceptible to selective inhibition by the SAH analogues . A mass spectrometry based enzymatic assay revealed that two compounds, N(6)-benzyl- and N(6)-naphthylmethyl-SAH, can inhibit the mutant enzyme over the wild-type with the selectivity greater than 20 . When the E117G mutation was introduced into the Saccharomyces cerevisiae chromosome, the methylation of Npl3p, a known in vivo Rmt1 substrate, could be moderately reduced by N(6)-naphthylmethyl-SAH in the resulting allele . In addition, an N(6)-benzyl-SAM analogue was found to serve as an orthogonal SAM cofactor . This analogue is preferentially utilized by the mutant methyltransferase relative to the wild-type enzyme with a selectivity greater than 67 . This specific enzyme/inhibitor and enzyme/substrate design should be applicable to other members of this protein family and facilitate the characterization of protein methyltransferase function in vivo when combined with RNA expression analysis. Biochem Biophys Res Commun, 2001 Nov 30, 289(2), 463 - 71 Mouse homologue of coq7/clk-1, longevity gene in Caenorhabditis elegans, is essential for coenzyme Q synthesis, maintenance of mitochondrial integrity, and neurogenesis; Nakai D et al.; coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, which showed sluggish behavior and an extended life span . Mouse coq7 is homologous to Saccharomyces cerevisiae coq7/cat5 that is required for biosynthesis of coenzyme Q (CoQ), an essential cofactor in mitochondrial respiration . Here we generated COQ7-deficient mice to investigate the biological role of COQ7 in mammals . COQ7-deficient mouse embryos failed to survive beyond embryonic day 10.5, exhibiting small-sized body and delayed embryogenesis . Morphological studies showed that COQ7-deficient neuroepithelial cells failed to show the radial arrangement in the developing cerebral wall, aborting neurogenesis at E10.5 . Electron microscopic analysis further showed the enlarged mitochondria with vesicular cristae and enlarged lysosomes filled with disrupted membranes, which is consistent with mitochondriopathy . Biochemical analysis demonstrated that COQ7-deficient embryos failed to synthesize CoQ(9), but instead yielded demethoxyubiquinone 9 (DMQ(9)) . Cultured embryonic cells from COQ7-deficient mice were rescued by adding bovine fetal serum in vitro, but exhibited slowed cell proliferation, which resembled to the phenotype of clk-1 with delayed cell divisions . The result implied the essential role of coq7 in CoQ synthesis, maintenance of mitochondrial integrity, and neurogenesis in mice. Arch Biochem Biophys, 2001 Dec 1, 396(1), 56 - 64 Nafenopin-, ciprofibroyl-, and palmitoyl-CoA conjugation in vitro: kinetic and molecular characterization of marmoset liver microsomes and expressed MLCL1; Drogemuller CJ et al.; Acyl-CoA conjugation of xenobiotic carboxylic acids is catalyzed by hepatic microsomal long-chain fatty acid CoA ligases (LCL, EC 6.2.1.3) . Marmosets (Callithrix jacchus) are considered genetically closer to humans than rodents and are used in pharmacological and toxicological studies . We have demonstrated that marmoset liver microsomes catalyze nafenopin-, ciprofibroyl-, and palmitoyl-CoA conjugation and that only palmitoyl-CoA conjugation is significantly upregulated (1.7-fold, P < 0.02) by a high fat diet . Additionally, the apparent C(50) values for nafenopin-, ciprofibroyl-, and palmitoyl-CoA conjugation of 149.7, 413.4, and 3.4 microM were comparable to those reported for human liver microsomes viz, 213.7, 379.8, and 3.4 microM, respectively . Comparison with human data was enabled by the cloning of a full-length marmoset cDNA (MLCL1) that encoded a 698-amino-acid protein sharing 83% similarity with rat liver acyl-CoA synthetase (ACS1) and 93 and 90% similarity with human liver LCL1 and LCL2, respectively . MLCL1 transiently expressed in COS-7 cells activated nafenopin (C(50) 192.9 microM), ciprofibrate (C(50) 168.7 microM), and palmitic acid (C(50) 4.5 microM) to their respective CoA conjugates . This study also demonstrated that the sigmoidal kinetics observed for nafenopin- and ciprofibroyl-CoA conjugation were not unique to human liver microsomes but were also characteristic of marmoset liver microsomes and recombinant MLCL1 . More extensive characterization of the substrate specificity of marmoset LCL isoforms will aid in determining further the suitability of marmosets as a model for human xenobiotic metabolism via acyl-CoA conjugation . (c)2001 Elsevier Science. Biochem Cell Biol, 2001, 79(5), 603 - 12 The dynamics of karyopherin-mediated nuclear transport; Marelli M et al.; The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs . Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport . Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process . Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC . The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport . These components and their potiential roles in nucleo-cytoplasmic transport are discussed. Biochem Cell Biol, 2001, 79(5), 557 - 68 Crowbars and ratchets: hsp100 chaperones as tools in reversing protein aggregation; Glover JR et al.; Molecular chaperones have the capacity to prevent inappropriate interactions between aggregation-prone folding or unfolding intermediates created in the cell during protein synthesis or in response to physical and chemical stress . What happens when surveillance by molecular chaperones is evaded or overwhelmed and aggregates accumulate? Recent progress in the elucidation of Hsp100/Clp function suggests that intracellular aggregates or stable complexes can be progressively dissolved by the action of chaperones that act as molecular crowbars or ratchets . These insights set the stage for new progress in the understanding and treatment of diseases of protein folding. Nat Rev Mol Cell Biol, 2001 Nov, 2(11), 815 - 26 Men and sin: what's the difference? Bardin AJ, Amon A. A conserved signalling cascade--termed the mitotic-exit network in budding yeast and the septation-initiation network in fission yeast--controls key events during exit from mitosis and cytokinesis . Although the components of these signalling networks are highly conserved between the two yeasts, the outputs seem quite different . How, then, do these two pathways function, and how are they regulated? Nat Cell Biol, 2001 Nov, 3(11), 966 - 72 Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1; Tanaka K et al.; Checkpoint responses change as cells proceed through the cell cycle . Here we describe a novel checkpoint gene in fission yeast, mrc1 (mediator of replication checkpoint), that confers activation of the checkpoint kinase Cds1 to DNA synthesis (S) phase . Mrc1 associates with Cds1 and is required for regulation of Cds1 by the checkpoint kinase Rad3 . Mrc1 is regulated by the cell cycle, with the appearance of Mrc1 mRNA and protein coinciding with S phase . We propose that coordinated expression of Mrc1 with replication control proteins helps to ensure activation of the appropriate checkpoint response during DNA replication. Nat Cell Biol, 2001 Nov, 3(11), 958 - 65 Mrc1 transduces signals of DNA replication stress to activate Rad53; Alcasabas AA et al.; Cells experiencing DNA replication stress activate a response pathway that delays entry into mitosis and promotes DNA repair and completion of DNA replication . The protein kinases ScRad53 and SpCds1 (in baker's and fission