S. cerevisiae

Nat Genet, 2001 Mar, 27(3), 271 - 6
Recombinational DNA double-strand breaks in mice precede synapsis; Mahadevaiah SK et al.; In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis . Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse . We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene . Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'.

Nature, 2001 Mar 8, 410(6825), 227 - 30
Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans; Tissenbaum HA et al.; In Caenorhabditis elegans, mutations that reduce the activity of an insulin-like receptor (daf-2) or a phosphatidylinositol-3-OH kinase (age-1) favour entry into the dauer state during larval development and extend lifespan in adults . Downregulation of this pathway activates a forkhead transcription factor (daf-16), which may regulate targets that promote dauer formation in larvae and stress resistance and longevity in adults . In yeast, the SIR2 gene determines the lifespan of mother cells, and adding an extra copy of SIR2 extends lifespan . Sir2 mediates chromatin silencing through a histone deacetylase activity that depends on NAD (nicotinamide adenine dinucleotide) as a cofactor . We have surveyed the lifespan of C . elegans strains containing duplications of chromosomal regions . Here we report that a duplication containing sir-2.1-the C . elegans gene most homologous to yeast SIR2-confers a lifespan that is extended by up to 50% . Genetic analysis indicates that the sir-2.1 transgene functions upstream of daf-16 in the insulin-like signalling pathway . Our findings suggest that Sir2 proteins may couple longevity to nutrient availability in many eukaryotic organisms.

Int J Cancer, 2001 Jan 20, 95(1), 23 - 8
TAP1 down-regulation in primary melanoma lesions: an independent marker of poor prognosis; Kamarashev J et al.; Melanoma tumor thickness is a major prognostic factor . Thin lesions, however, may metastasize, and sometimes thick tumors may not . To investigate the role of HLA class I-mediated antigen presentation, we correlated the expression of components of the antigen-processing machinery in primary melanoma lesions with their thickness and with the development of metastases . Seventeen formalin-fixed, paraffin-embedded primary melanomas thinner than 0.76 mm and 21 thicker than 1.50 mm were stained with anti-LMP2, -LMP7, -TAP1, -TAP2, -HLA class I and -beta2-microglobulin monoclonal antibodies . Twenty patients remained tumor-free in the follow-up period (10.5 +/- 1.8 years) . Eighteen patients relapsed within a median period of 15.0 months following tumor excision . Expression of all markers in the tested lesions was down-regulated, the frequency ranging from about 40% for LMP and TAP subunits to about 70% for HLA class I antigens . Expression of all markers was not correlated with tumor thickness . Only TAP1 and TAP2 down-regulation was significantly (p = 0.026 and 0.042, respectively) correlated with the development of metastases . This correlation was independent of tumor thickness for TAP1 . We suggest that TAP1 and probably TAP2 expression in primary lesions represents an independent prognostic marker in melanoma . Abnormalities in antigen presentation may account for the lack of absolute correlation between tumor thickness and prognosis .

Biopolymers, 2000, 55(5), 407 - 14
Engineering of the hydrophobic core of an alpha-helical coiled coil; Kiyokawa T et al.; The amino acid sequence that forms the alpha-helical coiled coil structure has a representative heptad repeat denoted by defgabc, according to their positions . Although the a and d positions are usually occupied by hydrophobic residues, hydrophilic residues at these positions sometimes play important roles in natural proteins . We have manipulated a few amino acids at the a and d positions of a de novo designed trimeric coiled coil to confer new functions to the peptides . The IZ peptide, which has four heptad repeats and forms a parallel triple-stranded coiled coil, has Ile at all of the a and d positions . We show three examples: (1) the substitution of one Ile at either the a or d position with Glu caused the peptide to become pH sensitive; (2) the metal ion induced alpha-helical bundles were formed by substitutions with two His residues at the d and a positions for a medium metal ion, and with one Cys residue at the a position for a soft metal ion; and (3) the AAB-type heterotrimeric alpha-helical bundle formation was accomplished by a combination of Ala and Trp residues at the a positions of different peptide chains . Furthermore, we applied these procedures to prepare an ABC-type heterotrimeric alpha-helical bundle and a metal ion-induced heterotrimeric alpha-helical bundle.

Mutat Res, 2001 Mar 1, 474(1-2), 93 - 103
Activation of Ty transposition by mutagens; Staleva Staleva L et al.; The induction of Ty1 transposition by mutagens (MMS and 4NQO) in asynchronous cultures and cells blocked in G1 and G2/M suggested G1 dependence of activation of Ty1 element by DNA damage . Northern blot analysis revealed immediate five-fold increase in levels of Ty1 transcript after 20min incubation of cells with 1 microg/ml 4NQO and four-fold increase in Ty1 RNA after treatment the cells with 0.1% MMS . Western blot analysis showed no difference in TyA protein in treated and untreated with mutagen cells . Quantitative mutagenicity assay and Northern blot analysis demonstrated dependence of induction of Ty1 element by DNA-damaging agents on the function of RAD9 gene and independence on DUN1 gene.