yeast, respectively) are central to this pathway . We describe a conserved protein Mrc1, mediator of the replication checkpoint, required for activation of ScRad53 and SpCds1 during replication stress . mrc1 mutants are sensitive to hydroxyurea and have a checkpoint defect similar to rad53 and cds1 mutants . Mrc1 may be the replicative counterpart of Rad9 and Crb2, which are required for activating ScRad53 and Chk1 in response to DNA damage. Genet Med, 2001 Nov-Dec, 3(6), 393 - 8 Functional significance of PMM2 mutations in mildly affected patients with congenital disorders of glycosylation Ia; Westphal V et al.; PURPOSE: Congenital disorders of glycosylation (CDG) result from mutations in N-glycan biosynthesis . Mutations in phosphomannomutase (PMM2) cause CDG-Ia . Here, we report four clinically mild patients and their mutations in PMM2 . METHODS: Analysis of the PMM2 cDNA and gene revealed the mutations affecting the glycosylation efficiency . RESULTS: The patients have 30% to 50% normal PMM activity in fibroblasts due to different mutations in PMM2, and we studied the effect of each mutation on the PMM activity in a Saccharomyces cerevisiae expression system . CONCLUSIONS: Each patient carried a severe mutation that decreased the PMM activity to less than 10% as well as a relatively mild mutation . A new mutation, deletion of base 24, changed the reading frame . The C9Y, C241S, and L32R mutations showed 27% to 45% activity when expressed in the eukaryotic expression system, and the more severe D148N was shown to be thermolabile. J Immunol, 2001 Dec 1, 167(11), 6366 - 73 The association of Aiolos transcription factor and Bcl-xL is involved in the control of apoptosis; Rebollo A et al.; We have analyzed the mechanism implicated in the control of the anti-apoptotic role of Bcl-xL . We show that IL-4 deprivation induces apoptosis, but does not modulate Bcl-xL expression . Because Bcl-xL does not promote cell survival in the absence of IL-4, we investigate the mechanism by which Bcl-xL was unable to inhibit apoptosis . Using yeast two-hybrid system, coimmunoprecipitation, and indirect immunofluorescence techniques, we found that Bcl-xL interacts with the transcription factor Aiolos in IL-4-stimulated cells, increasing upon IL-4 deprivation . IL-4 does not promote translocation of Aiolos or Bcl-xL, but induces tyrosine phosphorylation of Aiolos, which is required for dissociation from Bcl-xL . Transfection experiments confirm that cells overexpressing Bcl-xL are able to prevent apoptosis in the absence of IL-4 . On the contrary, cells that overexpress Bcl-xL and Aiolos are unable to block apoptosis in the absence of IL-4 . We propose a model for the regulation of the Bcl-xL anti-apoptotic role via Aiolos. J Biol Chem, 2002 Jan 25, 277(4), 3047 - 52 Epub 2001 Nov 19. Purification and characterization of the human elongator complex; Hawkes NA et al.; Human Elongator complex was purified to virtual homogeneity from HeLa cell extracts . The purified factor can exist in two forms: a six-subunit complex, holo-Elongator, which has histone acetyltransferase activity directed against histone H3 and H4, and a three-subunit core form, which does not have histone acetyltransferase activity despite containing the catalytic Elp3 subunit . Elongator is a component of early elongation complexes formed in HeLa nuclear extracts and can interact directly with RNA polymerase II in solution . Several human homologues of the yeast Elongator subunits were identified as subunits of the human Elongator complex, including StIP1 (STAT-interacting protein 1) and IKAP (IKK complex-associated protein) . Mutations in IKAP can result in the severe human disorder familial dysautonomia, raising the possibility that this disease might be due to compromised Elongator function and therefore could be a transcription disorder. J Biol Chem, 2002 Feb 15, 277(7), 5631 - 6 Epub 2001 Nov 19. Gemin5, a novel WD repeat protein component of the SMN complex that binds Sm proteins; Gubitz AK et al.; The survival of motor neurons (SMN) protein is the product of the disease gene of spinal muscular atrophy and is found both in the cytoplasm and the nucleus, where it is concentrated in gems . SMN is part of a multi-protein complex that includes Gemin2, Gemin3, and Gemin4 . The SMN complex plays an important role in the cytoplasmic assembly of small nuclear ribonucleoproteins (snRNPs) and likely other RNPs in pre-mRNA splicing and in the assembly of transcriptosomes . Here, we report the identification of an additional component of the SMN complex, a novel WD repeat protein termed Gemin5 . Gemin5 binds SMN directly and is a component of the SMN complex . Furthermore, Gemin5 interacts with several of the snRNP core proteins including SmB, SmD1, SmD2, SmD3, and SmE, suggesting that it participates in the activities of the SMN complex in snRNP assembly . Immunolocalization studies demonstrate that Gemin5 is found in the cytoplasm and in the nucleus, where it colocalizes with SMN in gems . The presence of 13 WD repeat domains in the amino-terminal half of Gemin5 and a coiled-coil motif near its carboxyl terminus indicate that it may form a large heteromeric complex and engage in multiple interactions. J Biol Chem, 2002 Feb 15, 277(7), 4797 - 805 Epub 2001 Nov 19. Splitting the two pore domains from TOK1 results in two cationic channels with novel functional properties; Saldana C et al.; Potassium channels are membrane-spanning proteins with several transmembrane segments and a single pore region where ion conduction takes place (Biggin, P . C., Roosild, T., and Choe, S . (2000) Curr . Opin . Struct . Biol . 4, 456-461; Doyle, D . A., Morais Cabral, J., Pfuetzner, R . A., Kuo, A., Gulbis, J . M., Cohen, S . L., Chait, B . T., and MacKinnon, R . (1998) Science 280, 69-77) . TOK1, a potassium channel identified in the yeast Saccharomyces cerevisiae, was the first described member from a growing new family of potassium channels with two pore domains in tandem (2P) (Ketchum, K . A., Joiner, W . J., Sellers, A . J., Kaczmarek, L . K., and Goldstein, S . A . (1995) Nature 376, 690-695) . In an attempt to understand the relative contribution of each one of the 2P from TOK1 to the functional properties of this channel, we split and expressed the pore domains separately or in combination . Expression of the two domains separately rescued a potassium transport-deficient yeast mutant, suggesting that each domain forms functional potassium-permeable channels in yeast . In Xenopus laevis oocytes expression of each pore domain resulted in the appearance of unique inwardly rectifying cationic channels with novel gating and pharmacological properties . Both pore domains were poorly selective to potassium; however, upon co-expression they partially restored TOK1 channel selectivity . The single channel conductance was different in both pore domains with 7 +/- 1 (n = 12) and 15 +/- 2 (n = 12) picosiemens for the first and second domain, respectively . In light of the known structure of the Streptomyces lividans KcsA potassium channel pore (see Doyle et al . above), these results suggest a novel non-four-fold-symmetric architecture for 2P potassium-selective channels. J Biol Chem, 2002 Feb 1, 277(5), 3599 - 605 Epub 2001 Nov 19. Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response; Honda Y et al.; Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation . The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain . The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase . Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage . A yeast two-hybrid screen showed that DNA topoisomerase IIbeta-binding protein 1 (TopBP1) interacted with hHYD . Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators . Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1 . TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with gamma-H2AX nuclear foci in DNA breaks . These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response. Mol Genet Genomics, 2001 Nov, 266(3), 363 - 73 Characterization of Chlamydomonas mutants defective in the H subunit of Mg-chelatase; Chekounova E et al.; Two chlorophyll-deficient mutants of Chlamydomonas reinhardtii, chl1 and brs-1, are light sensitive and, when grown heterotrophically in the dark, accumulate protoporphyrin IX and exhibit yellow/orange pigmentation . The lesions in both mutants were mapped to the gene (CHLH) for the plastid-localized H subunit of the heterotrimeric magnesium chelatase that catalyzes the insertion of magnesium into protoporphyrin IX . The genetic defects in the mutants could be assigned to +1 frameshift mutations in exon 9 (chl1) and exon 10 (brs-1) of the CHLH gene . In both mutants, the H subunit of magnesium chelatase was undetectable, but, as shown for chl1, the steady-state levels of the I and D subunits were unaltered in comparison to wild type . The CHLH gene exhibits marked light inducibility: levels of both the mRNA and the protein product are strongly increased when cultures are shifted from from the dark into the light, suggesting that this protein may play a crucial role in the light regulation of chlorophyll biosynthesis. Int J Oncol, 2001 Dec, 19(6), 1325 - 32 Interaction of SV40 large T antigen with components of the nucleo/cytoskeleton; Klawitz I et al.; The SV40 large T antigen is a viral oncoprotein which performs multiple interactions with cellular factors to achieve a proliferative state required for viral replication as well as for transformation . The major targets in this scenario are members of the Rb family, pRb, p107, and p130, and tumor suppressor protein p53 . These interactions of large T with Rb proteins and p53 are required but not sufficient for transformation . To search for unknown interaction partners of large T that might participate in its transforming activity we employed the yeast two-hybrid system . Screening a cDNA library from a large T-induced brain tumor cell line revealed a total of 86 positive clones representing 37 individual clones . Of these, four clones were selected for further analyses . Interestingly, the cDNA inserts of these clones coded for different components of the cytoskeleton, lamin C, laminin gamma1, thymosin beta4, and gelsolin . Complex formation between large T and these proteins was confirmed in vitro . Interaction of large T with these components might influence activities such as intracellular transport, signal transduction, adhesion, or migration. Int J Oncol, 2001 Dec, 19(6), 1249 - 54 Identity between the PCPH proto-oncogene and the CD39L4 (ENTPD5) ectonucleoside triphosphate diphosphohydrolase gene; Paez JG et al.; PCPH was initially defined as a proto-oncogene on the basis of its frequent detection as an activated oncogene in tumorigenic Syrian hamster embryo fibroblast cell lines converted to the neoplastic state by a single treatment with the carcinogen 3-methylcholanthrene (MC) . Further studies identified the translation product of the PCPH gene as a ribonucleotide-binding protein with special affinity for ribonucleoside diphosphates . Later, we showed that the PCPH protein was homologous to the product of the yeast GDA1 gene and demonstrated that it had intrinsic guanosine diphosphatase activity, although it did not complement the disrupted phenotype when expressed in gda1 null Saccharomyces cerevisiae strains . These results indicated that the primary function of PCPH was unlikely to be related to the ribonucleotide recycling function that its yeast counterpart performs in the Golgi during the process of protein glycosylation . However, taken together, our data strongly suggested that the normal cellular function of PCPH was related to ribonucleotide metabolism . We now report that PCPH is structurally and functionally identical to the mammalian ectonucleoside triphosphate diphosphohydrolase CD39L4 (ENTPD5), recently described as a member of the lymphoid activation antigen (<cluster of differentiation>) CD39 protein family . These results may help to establish the normal cellular function of the PCPH proto-oncogene product and its role in neoplastic development during carcinogenesis. Nat Struct Biol, 2001 Dec, 8(12), 1074 - 82 A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23; Truscott KN et al.; Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes . The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel . Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase . The Tim23 channel is cation selective and activated by a membrane potential and presequences . The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction . Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences. Nucleic Acids Res, 2001 Nov 15, 29(22), 4570 - 80 RNA polymerase II and III transcription factors can stimulate DNA replication by modifying origin chromatin structures; Bodmer-Glavas M et al.; Many transcription factors are multifunctional and also influence DNA replication . So far, their mechanism of action has remained elusive . Here we show that a DNA-binding protein could rely on the same biochemical activity that activates transcription to stimulate replication from the yeast chromosomal ARS1 origin . Unexpectedly, the ability to stimulate replication from this origin was not restricted to polymerase II transcription factors, but was a property shared by polymerase III factors . Furthermore, activation of replication did not depend on the process of transcription, but rather on the ability of DNA-binding transcription factors to remodel chromatin . The natural ARS1 activator Abf1 and the other transcription factors that stimulated replication remodeled chromatin in a very similar manner . Moreover, the presence of a histone H3 mutant that was previously shown to generally increase transcription also facilitated replication from ARS1 and partially compensated for the absence of a transcription factor . We propose that multifunctional transcription factors work by influencing the chromatin architecture at replication origins so as to generate a structure that is favorable to the initiation of replication. Nucleic Acids Res, 2001 Nov 15, 29(22), 4551 - 60 Identification of factors mediating the developmental regulation of the early acting -3.9 kb chicken lysozyme enhancer element; Lefevre P et al.; The chicken lysozyme gene -3.9 kb enhancer forms a DNase I hypersensitive site (DHS) early in macrophage differentiation, but not in more primitive multipotent myeloid precursor cells . A nucleosome becomes precisely positioned across the enhancer in parallel with DHS formation . In transfection assays, the 5'-part of the -3.9 kb element has ubiquitous enhancer activity . The 3'-part has no stimulatory activity, but is necessary for enhancer repression in lysozyme non-expressing cells . Recent studies have shown that the chromatin fine structure of this region is affected