Biol Psychiatry, 2001 Feb 15, 49(4), 333 - 9
Haplotype study of three polymorphisms at the dopamine transporter locus confirm linkage to attention-deficit/hyperactivity disorder; Barr CL et al.; BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is often treated using methylphenidate, a psychostimulant that inhibits the dopamine transporter . This led E.H . Cook and colleagues to consider the dopamine transporter locus (DAT1) as a primary candidate gene for ADHD . That group reported a significant association between ADHD and the 480-base pair (bp) allele of the variable number of tandem repeats (VNTR) polymorphism located in the 3' untranslated region of the DAT1 gene . This association was later replicated in additional studies . METHODS: The DAT1 gene has additional common polymorphisms in intron 9 and exon 9 . We investigated the possibility of linkage of DAT1 and ADHD using the VNTR polymorphism and two additional common polymorphisms in 102 nuclear families with an ADHD proband . Using the transmission disequilibrium test, we examined the transmission of the alleles of each of these polymorphisms, as well as the haplotypes of the polymorphisms . RESULTS: We did not observe significant evidence for the biased transmission of the alleles of either the VNTR or the additional two polymorphisms when examined individually, although there was a trend for the biased transmission of the 480-bp allele of the VNTR . When we examined the haplotypes of the three polymorphisms we found significant evidence for biased transmission of one of the haplotypes containing the 480-bp VNTR allele . We also genotyped six additional DNA sequence variants of the DAT1 gene . However, these variants were not sufficiently polymorphic in our sample to be informative . Two of the DNA variants that result in an amino acid change, Ala559Val and Glu602Gly, were not observed in our sample . CONCLUSIONS: Our results support previous findings of an association between the DAT1 gene and ADHD.

Mol Cell, 2001 Feb, 7(2), 433 - 42
TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport; Sacher M et al.; TRAPP is a conserved protein complex required early in the secretory pathway . Here, we report two forms of TRAPP, TRAPP I and TRAPP II, that mediate different transport events . Using chemically pure TRAPP I and COPII vesicles, we have reconstituted vesicle targeting in vitro . The binding of COPII vesicles to TRAPP I is specific, blocked by GTPgammaS, and, surprisingly, does not require other tethering factors . Our findings imply that TRAPP I is the receptor on the Golgi for COPII vesicles . Once the vesicle binds to TRAPP I, the small GTP binding protein Ypt1p is activated and other tethering factors are recruited.

Mol Cell, 2001 Feb, 7(2), 319 - 29
A biochemical function for the Sm complex; Zhang D et al.; Within the yeast commitment complex, SmB, SmD1, and SmD3 make direct contact with the pre-mRNA substrate, close to the 5' splice site . Only these three Sm proteins have long and highly charged C-terminal tails, in metazoa as well as in yeast . We replaced these proteins with tail-truncated versions . Genetic assays demonstrate that the tails contribute to similar and overlapping functions, and cross-linking assays show that the tails make direct contact with the pre-mRNA in a largely sequence-independent manner . Other biochemical assays indicate that they function at least in part to stabilize the U1 snRNP-pre-mRNA interaction . We speculate that this role may be general, and may have even evolved to aid weak intermolecular nucleic acid interactions of only a few base pairs.

Cell, 2001 Feb 9, 104(3), 387 - 96
Cdc13 delivers separate complexes to the telomere for end protection and replication; Pennock E et al.; In Saccharomyces cerevisiae, the telomere binding protein Cdc13 mediates telomere replication by recruiting telomerase, and also performs an essential function in chromosome end protection . We show here that delivery of the Stn1 protein to the telomere, by fusing the DNA binding domain of Cdc13 (DBD(CDC13)) to Stn1, is sufficient to rescue the lethality of a cdc13 null strain and, hence, provide end protection . Telomere replication is still defective in this strain, but can be restored by delivering telomerase to the telomere as a DBD(CDC13)-telomerase fusion . These results establish Stn1 as the primary effector of chromosome end protection, whereas the principal function of Cdc13 is to provide a loading platform to recruit complexes that provide end protection and telomere replication.

Nucleic Acids Res . 2001 Mar 15;29(6):E32.
Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning; Noskov VN et al.; The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes . The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest . The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest . To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region . For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning . When vectors with a specific hook that was >/=60 bp were utilized, approximately 2% of transformants contained circular YACs with the Tg.AC transgene sequences . Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less . Thus, the minimal length of a unique sequence required for gene isolation by TAR is approximately 60 bp . No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

Nucleic Acids Res, 2001 Mar 15, 29(6), 1341 - 51
Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells; Balajee AS et al.; Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is involved in DNA replication as well as in diverse DNA repair pathways . In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases delta and epsilon . In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques . Exposure of normal cells to gamma-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40-45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling . The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h . The PCNA foci formed after gamma-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion . Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after gamma-irradiation . We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks . The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells . Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks . Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.