by inhibition of histone deacetylase activity after Trichostatin A (TSA) treatment, but only in lysozyme non-expressing cells . These results indicated a developmental modification of chromatin structure from a dynamic, but inactive, to a stabilised, possibly hyperacetylated, active state . Here we have identified positively and negatively acting transcription factors binding to the -3.9 kb enhancer and determined their contribution to enhancer activity . Furthermore, we examined the influence of TSA treatment on enhancer activity in macrophage cells and lysozyme non-expressing cells, including multipotent macrophage precursors . Interestingly, TSA treatment was able to restore enhancer activity fully in macrophage precursor cells, but not in non-macrophage lineage cells . These results suggest (i) that the transcription factor complement of multipotent progenitor cells is similar to that of lysozyme-expressing cells and (ii) that developmental regulation of the -3.9 kb enhancer is mediated by the interplay of repressing and activating factors that respond to or initiate changes in the chromatin acetylation state. Mol Cell Biol, 2001 Dec, 21(24), 8371 - 84 Domain structure of the NRIF3 family of coregulators suggests potential dual roles in transcriptional regulation; Li D et al.; The identification of a novel coregulator for nuclear hormone receptors, designated NRIF3, was recently reported (D . Li et al., Mol . Cell . Biol . 19:7191-7202, 1999) . Unlike most known coactivators, NRIF3 exhibits a distinct receptor specificity in interacting with and potentiating the activity of only TRs and RXRs but not other examined nuclear receptors . However, the molecular basis underlying such specificity is unclear . In this report, we extended our study of NRIF3-receptor interactions . Our results suggest a bivalent interaction model, where a single NRIF3 molecule utilizes both the C-terminal LXXIL (receptor-interacting domain 1 {RID1}) and the N-terminal LXXLL (RID2) modules to cooperatively interact with TR or RXR (presumably a receptor dimer), with the spacing between RID1 and RID2 playing an important role in influencing the affinity of the interactions . During the course of these studies, we also uncovered an NRIF3-NRIF3 interaction domain . Deletion and mutagenesis analyses mapped the dimerization domain to a region in the middle of NRIF3 (residues 84 to 112), which is predicted to form a coiled-coil structure and contains a putative leucine zipper-like motif . By using Gal4 fusion constructs, we identified an autonomous transactivation domain (AD1) at the C terminus of NRIF3 . Somewhat surprisingly, full-length NRIF3 fused to the DNA-binding domain of Gal4 was found to repress transcription of a Gal4 reporter . Further analyses mapped a novel repression domain (RepD1) to a small region at the N-terminal portion of NRIF3 (residues 20 to 50) . The NRIF3 gene encodes at least two additional isoforms due to alternative splicing . These two isoforms contain the same RepD1 region as NRIF3 . Consistent with this, Gal4 fusions of these two isoforms were also found to repress transcription . Cotransfection of NRIF3 or its two isoforms did not relieve the transrepression function mediated by their corresponding Gal4 fusion proteins, suggesting that the repression involves a mechanism(s) other than the recruitment of a titratable corepressor . Interestingly, a single amino acid residue change of a potential phosphorylation site in RepD1 (Ser(28) to Ala) abolishes its transrepression function, suggesting that the coregulatory property of NRIF3 (or its isoforms) might be subjected to regulation by cellular signaling . Taken together, our results identify NRIF3 as an interesting coregulator that possesses both transactivation and transrepression domains and/or functions . Collectively, the NRIF3 family of coregulators (which includes NRIF3 and its other isoforms) may play dual roles in mediating both positive and negative regulatory effects on gene expression. Int J Mol Med, 2001 Dec, 8(6), 595 - 601 An essential role of histone deacetylases in postembryonic organ transformations in Xenopus laevis; Sachs LM et al.; Amphibian metamorphosis is the result of thyroid hormone (TH)-induced organ transformations including de novo morphogenesis, tissue remodeling and resorption through programmed cell death (apoptosis) . All changes during metamorphosis are presumed to be mediated through gene regulation cascades initiated by TH . Numerous studies have implicated important roles of chromatin remodeling in transcriptional regulation . In particular, several lines of evidence support the view that histone acetylation is associated with transcriptional activation and histone deacetylation leads to gene repression . Here we address the physiological roles of histone deacetylases during vertebrate postembryonic development by using amphibian metamorphosis as a model . We first demonstrate that Xenopus laevis Rpd3 (a histone deacetylase) and Sin3 (a corepressor associated to Rpd3) are expressed in premetamorphic and metamorphic tadpole tissues, suggesting their involvement in these postembryonic processes . To test this possibility, we use a histone deacetylase inhibitor, trichostatin A, to block histone deacetylases and examine the development of the tadpoles . Our results indicate both natural and T3-induced metamorphosis are blocked by the inhibitor . We further show that this drug inhibits metamorphosis in different tissues, whether they involve de novo development or resorption through apoptosis, and that it functions in a stage-dependent but organ-autonomous manner . The data thus support an important role of histone deacetylases in the gene regulation cascades induced by T3 during metamorphosis. Science, 2001 Nov 16, 294(5546), 1528 - 31 Role of Erv29p in collecting soluble secretory proteins into ER-derived transport vesicles; Belden WJ et al.; Proteins are transported from the endoplasmic reticulum (ER) in vesicles formed by coat protein complex II (COPII) . Soluble secretory proteins are thought to leave the ER in these vesicles by "bulk flow" or through recognition by hypothetical shuttling receptors . We found that Erv29p, a conserved transmembrane protein, was directly required for packaging glycosylated pro-alpha-factor (gpalphaf) into COPII vesicles in Saccharomyces cerevisiae . Further, an Erv29p-gpalphaf complex was isolated from ER-derived transport vesicles . In vivo, export of gpalphaf from the ER was saturable and depended on the expression level of Erv29p . These results indicate that membrane receptors can link soluble cargo proteins to the COPII coat. J Virol, 2001 Dec, 75(24), 12370 - 81 Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes; den Boon JA et al.; RNA replication of all positive-strand RNA viruses is closely associated with intracellular membranes . Brome mosaic virus (BMV) RNA replication occurs on the perinuclear region of the endoplasmic reticulum (ER), both in its natural plant host and in the yeast Saccharomyces cerevisiae . The only viral component in the BMV RNA replication complex that localizes independently to the ER is 1a, a multifunctional protein with an N-terminal RNA capping domain and a C-terminal helicase-like domain . The other viral replication components, the RNA polymerase-like protein 2a and the RNA template, depend on 1a for recruitment to the ER . We show here that, in