Nucleic Acids Res, 2001 Mar 15, 29(6), 1300 - 7
Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase; Romero F et al.; We have previously isolated the hpttg proto-oncogene, which is expressed in normal tissues containing proliferating cells and in several kinds of tumors . In fact, expression of hPTTG correlates with cell proliferation in a cell cycle-dependent manner . Recently it was reported that PTTG is a vertebrate analog of the yeast securins Pds1 and Cut2, which are involved in sister chromatid separation . Here we show that hPTTG binds to Ku, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) . hPTTG and Ku associate both in vitro and in vivo and the DNA-PK catalytic subunit phosphorylates hPTTG in vitro . Furthermore, DNA double-strand breaks prevent hPTTG-Ku association and disrupt the hPTTG-Ku complexes, indicating that genome damaging events, which result in the induction of pathways that activate DNA repair mechanisms and halt cell cycle progression, might inhibit hPTTG-Ku interaction in vivo . We propose that hPTTG might connect DNA damage-response pathways with sister chromatid separation, delaying the onset of mitosis while DNA repair occurs.

Mol Cell Biol, 2001 Mar, 21(6), 2184 - 91
Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization; Desai-Mehta A et al.; The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin . Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50 . Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair . In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic . In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts . Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells . However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci . Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50 . Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci . These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation . However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation . Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction.

Mol Cell Biol, 2001 Mar, 21(6), 2085 - 97
The neuron-restrictive silencer element-neuron-restrictive silencer factor system regulates basal and endothelin 1-inducible atrial natriuretic peptide gene expression in ventricular myocytes; Kuwahara K et al.; Induction of the atrial natriuretic peptide (ANP) gene is a common feature of ventricular hypertrophy . A number of cis-acting enhancer elements for several transcriptional activators have been shown to play central roles in the regulation of ANP gene expression, but much less is known about contributions made by transcriptional repressors . The neuron-restrictive silencer element (NRSE), also known as repressor element 1, mediates repression of neuronal gene expression in nonneuronal cells . We found that NRSE, which is located in the 3' untranslated region of the ANP gene, mediated repression of ANP promoter activity in ventricular myocytes and was also involved in the endothelin 1-induced increase in ANP gene transcription . The repression was conferred by a repressor protein, neuron-restrictive silencer factor (NRSF) . NRSF associated with the transcriptional corepressor mSin3 and formed a complex with histone deacetylase (HDAC) in ventricular myocytes . Trichostatin A (TSA), a specific HDAC inhibitor, relieved NRSE-mediated repression of ANP promoter activity, and chromatin immunoprecipitation assays revealed the involvement of histone deacetylation in NRSE-mediated repression of ANP gene expression . Furthermore, in myocytes infected with recombinant adenovirus expressing a dominant-negative form of NRSF, the basal level of endogenous ANP gene expression was increased and a TSA-induced increase in ANP gene expression was apparently attenuated, compared with those in myocytes infected with control adenovirus . Our findings show that an NRSE-NRSF system plays a key role in the regulation of ANP gene expression by HDAC in ventricular myocytes and provide a new insight into the role of the NRSE-NRSF system outside the nervous system.

Mol Cell Biol, 2001 Mar, 21(6), 2048 - 56
Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break; Signon L et al.; Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR) . In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion . Previously, we have shown that in the absence of RAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR . We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as with rad51Delta cells . DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1 (RDH54), SRS2, or SGS1 is deleted . Various double mutations largely eliminate both gene conversion and BIR, including rad51Delta rad50Delta, rad51Delta rad59Delta, and rad54Delta tid1Delta . These results demonstrate that there is a RAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumably MRE11 and XRS2 . The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.

Mol Cell Biol, 2001 Mar, 21(6), 2026 - 37
Fip1 regulates the activity of Poly(A) polymerase through multiple interactions; Helmling S et al.; Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation machinery and the only protein known to interact directly with poly(A) polymerase (Pap1) . Its association with Pap1 inhibits the extension of an oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA binding domain (C-RBD) of Pap1 . We present here the identification of separate functional domains of Fip1 . Amino acids 80 to 105 are required for binding to Pap1 and for the inhibition of Pap1 activity . This region is also essential for viability, suggesting that Fip1-mediated repression of Pap1 has a crucial physiological function . Amino acids 206 to 220 of Fip1 are needed for the interaction with the Yth1 subunit of the complex and for specific polyadenylation of the cleaved mRNA precursor . A third domain within amino acids 105 to 206 helps to limit RNA binding at the C-RBD of Pap1 . Our data demonstrate that the C terminus of Fip1 is required to relieve the Fip1-mediated repression of Pap1 in specific polyadenylation . In the absence of this domain, Pap1 remains in an inhibited state . These findings show that Fip1 has a crucial regulatory function in the polyadenylation reaction by controlling the activity of poly(A) tail synthesis through multiple interactions within the polyadenylation complex.

Mol Cell Biol, 2001 Mar, 21(6), 1997 - 2007
Dosage suppressors of pds1 implicate ubiquitin-associated domains in checkpoint control; Clarke DJ et al.; In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p . Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints . Though some components of these pathways are known, others remain to be identified . Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated . To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37 degrees C . Genetic and functional interactions of two suppressors are described . RAD23 and DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128 . rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints . Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls . Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128 . UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them . Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling . Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.