membrane extracts, 1a is fully susceptible to proteolytic digestion in the absence of detergent and thus, a finding consistent with its roles in RNA replication, is wholly or predominantly on the cytoplasmic face of the ER with no detectable lumenal protrusions . Nevertheless, 1a association with membranes is resistant to high-salt and high-pH treatments that release most peripheral membrane proteins . Membrane flotation gradient analysis of 1a deletion variants and 1a segments fused to green fluorescent protein (GFP) showed that sequences in the N-terminal RNA capping module of 1a mediate membrane association . In particular, a region C-terminal to the core methyltransferase homology was sufficient for high-affinity ER membrane association . Confocal immunofluorescence microscopy showed that even though these determinants mediate ER localization, they fail to localize GFP to the narrow region of the perinuclear ER, where full-length 1a normally resides . Instead, they mediate a more globular or convoluted distribution of ER markers . Thus, additional sequences in 1a that are distinct from the primary membrane association determinants contribute to 1a's normal subcellular distribution, possibly through effects on 1a conformation, orientation, or multimerization on the membrane. J Biol Chem, 2002 Jan 25, 277(4), 3040 - 6 Epub 2001 Nov 15. Mechanisms of dCMP transferase reactions catalyzed by mouse Rev1 protein; Masuda Y et al.; The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction . Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus . The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides . Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the V(max) value . Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at a V(max) similar to that for dCMP. J Biol Chem, 2002 Jan 25, 277(4), 2629 - 36 Epub 2001 Nov 15. Two clusters of residues at the docking groove of mitogen-activated protein kinases differentially mediate their functional interaction with the tyrosine phosphatases PTP-SL and STEP; Tarrega C et al.; Regulated function of mitogen-activated protein (MAP) kinases involves their selective association through docking sites with both activating MAP kinase kinases and inactivating phosphatases, including dual specificity and protein-tyrosine phosphatases (PTP) . Site-directed mutagenesis on the mammalian MAP kinases ERK2 and p38alpha identified within their C-terminal docking grooves two clusters of residues important for association with their regulatory PTPs, PTP-SL and STEP . ERK2 and p38alpha mutations that resembled the sevenmaker gain-of-function mutation in the Rolled D . melanogaster ERK2 homologue failed to associate with PTP-SL, were not retained in the cytosol, and were poorly inactivated by this PTP . Additional ERK2 mutations at the docking groove showed deficient association and dephosphorylation by PTP-SL, although their cytosolic retention was unaffected . Other ERK2 mutations, resembling gain-of-function mutations in the FUS3 yeast ERK2 homologue, associated to PTP-SL and were inactivated normally by this PTP . Our results demonstrate that mutations at distinct regions of the docking groove of ERK2 and p38alpha differentially affect their association and regulation by the PTP-SL and STEP PTPs. Phytochemistry, 1998 Nov 20, 49(6), 1659 - 1662 Acetylated DNA-damaging clerodane diterpenes from Casearia sylvestris; de Carvalho PR et al.; In addition to the known diterpene casearin G (1), two new clerodane diterpene casearins type, casearin S (2) and casearin T (3), were isolated from an acetylated bioactive CH(2)Cl(2)/MeOH extract from leaves of Casearia sylvestris . The diterpenes 1-3 exhibited moderate but selective activity towards the DNA-repair deficient yeast Saccharomyces cerevisiae mutants RAD 52YK and RS 321 . The structures of 1-3 were established on the basis of NMR spectroscopic experiments Mol Cells, 2001 Oct 31, 12(2), 149 - 57 Replication of the lagging strand: a concert of at least 23 polypeptides; Hubscher U et al.; DNA replication is one of the most important events in living cells, and it is still a key problem how the DNA replication machinery works in its details . A replication fork has to be a very dynamic apparatus since frequent DNA polymerase switches from the initiating DNA polymerase alpha to the processive elongating DNA polymerase delta occur at the leading strand (about 8 x 10(4) fold on both strands in one replication round) as well as at the lagging strand (about 2 x 10(7) fold on both strands in one replication round) in mammalian cells . Lagging strand replication involves a very complex set of interacting proteins that are able to frequently initiate, elongate and process Okazaki fragments of 180 bp . Moreover, key proteins of this important process appear to be controlled by S-phase check-point proteins . It became furthermore clear in the last few years that DNA replication cannot be considered uncoupled from DNA repair, another very important event for any living organism . The reconstitution of nucleotide excision repair and base excision repair in vitro with purified components clearly showed that the DNA synthesis machinery of both of these macromolecular events are similar and do share many components of the lagging strand DNA synthesis machinery . In this minireview we summarize our current knowledge of the components involved in the execution and regulation of DNA replication at the lagging strand of the replication fork. J Biol Chem, 2002 Jan 25, 277(4), 2589 - 96 Epub 2001 Nov 14. PITSLRE p110 protein kinases associate with transcription complexes and affect their activity; Trembley JH et al.; Although the PITSLRE protein kinases are members of the cyclin-dependent kinase superfamily, their cellular function is unclear . Previously we demonstrated that the general RNA splicing factor RNPS1 is a specific PITSLRE p110 kinase interactor in vivo . This suggests that the PITSLRE family of protein kinases is involved in some aspect of RNA processing or transcription . Here we identify multiple transcriptional elongation factors, including ELL2, TFIIF(1), TFIIS, and FACT, as PITSLRE kinase-associated proteins . We demonstrate that PITSLRE p110 protein kinases co-immunoprecipitate and/or co-purify with these elongation factors as well as with RNA polymerase II . Antibody-mediated inhibition of PITSLRE kinase specifically suppressed RNA polymerase II-dependent in vitro transcription initiated at a GC-rich (adenosine deaminase) or TATA box-dependent (Ad2ML) promoter, and this suppression was rescued by readdition of purified PITSLRE p110 kinase . Together, these data strongly suggest that PITSLRE protein kinases participate in a signaling pathway that potentially regulates or links transcription and RNA processing events. Biochem Soc Trans, 2001 Nov, 29(Pt 6), 655 - 61 Detecting, signalling and repairing DNA double-strand breaks; Jackson SP; DNA double-strand breaks (DSBs) can be generated by a variety of genotoxic agents, including ionizing radiation and radiomimetic chemicals . They can also occur when DNA replication complexes encounter other forms of DNA damage, and are produced as intermediates during certain site-specific recombination processes . It is crucial that cells recognize DSBs and bring about their efficient repair, because a single unrepaired cellular DSB can induce cell death, and defective DSB repair can lead to mutations or the loss of significant segments of chromosomal material . Eukaryotic cells have evolved a variety of systems to detect DNA DSBs, repair them, and signal their presence to the transcription, cell cycle and apoptotic machineries . In this review, I describe how work on mammalian cells and also on model organisms such as yeasts has revealed that such systems are highly conserved throughout evolution, and has provided insights into the molecular mechanisms by which DNA DSBs are recognized, signalled and repaired . I also explain how defects in the proteins that function in these pathways are associated with a variety of human pathological states. Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 181 - 90 Crosstalk of prolyl isomerases, Pin1/Ess1, and cyclophilin A; Fujimori F et al.; Previous studies have indicated that Ess1/Pin1, a gene in the parvulin family of peptidyl-prolyl isomerases (PPIases), plays an important role in regulating the G(2)/M transition of the cell cycle by binding cell-cycle-regulating proteins in eukaryotic cells . Although the ess1 gene has been considered to be essential in yeast, we have isolated viable ess1 deletion mutants and demonstrated, via analysis of yeast gene expression profiles using microarray techniques, a novel regulatory role for ESS1 in the G(1) phase . Although the overall expression profiles in the tested strains (C110-1, W303, S288c, and RAY-3AD) were similar, marked changes were detected for a number of genes involved in the molecular action of ESS1 . Among these, the expression levels of a cyclophilin A gene, also a member of the PPIase family, increased in the ess1 null mutant derived from C110-1 . Subsequent treatment with cyclosporin A significantly retarded growth, which suggests that ESS1 and cyclophilin A are functionally linked in yeast cells and play important roles at the G(1) phase of the cell cycle . J Biol Chem, 2002 Feb 1, 277(5), 3520 - 9 Epub 2001 Nov 13. A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression; Cheng J et al.; We identified a novel cystic fibrosis transmembrane conductance regulator (CFTR)-associating, PDZ domain-containing protein, CAL (CFTR associated ligand) containing two predicted coiled-coiled domains and one PDZ domain . The PDZ domain of CAL binds to the C terminus of CFTR . Although CAL does not have any predicted transmembrane domains, CAL is associated with membranes mediated by a region containing the coiled-coil domains . CAL is located primarily at the Golgi apparatus, co-localizing with trans-Golgi markers and is sensitive to Brefeldin A treatment . Immunoprecipitation experiments suggest that CAL exists as a multimer . Overexpression of CAL reduces CFTR chloride currents in mammalian cells and decreases expression, rate of insertion and half-life of CFTR in the plasma membrane . The Na(+)/H(+) exchanger regulatory factor, NHE-RF, a subplasma membrane PDZ domain protein, restores cell surface expression of CFTR and chloride currents . In addition, NHE-RF inhibits the binding of CAL to CFTR . CAL modulates the surface expression of CFTR . CAL favors retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR . Thus, the regulation of CFTR in the plasma membrane involves the dynamic interaction between at least two PDZ domain proteins. EMBO J, 2001 Nov 15, 20(22), 6499 - 508 Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells; Sudo T et al.; Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1 . In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G1 phase . To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci . Cdh1 was dispensable for viability and cell cycle progression . However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27(Kip1) . Further more, we found that Cdh1(-/-) cells fail to maintain DNA damage-induced G2 arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage . Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest. EMBO J, 2001 Nov 15, 20(22), 6453 - 63 Physical evidence for distinct mechanisms of translational control by upstream open reading frames; Gaba A et al.; The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream open reading frames (uORFs) and the CPA1 leader contains a single uORF . To determine how these uORFs control translation, we examined mRNAs containing these leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translation . Ribosomes predominantly loaded first at GCN4 uORF1 . Following its translation, but not the translation of uORF4, they efficiently reinitiated protein synthesis at Gcn4p . Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p . This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro . In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstream start codon by scanning past the uORF . Addition of arginine caused ribosomes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocking scanning ribosomes, and not by affecting reinitiation . The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways. EMBO J, 2001 Nov 15, 20(22), 6404 - 13 hADA3 is required for p53 activity; Wang T et al.; The tumor suppressor protein p53 is a transcription factor that is frequently mutated in human cancers . In response to DNA damage, p53 protein is stabilized and activated by post-translational modifications that enable it to induce either apoptosis or cell cycle arrest . Using a novel yeast p53 dissociator assay, we identify hADA3, a part of histone acetyltransferase complexes, as an important cofactor for p53 activity . p53 and hADA3 physically interact in human cells . This interaction is enhanced dramatically after DNA damage due to phosphorylation event(s) in the p53 N-terminus . Proper hADA3 function is essential for full transcriptional activity of p53 and p53-mediated apoptosis. EMBO J, 2001 Nov 15, 20(22), 6359 - 70 Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex; Pereira G et al.; Yeast spindle pole bodies (SPBs) duplicate once per cell cycle by a conservative mechanism resulting in a pre-existing 'old' and a newly formed SPB . The two SPBs of yeast cells are functionally distinct . It is only the SPB that migrates into the daughter cell, the bud, which carries the Bfa1p-Bub2p GTPase-activating protein (GAP) complex, a component of the spindle positioning checkpoint . We investigated whether the functional difference of the two SPBs correlates with the time of their assembly . We describe that in unperturbed cells the 'old' SPB always migrates into the bud . However, Bfa1p localization is not determined by SPB inheritance . It is the differential interaction of cytoplasmic microtubules with the mother and bud cortex that directs the Bfa1p-Bub2p GAP to the bud-ward-localized SPB . In response to defects of cytoplasmic microtubules to interact with the cell cortex, the Bfa1p-Bub2p complex binds to both SPBs . This may provide a mechanism to delay cell cycle progression when cytoplasmic microtubules fail to orient the spindle . Thus, SPBs are able to sense cytoplasmic microtubule properties and regulate the Bfa1p-Bub2p GAP accordingly. EMBO J, 2001 Nov 15, 20(22), 6236 - 45 Strains of {PSI(+)} are distinguished by their efficiencies of prion-mediated conformational conversion; Uptain SM et al.; Yeast prions are protein-based genetic elements that produce phenotypes through self-perpetuating changes in protein conformation . For the prion {PSI(+)} this protein is Sup35, which is comprised of a prion-determining region (NM) fused to a translational termination region . {PSI(+)} strains (variants) with different heritable translational termination defects (weak or strong) can exist in the same genetic background . {PSI(+)} variants are reminiscent of mammalian prion strains, which can be passaged in the same mouse strain yet have different disease latencies and brain pathologies . We found that {PSI(+)} variants contain different ratios of Sup35 in the prion and non-prion state that correlate with different translation termination efficiencies . Indeed, the partially purified prion form of Sup35 from a strong {PSI(+)} variant converted purified NM much more efficiently than that of several weak variants . However, this difference was lost in a second round of conversion in vitro . Thus, {PSI(+)} variants result from differences in the efficiency of prion-mediated conversion, and the maintenance of {PSI(+)} variants involves more than nucleated conformational conversion (templating) to NM alone. Gene, 2001 Oct 31, 278(1-2), 185 - 92 Genomic organization and transcription units of the human acyl-CoA synthetase 3 gene; Minekura H et al.; Acyl-CoA synthetases (ACSs) play an essential role in fatty acid metabolism . ACS3 is an arachidonate-preferring enzyme expressed in a wide range of human tissues including brain, heart, placenta, prostate, skeletal muscle, testis and thymus . As an initial step to understanding the transcriptional regulation of the human ACS3 gene, we analyzed the genomic organization and transcription units of the human ACS3 gene . Sequence analysis of genomic clones demonstrates that the human ACS3 gene spans at least 80.6 kb and contains 17 exons . The human ACS3 gene was mapped between the sequence-tagged site markers D2S360 and WI-21901 . Sequence inspection of the 5'-flanking region revealed potential DNA elements including CCAAT, AP-1, Oct-1, GATAs, SRY, CdxA, Nkx-2.5, c-Myb, HSF2, NF-AT, AP-2, NF-Y, and p300 . A minimal promoter region required for the expression of the human ACS3 gene in melanoma G361 cells was determined. FEBS Lett, 2001 Nov 9, 508(1), 23 - 8 Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p; Barth H et al.; We here report the identification of AUT10 as a novel gene required for both the cytoplasm to vacuole targeting of proaminopeptidase I and starvation-induced autophagy . aut10Delta cells are impaired in maturation of proaminopeptidase I under starvation and non-starvation conditions . A lack of Aut10p causes a defect in autophagy prior to vacuolar uptake of autophagosomes . Homozygous aut10Delta diploids do not sporulate . Vacuolar acidification indicated by accumulation of quinacrine is normal in aut10Delta cells and mature vacuolar proteinases are present . A biologically active Ha-tagged Aut10p, chromosomally expressed from its endogenous promoter, localizes in indirect immunofluorescence microscopy in the cytosol and on granulated structures, which appear clustered around the vacuolar membrane . This localization differs from known autophagy proteins. Plant Physiol, 2001 Nov, 127(3), 1266 - 78 The Arabidopsis pxa1 mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta-oxidation; Zolman BK et al.; Peroxisomes are important organelles in plant metabolism, containing all the enzymes required for fatty acid beta-oxidation . More than 20 proteins are required for peroxisomal biogenesis and maintenance . The Arabidopsis pxa1 mutant, originally isolated because it is resistant to the auxin indole-3-butyric acid (IBA), developmentally arrests when germinated without supplemental sucrose, suggesting defects in fatty acid beta-oxidation . Because IBA is converted to the more abundant auxin, indole-3-acetic acid (IAA), in a mechanism that parallels beta-oxidation, the mutant is likely to be IBA resistant because it cannot convert IBA to IAA . Adult pxa1 plants grow slowly compared with wild type, with smaller rosettes, fewer leaves, and shorter inflorescence stems, indicating that PXA1 is important throughout development . We identified the molecular defect in pxa1 using a map-based positional approach . PXA1 encodes a predicted peroxisomal ATP-binding cassette transporter that is 42% identical to the human adrenoleukodystrophy (ALD) protein, which is defective in patients with the demyelinating disorder X-linked ALD . Homology to ALD protein and other human and yeast peroxisomal transporters suggests that PXA1 imports coenzyme A esters of fatty acids and IBA into the peroxisome for beta-oxidation . The pxa1 mutant makes fewer lateral roots than wild type, both in response to IBA and without exogenous hormones, suggesting that the IAA derived from IBA during seedling development promotes lateral root formation. Plant Physiol, 2001 Nov, 127(3), 1020 - 9 Regulation of CAX1, an Arabidopsis Ca(2+)/H+ antiporter . Identification of an N-terminal autoinhibitory domain; Pittman JK et al.; Regulation of Ca(2+) transport determines the duration of a Ca(2+) signal, and hence, the nature of the biological response . Ca(2+)/H+ antiporters such as CAX1 (cation exchanger 1), play a key role in determining cytosolic Ca(2+) levels . Analysis of a full-length CAX1 clone suggested that the CAX1 open reading frame contains an additional 36 amino acids at the N terminus that were not found in the original clone identified by suppression of yeast (Saccharomyces cerevisiae) vacuolar Ca(2+) transport mutants . The long CAX1 (lCAX1) could not suppress the yeast Ca(2+) transport defects despite localization to the yeast vacuole . Calmodulin could not stimulate lCAX1 Ca(2+)/H+ transport in yeast; however, minor alterations in the 36-amino acid region restored Ca(2+)/H+ transport . Sequence analysis suggests that a 36-amino acid N-terminal regulatory domain may be present in all Arabidopsis CAX-like genes . Together, these results suggest a structural feature involved in regulation of Ca(2+)/H+ antiport. Plant Physiol, 2001 Nov, 127(3), 949 - 62 Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases; Koizumi N et al.; A major response of eukaryotic cells to the presence of unfolded proteins in the lumen of the endoplasmic reticulum (ER) is to activate genes that encode ER-located molecular chaperones, such as the binding protein . This response, called the unfolded protein response, requires the transduction of a signal from the ER to the nucleus . In yeast (Saccharomyces cerevisiae) and mammalian cells, an ER-located transmembrane receptor protein kinase/ribonuclease called Ire1, with a sensor domain in the lumen of the ER, is the first component of this pathway . Here, we report the cloning and derived amino acid sequences of AtIre1-1 and AtIre1-2, two Arabidopsis homologs of Ire1 . The two proteins are located in the perinuclear ER (based on heterologous expression of fusions with green fluorescent protein) . The expression patterns of the two genes (using beta-glucuronidase fusions) are nearly nonoverlapping . We also demonstrate functional complementation of the sensor domains of the two proteins in yeast and show that the Ire1-2 protein is capable of autotransphosphorylation . These and other findings are discussed in relation to the involvement of these genes in unfolded protein response signaling in plants. Plant Physiol, 2001 Nov, 127(3), 777 - 91 Developmental and thermal regulation of the maize heat shock protein, HSP101; Young TE et al.; The plant heat stress protein, Hsp101, and the yeast