Mol Cell Biol, 2001 Mar, 21(5), 1819 - 27
Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events; Chen Q et al.; Yeast cells can survive in the absence of telomerase RNA, TLC1, by recombination-mediated telomere elongation . Two types of survivors, type I and type II, can be distinguished by their characteristic telomere patterns . RAD52 is essential for the generation of both types of survivors . Deletion of both RAD50 and RAD51 produces a phenotype similar to that produced by deletion of RAD52 . Here we examined the effects of the RAD50 and the RAD51 epistasis groups as well as the RAD52 homologue, RAD59, on the types of survivors generated in the absence of telomerase . rad59 mutations completely abolished the ability to generate type II survivors, while rad50 mutations decreased the growth viability of type II survivors but did not completely eliminate their appearance . Mutations in RAD51, RAD54, and RAD57 had the converse affect: they eliminated the ability of cells to generate type I survivors in a tlc1 strain . The triple mutant, tlc1 rad51 rad59, was not able to generate survivors . Thus either type I or type II recombination pathways can allow cells to survive in the absence of telomerase; however, elimination of both pathways in a telomerase mutant leads to the inability to elongate telomeres and ultimately cell death.

Mol Cell Biol, 2001 Mar, 21(5), 1784 - 94
CAC3(MSI1) suppression of RAS2(G19V) is independent of chromatin assembly factor I and mediated by NPR1; Johnston SD et al.; Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA . CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism . We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function . CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function . Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm . In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway . Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway . Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p . Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p . Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway . Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.

Mol Cell Biol, 2001 Mar, 21(5), 1719 - 29
Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage; Anderson L et al.; 53BP1 is a human BRCT protein that was originally identified as a p53-interacting protein by the Saccharomyces cerevisiae two-hybrid screen . Although the carboxyl-terminal BRCT domain shows similarity to Crb2, a DNA damage checkpoint protein in fission yeast, there is no evidence so far that implicates 53BP1 in the checkpoint . We have identified a Xenopus homologue of 53BP1 (XL53BP1) . XL53BP1 is associated with chromatin and, in some cells, localized to a few large foci under normal conditions . Gamma-ray irradiation induces increased numbers of the nuclear foci in a dose-dependent manner . The damage-induced 53BP1 foci appear rapidly (in 30 min) after irradiation, and de novo protein synthesis is not required for this response . In human cells, 53BP1 foci colocalize with Mrel1 foci at later stages of the postirradiation period . XL53BP1 is hyperphosphorylated after X-ray irradiation, and inhibitors of ATM-related kinases delay the relocalization and reduce the phosphorylation of XL53BP1 in response to X-irradiation . In AT cells, which lack ATM kinase, the irradiation-induced responses of 53BP1 are similarly affected . These results suggest a role for 53BP1 in the DNA damage response and/or checkpoint control which may involve signaling of damage to p53.

Mol Cell Biol, 2001 Mar, 21(5), 1515 - 30
Upf1p, Nmd2p, and Upf3p regulate the decapping and exonucleolytic degradation of both nonsense-containing mRNAs and wild-type mRNAs; He F et al.; In Saccharomyces cerevisiae, rapid degradation of nonsense-containing mRNAs requires the decapping enzyme Dcp1p, the 5'-to-3' exoribonuclease Xrn1p, and the three nonsense-mediated mRNA decay (NMD) factors, Upf1p, Nmd2p, and Upf3p . To identify specific functions for the NMD factors, we analyzed the mRNA decay phenotypes of yeast strains containing deletions of DCP1 or XRN1 and UPF1, NMD2, or UPF3 . Our results indicate that Upf1p, Nmd2p, and Upf3p regulate decapping and exonucleolytic degradation of nonsense-containing mRNAs . In addition, we show that these factors also regulate the same processes in the degradation of wild-type mRNAs . The participation of the NMD factors in general mRNA degradation suggests that they may regulate an aspect of translation termination common to all transcripts.

J Virol, 2001 Apr, 75(7), 3207 - 19
Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal; Chen J et al.; Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors . Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions . 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain . In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane . Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication . To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo . In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a . In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane . Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5'-proximal third of RNA2 . The RNA2 5' untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA . However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA . A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TPsiC stem-loop of host tRNAs . The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates . These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.

J Cell Biol, 2001 Mar 5, 152(5), 935 - 44
Rer1p, a retrieval receptor for endoplasmic reticulum membrane proteins, is dynamically localized to the Golgi apparatus by coatomer; Sato K et al.; Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins . We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal . A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER . Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole . The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits . These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

Bioinformatics, 2001 Feb, 17(2), 196 - 7
MOSAIC: segmenting multiple aligned DNA sequences; Andre C et al.; MOSAIC is a set of tools for the segmentation of multiple aligned DNA sequences into homogeneous zones . The segmentation is based on the distribution of mutational events along the alignment . As an example, the analysis of one repeated sequence belonging to the subtelomeric regions of the yeast genome is presented . AVAILABILITY: Free access from ftp://ftp.biomath.jussieu.fr/pub/papers/MOSAIC

J Mol Biol, 2001 Mar 9, 306(5), 957 - 68
MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp; Bowers J et al.; In the yeast Saccharomyces cerevisiae, mismatch repair (MMR) is initiated by the binding of heterodimeric MutS homolog (MSH) complexes to mismatches that include single nucleotide and loop insertion/deletion mispairs . In in vitro experiments, the mismatch binding specificity of the MSH2-MSH6 heterodimer is eliminated if ATP is present . However, addition of the MutL homolog complex MLH1-PMS1 to binding reactions containing MSH2-MSH6, ATP, and mismatched substrate results in the formation of a stable ternary complex . The stability of this complex suggests that it represents an intermediate in MMR that is subsequently acted upon by other MMR factors . In support of this idea, we found that the replication processivity factor proliferating cell nuclear antigen (PCNA), which plays a critical role in MMR at step(s) prior to DNA resynthesis, disrupted preformed ternary complexes . These observations, in conjunction with experiments performed with streptavidin end-blocked mismatch substrates, suggested that PCNA interacts with an MSH-MLH complex formed on DNA mispairs.

Dev Biol, 2001 Mar 15, 231(2), 410 - 9
Formation of the middle ear: recent progress on the developmental and molecular mechanisms; Mallo M; The middle ear allows animals to hear while moving in an aerial medium . It is composed of a cavity harbouring a chain of three ossicles that transmit vibrations produced by airborne sound in the tympanic membrane into the inner ear, where they are converted into neural impulses . The middle ear develops in the branchial arches, and this requires sequential interactions between the epithelia and the underlying mesenchyme . Gene-inactivation experiments have identified genes required for the formation of different middle ear components . Some encode for signalling molecules, including Endothelin1 and Fgf8, probable mediators of epithelial-mesenchymal interactions . Other genes, including Eya1, Prx1, Hoxa1, Hoxa2, Dlx1, Dlx2, Dlx5, and Gsc, are most likely involved in patterning and morphogenetic processes in the neural crest-derived mesenchyme . Mechanisms controlling formation of a functional tympanic membrane are also discussed . Basically, the tympanic ring, which serves as support for the tympanic membrane, directs invagination of the first pharyngeal cleft ectoderm to form the external acoustic meatus (EAM), which provides the outer layer of the membrane . Gsc and Prx1 are essential for tympanic ring development . While invaginating, the EAM controls skeletogenesis in the underlying mesenchyme to form the manubrium of the malleus, the link between the membrane and the middle ear ossicles .

Nature, 2001 Feb 15, 409(6822), 839 - 41
A genomic perspective on membrane compartment organization; Bock JB et al.; Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families . To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes . Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm . All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits . In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.

Shock, 2001 Mar, 15(3), 165 - 70
Injury in the era of genomics; Cobb JP et al.; The traditional approach to the study of biology employs small-scale experimentation that results in the description of a molecular sequence of known function or relevance . In the era of the genome the reverse is true, as large-scale cloning and gene sequencing come first, followed by the use of computational methods to systematically determine gene function and regulation . The overarching goal of this new approach is to translate the knowledge learned from a systematic, global analysis of genomic data into a complete understanding of biology . For investigators who study shock, the specific goal is to increase understanding of the adaptive response to injury at the level of the entire genome . This review describes our initial experience using DNA microarrays to profile stress-induced changes in gene expression . We conclude that efforts to apply genomics to the study of injury are best coordinated by multi-disciplinary groups, because of the extensive expertise required.

Biochem Cell Biol, 2001, 79(1), 83 - 91
A tale of two charges: distinct roles for an acidic and a basic amino acid in the structure and function of cytochrome c; Parrish JC et al.; Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria . As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history . Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp . There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined . Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electrostatically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular) . This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86 . Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90.

RNA, 2001 Feb, 7(2), 275 - 84
Programmed +1 frameshifting stimulated by complementarity between a downstream mRNA sequence and an error-correcting region of rRNA; Li Z et al.; Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3) . The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs . We showed previously that the shift occurs by out-of-frame binding of a normal aminoacyl-tRNA in the ribosomal A site caused by an aberrant codonoanticodon interaction in the P site . This event is unlike all previously described programmed translational frameshifts because it does not require tRNA slippage between cognate or near-cognate codons in the mRNA . A sequence of 15 nt distal to the frameshift site stimulates frameshifting 7.5-fold . Here we show that the Ty3 stimulator acts as an unstructured region to stimulate frameshifting . Its function depends on strict spacing from the site of frameshifting . Finally, the stimulator increases frameshifting dependent on sense codon-induced pausing, but has no effect on frameshifting dependent on pauses induced by nonsense codons . Complementarity between the stimulator and a portion of the accuracy center of the ribosome, Helix 18, implies that the stimulator may directly disrupt error correction by the ribosome.

Yi Chuan Xue Bao, 2001, 28(2), 144 - 51
{Differential accumulation of the new high-affinity phosphate transporter candidated gene fragment in rice roots in response to phosphorus deficiency stress}; Yu FT et al.; Phosphate is a major constraint to crop production, and phosphate uptake in plant is mainly by high-affinity phosphate transporter under phosphate deficiency condition . Using RT-PCR, a 1,178 bp phosphate transporter gene fragment OjPT1 was cloned from roots of Jingxi17 (Oryza sativa L . ssp . japanica) supplied with no phosphate . The comparison of this sequence with ones in GenBank indicated that it shared about 70% similarity at amino acid level with other phosphate transporters in higher plants, such as Arabidopsis thaliana, potate, tamato, Medicago truncatula and Catharanthus roseus, and high similarity with phosphate transporters in Saccharomyces cerevisiae and Neurospora crassa . RT-PCR assay showed that the OjPT1 transcripts were induced under phosphate deficiency condition . This gene fragment OjPT1 has been deposited in GenBank (accession No . AF249619).