ortholog, Hsp104, are required to confer thermotolerance in plants and yeast (Saccharomyces cerevisiae), respectively . In addition to its function during stress, Hsp101 is developmentally regulated in plants although its function during development is not known . To determine how the expression of Hsp101 is regulated in cereals, we investigated the Hsp101 expression profile in developing maize (Zea mays) . Hsp101 protein was most abundant in the developing tassel, ear, silks, endosperm, and embryo . It was less abundant in the vegetative and floral meristematic regions and was present at only a low level in the anthers and tassel at anthesis, mature pollen, roots, and leaves . As expected, heat treatment resulted in an increase in the level of Hsp101 protein in several organs . In expanding foliar leaves, husk leaves, the tassel at the premeiosis stage of development, or pre-anthesis anthers, however, the heat-mediated increase in protein was not accompanied by an equivalent increase in mRNA . In contrast, the level of Hsp101 transcript increased in the tassel at anthesis following a heat stress without an increase in Hsp101 protein . In other organs such as the vegetative and floral meristematic regions, fully expanded foliar leaves, the young ear, and roots, the heat-induced increase in Hsp101 protein was accompanied by a corresponding increase in Hsp101 transcript level . However, anthers at anthesis, mature pollen, developing endosperm, and embryos largely failed to mount a heat stress response at the level of Hsp101 protein or mRNA, indicating that Hsp101 expression is not heat inducible in these organs . In situ RNA localization analysis revealed that Hsp101 mRNA accumulated in the subaleurone and aleurone of developing kernels and was highest in the root cap meristem and quiescent center of heat-stressed roots . These data suggest an organ-specific control of Hsp101 expression during development and following a heat stress through mechanisms that may include posttranscriptional regulation. Plant Physiol, 2001 Nov, 127(3), 765 - 76 Identification and analysis of Arabidopsis expressed sequence tags characteristic of non-coding RNAs; MacIntosh GC et al.; Sequencing of the Arabidopsis genome has led to the identification of thousands of new putative genes based on the predicted proteins they encode . Genes encoding tRNAs, ribosomal RNAs, and small nucleolar RNAs have also been annotated; however, a potentially important class of genes has largely escaped previous annotation efforts . These genes correspond to RNAs that lack significant open reading frames and encode RNA as their final product . Accumulating evidence indicates that such "non-coding RNAs" (ncRNAs) can play critical roles in a wide range of cellular processes, including chromosomal silencing, transcriptional regulation, developmental control, and responses to stress . Approximately 15 putative Arabidopsis ncRNAs have been reported in the literature or have been annotated . Although several have homologs in other plant species, all appear to be plant specific, with the exception of signal recognition particle RNA . Conversely, none of the ncRNAs reported from yeast or animal systems have homologs in Arabidopsis or other plants . To identify additional genes that are likely to encode ncRNAs, we used computational tools to filter protein-coding genes from genes corresponding to 20,000 expressed sequence tag clones . Using this strategy, we identified 19 clones with characteristics of ncRNAs, nine putative peptide-coding RNAs with open reading frames smaller than 100 amino acids, and 11 that could not be differentiated between the two categories . Again, none of these clones had homologs outside the plant kingdom, suggesting that most Arabidopsis ncRNAs are likely plant specific . These data indicate that ncRNAs represent a significant and underdeveloped aspect of Arabidopsis genomics that deserves further study. J Cell Biol, 2001 Nov 12, 155(4), 557 - 70 Epub 2001 Nov 12. Maintenance of Golgi structure and function depends on the integrity of ER export; Ward TH et al.; The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells . Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle . We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model . Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm . When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1{T31N}), the Golgi structure disassembles, leaving no residual Golgi membranes . Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos . A similar phenomenon is induced by the constitutively active Sar1{H79G} mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region . In cells expressing Sar1{T39N}, a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER . These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis . Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems. J Cell Biol, 2001 Nov 12, 155(4), 498 - 9 Epub 2001 Nov 12. Let's make Golgi; Wells WA; Does the Golgi self-organize or does it form around an instructive template? Evidence on both sides is piling up, but a definitive conclusion is proving elusive. Infect Immun, 2001 Dec, 69(12), 7517 - 26 WdChs2p, a class I chitin synthase, together with WdChs3p (class III) contributes to virulence in Wangiella (Exophiala) dermatitidis; Wang Z et al.; The chitin synthase structural gene WdCHS2 was isolated by screening a subgenomic DNA library of Wangiella dermatitidis by using a 0.6-kb PCR product of the gene as a probe . The nucleotide sequence revealed a 2,784-bp open reading frame, which encoded 928 amino acids, with a 59-bp intron near its 5' end . Derived protein sequences showed highest amino acid identities with those derived from the CiCHS1 gene of Coccidioides immitis and the AnCHSC gene of Aspergillus nidulans . The derived sequence also indicated that WdChs2p is an orthologous enzyme of Chs1p of Saccharomyces cerevisiae, which defines the class I chitin synthases . Disruptions of WdCHS2 produced strains that showed no obvious morphological defects in yeast vegetative growth or in ability to carry out polymorphic transitions from yeast cells to hyphae or to isotropic forms . However, assays showed that membranes of wdchs2Delta mutants were drastically reduced in chitin synthase activity . Other assays of membranes from a wdchs1Deltawdchs3Deltawdchs4Delta triple mutant showed that their residual chitin synthase activity was extremely sensitive to trypsin activation and was responsible for the majority of zymogenic activity . Although no loss of virulence was detected when wdchs2Delta strains were tested in a mouse model of acute infection, wdchs2Deltawdchs3Delta disruptants were considerably less virulent in the same model, even though wdchs3Delta strains also had previously shown no loss of virulence . This virulence attenuation in the wdchs2Deltawdchs3Delta mutants was similarly documented in a limited fashion in more-sensitive cyclophosphamide-induced immunocompromised mice . The importance of WdChs2p and WdChs3p to the virulence of W . dermatitidis was then confirmed by reconstituting virulence in the double mutant by the reintroduction of either WdCHS2 or WdCHS3 into the wdchs2Deltawdchs3Delta mutant background. Biochemistry, 2001 Nov 20, 40(46), 13933 - 40 Site-specific mutations in the myosin binding sites of actin affect structural transitions that control myosin binding; Prochniewicz E et al.; We have examined