Indian J Exp Biol, 2000 Jan, 38(1), 1 - 5
Ageing, gerontogenes, and hormesis; Rattan SI; Evolutionary theories of ageing and longevity argue against the existence of specific genes that cause ageing . However, genes whose altered activity influences ageing and longevity, may be termed gerontogenes . Several putative gerontogenes have been identified in various ageing systems, including the Drosophila, budding yeast, nematodes and cells in culture . Since ageing is characterized by a progressive failure of maintenance and repair, it is reasoned that genes involved in homeodynamic repair pathways are the most likely candidate gerontogenes . A promising approach for the identification of critical gerontogenic processes is hormesis-like positive effects of stress . Stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms.

Eur J Biochem, 2001 Mar, 268(5), 1238 - 49
Phosphoinositide fatty acids regulate phosphatidylinositol 5-kinase, phospholipase C and protein kinase C activities; Carricaburu V et al.; PtdIns(4,5)P(2) generally results from phosphorylation of PtdIns(4)P by the phosphatidylinositol 5-kinase (PtdIns5-K) . Its hydrolysis by phospholipase C (PLC) yields inositol 1,4,5-trisphosphate and diacylglycerol, which stimulates protein kinase C (PKC) . We show that epithelial cells of the cockroach rectum contain three different inositol lipids: PtdIns(4,5)P(2), PtdIns(4)P, and PtdIns . They are composed of six major fatty acids: palmitic (16:0) stearic (18:0), oleic (18:1n--9), linoleic (18:2n--6), linolenic (18:3n--3), and arachidonic (20:4n-6) acids . The fatty acid preference of each of the above enzymes was evaluated by incorporating different fatty acids in pairs into membrane lipids . Incorporation of 16:0 plus 18:1n--9 provoked an increase in PtdIns(4,5)P2-PLC activity and a decrease in PtdIns5-K activity . In contrast, incorporation of 16:0 plus 18:3n--3 led to a potentiation of PtdIns5-K activity and a decrease in PtdIns(4,5)P(2)-PLC activity . Furthermore, PLC and PtdIns5-K acted preferentially on substrates containing 18:3n--3, and 18:3n--3-containing diacylglycerol specifically potentiated PKC activity . Thus, we propose that the fatty acids that make up the phosphoinositides function as intracellular modulators of the activity of certain enzymes.

Curr Biol, 2001 Feb 6, 11(3), R109 - 11
Membrane transport: retromer to the rescue; Pfeffer SR; Genetic analysis in yeast has led to the discovery of a complex that retrieves proteins selectively from the prevacuolar compartment and transports them to the Golgi . Orthologs of these proteins in mammalian cells are likely to play a similar role but their cargoes are yet to be identified.

Curr Biol, 2001 Jan 23, 11(2), R45 - 8
Meiotic recombination: Making and breaking go hand in hand; Baudat F et al.; Accurate segregation of homologous chromosomes at the first meiotic division requires the tight coordination of DNA replication, homologous recombination and chromosome organization . Recent studies suggest that the initiation of meiotic recombination is mechanistically coupled to premeiotic DNA replication.

Curr Biol, 2001 Jan 23, 11(2), 99 - 104
enok encodes a Drosophila putative histone acetyltransferase required for mushroom body neuroblast proliferation; Scott EK et al.; Mushroom bodies in the Drosophila brain are centers for olfactory learning and memory . We have previously shown that the mushroom bodies comprise three types of neurons with distinct axonal projections . These three types of neurons are generated sequentially from common neuroblasts . We report here the identification of a gene that we have named enoki mushroom (enok), which when it is mutated gives rise to mushroom bodies with reduced axonal structures . enok encodes a putative histone acetyltransferase (HAT) of the MYST family, members of which have been implicated as important modulators of transcriptional activity . A single amino acid change in the zinc finger motif of the putative catalytic HAT domain gives the same phenotype as a null allele, and this finding indicates the importance of HAT activity to Enok's function . Further phenotypic analysis demonstrates that the mushroom body defect is due to an arrest in neuroblast proliferation rather than a failure of either cell fate switching or axon branching . Clonal analyses in the wing discs and the ovaries suggest that enok is essential for normal cell proliferation in some, but not all, tissues . Our results provide in vivo evidence for essential functions of a histone acetyltransferase in the construction of the Drosophila brain.

Plant Cell Physiol, 2001 Feb, 42(2), 231 - 5
Novel family of sensor histidine kinase genes in Arabidopsis thaliana; Ueguchi C et al.; We identified three novel, highly homologous, sensor histidine kinases that possibly function in the plasma membrane of Arabidopsis thaliana, i.e . AHK2, 3 and 4 . While AHK2 and 3 are expressed in several organs, AHK4 is mainly expressed in roots . AHK3 suppresses a sensor histidine kinase mutant of yeast.

Mol Biol Evol, 2001 Mar, 18(3), 330 - 43
Tracing the origin of the compensasome: evolutionary history of DEAH helicase and MYST acetyltransferase gene families; Sanjuan R et al.; Dosage compensation in Drosophila is mediated by a complex of proteins and RNAs called the "compensasome." Two of the genes that encode proteins of the complex, maleless (mle) and males-absent-on-the-first (mof), respectively, belong to the DEAH helicase and MYST acetyltransferase gene families . We performed comprehensive phylogenetic and structural analyses to determine the evolutionary histories of these two gene families and thus to better understand the origin of the compensasome . All of the members of the DEAH and MYST families of the completely sequenced Saccharomyces cerevisiae and Caenorhabditis elegans genomes, as well as those so far (June 2000) found in Drosophila melanogaster (for which the euchromatic part of the genome has also been fully sequenced) and Homo sapiens, were analyzed . We describe a total of 39 DEAH helicases in these four species . Almost all of them can be grouped in just three main branches . The first branch includes the yeast PRP2, PRP16, PRP22, and PRP43 splicing factors and their orthologs in animal species . Each PRP gene has a single ortholog in metazoans . The second branch includes just four genes, found in yeast (Ecm16) and Drosophila (kurz) and their orthologs in humans and Caenorhabditis . The third branch includes (1) a single yeast gene (YLR419w); (2) six Drosophila genes, including maleless and spindle-E/homeless; (3) four human genes, among them the ortholog of maleless, which encodes RNA helicase A; and (4) three C . elegans genes, including orthologs of maleless and spindle-E . Thus, this branch has largely expanded in metazoans . We also show that, for the whole DEAH family, only MLE and its metazoan orthologs have acquired new protein domains since the fungi/animals split . We found a total of 17 MYST family proteins in the four analyzed species . We determined putative orthologs of mof in both C . elegans and H . sapiens, and we show that the most likely ortholog in yeast is the Sas2 gene . Moreover, a paralog of mof exists in Drosophila . All of these results, together with those found for a third member of the compensasome, msl-3, suggest that this complex emerged after the fungi/animals split and that it may be present in mammalian species . Both gene duplication and the acquisition of new protein modules may have played important roles in the origin of the compensasome.

Hum Mol Genet, 2001 Mar 15, 10(6), 635 - 43
Mutations in the regulatory domain of cystathionine beta synthase can functionally suppress patient-derived mutations in cis; Shan X et al.; Human cystathionine beta--synthase (CBS) is an S-adenosylmethionine-regulated enzyme that plays a key role in the metabolism of homocysteine . Mutations in CBS are known to cause homocystinuria, an inborn error in metabolism . We previously developed a yeast functional assay for CBS and used it to characterize mutations found in homocystinuric patients . We discovered that many patient-derived mutations are functionally suppressed by deletion of the C-terminal 142 amino acids, which contain a 53 amino acid motif known as the CBS domain . This domain is found in a wide variety of proteins of diverse biological function . Here we have used a genetic screen to identify missense mutations in the C-terminal region of CBS that can suppress the most common patient mutation, I278T . Seven suppressor mutations were identified, four of which map to the CBS domain . When combined in cis with another pathogenic mutation, V168M, six of seven of the suppressor mutations rescued the yeast phenotype . Enzyme activity analyses indicate that the suppressors restore activity from <2% to 17--64% of the wild-type levels . Analysis of the suppressor mutations in the absence of the pathogenic mutation shows that six of the seven suppressor alleles have lost enzymatic responsiveness to S-adenosylmethionine . Using homology modeling, we show that the suppressor mutations appear to map on one face of the CBS domain . Our results indicate that subtle changes to the C-terminus of CBS can restore activity to mutant proteins and provide a rationale for screening for compounds that can activate mutant CBS alleles.

Genome Res, 2001 Mar, 11(3), 373 - 81
Gene duplication and the structure of eukaryotic genomes; Friedman R et al.; A simple method for understanding how gene duplication has contributed to genomic structure was applied to the complete genomes of Caenorhabditis elegans, Drosophila melanogaster, and yeast Saccharomyces cerevisiae . By this method, the genes belonging to gene families (the paranome) were identified, and the extent of sharing of two or more families between genomic windows was compared with that expected under a null model . The results showed significant evidence of duplication of genomic blocks in both C . elegans and yeast . In C . elegans, the five block duplications identified all occurred intra-chromosomally, and all but one occurred quite recently . In yeast, by contrast, 39 duplicated blocks were identified, and all but one of these was inter-chromosomal . Of these 39 blocks, 28 showed evidence of ancient duplication, possibly as a result of an ancient polyploidization event . By contrast, three blocks showed evidence of very recent duplication, while seven others showed a mixture of ancient and recent duplication events . Thus, duplication of genomic blocks has been an ongoing feature of yeast evolution over the past 200--300 million years.

EMBO J, 2001 Mar 1, 20(5), 1173 - 83
Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13; Grandin N et al.; In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends . Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance . Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13 . A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells . Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed . Several ten1 mutations were found to confer telomerase-dependent telomere lengthening . Other, temperature-sensitive, mutants of TEN1 arrested at G(2)/M via activation of the Rad9-dependent DNA damage checkpoint . These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes . We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA.

EMBO J, 2001 Mar 1, 20(5), 1134 - 43
A novel mRNA-decapping activity in HeLa cytoplasmic extracts is regulated by AU-rich elements; Gao M et al.; While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar activity in mammalian cells has been elusive . We have now identified a decapping activity in HeLa cytoplasmic extracts that releases (7me)GDP from capped transcripts . Decapping is activated in extracts by the addition of (7me)GpppG, which specifically sequesters cap-binding proteins such as eIF4E and the deadenylase DAN/PARN . Similar to in vivo observations, the presence of a poly(A) tail represses decapping of RNAs in vitro in a poly(A)-binding protein-dependent fashion . AU-rich elements (AREs), which act as regulators of mRNA stability in vivo, are potent stimulators of decapping in vitro . The stimulation of decapping by AREs requires sequence-specific ARE-binding proteins . These data suggest that cap recognition and decapping play key roles in mediating mRNA turnover in mammalian cells.

EMBO J, 2001 Mar 1, 20(5), 1123 - 33
Regulation of the Sko1 transcriptional repressor by the Hog1 MAP kinase in response to osmotic stress; Proft M et al.; Exposure of yeast to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which is essential for the induction of gene expression required for cell survival upon osmotic stress . Several genes are regulated in response to osmotic stress by Sko1, a transcriptional repressor of the ATF/CREB family . We show by in vivo coprecipitation and phosphorylation studies that Sko1 and Hog1 interact and that Sko1 is phosphorylated upon osmotic stress in a Hog1-dependent manner . Hog1 phosphorylates Sko1 in vitro at multiple sites within the N-terminal region . Phosphorylation of Sko1 disrupts the Sko1-Ssn6-Tup1 repressor complex, and consistently, a mutant allele of Sko1, unphosphorylatable by Hog1, exhibits less derepression than the wild type . Interestingly, Sko1 repressor activity is further enhanced in strains with high protein kinase A (PKA) activity . PKA phosphorylates Sko1 near the bZIP domain and mutation of these sites eliminates modulation of Sko1 responses to high PKA activity . Thus, Sko1 transcriptional repression is controlled directly by the Hog1 MAPK in response to stress, and this effect is further modulated by an independent signaling mechanism through the PKA pathway.

EMBO J, 2001 Mar 1, 20(5), 951 - 60
The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria; Wiedemann N et al.; The ADP/ATP carrier (AAC) is a major representative of mitochondrial preproteins lacking an N-terminal presequence . AAC contains targeting information in each of its three modules, which has led to a search for the dominant targeting region . An alternative, not yet tested model would be that several distinct targeting signals function simultaneously in import of the preprotein . We report that the three AAC modules cooperate in binding to the receptor Tom70 such that three Tom70 dimers are recruited to one preprotein . The modules are transferred to the import pore in a stepwise manner and cooperate again in the accumulation of AAC in the general import pore complex . AAC can cross the outer membrane with an internal segment first, i.e . in a loop formation . Each module of AAC is required for dimerization in the inner membrane . We propose a new concept for import of the hydrophobic carrier proteins into mitochondria where multiple signals cooperate in receptor recruitment, outer membrane translocation via loop formation and assembly in the inner membrane.

EMBO J, 2001 Mar 1, 20(5), 941 - 50
The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation; Lim JH et al.; Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding . We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates . Preproteins with short N-terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins . The lag phase is shortened by extending the N-terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein . A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity . A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is significantly faster, proving an active unfolding process . We conclude that access of mtHsp70 to N-terminal preprotein segments is critical for active unfolding and initiation of translocation.

Antioxid Redox Signal, 2000 Fall, 2(3), 397 - 404
Tocopherol-binding proteins: their function and physiological significance; Stocker A et al.; The present review is a continuation of earlier essays on the uptake mechanisms and the biological function of vitamin E . There are eight naturally occurring homologues of vitamin E, which differ in their structure and in biological activity in vivo and in vitro . Various studies have suggested that after normal gastrointestinal absorption of dietary vitamin E specific mechanisms favor the preferential accumulation of one of its homologues, alpha-tocopherol, in the human body . This process is thought to be mediated in part by the alpha-tocopherol transfer protein (alpha-TTP) in the liver cytoplasm . The mechanism and pathway by which alpha-TTP specifically incorporates alpha-tocopherol into plasma lipoproteins is not yet fully understood . Because alpha-tocopherol is widely distributed in tissues in various concentrations but alpha-TTP resides only in liver, its role as intracellular carrier of alpha-tocopherol seems unlikely . However, recent data indicate that a system of alpha-tocopherol-binding proteins is involved in these processes that favor the localization of alpha-tocopherol at the sites where it is required . The current status of the evidence for the regulation of alpha-tocopherol levels and their impact on cellular signaling is discussed.

Leuk Res, 2001 Mar, 25(3), 241 - 7
Phorbol ester responsiveness of the glutathione S-transferase P1 gene promoter involves an inducible c-jun binding in human K562 leukemia cells; Borde-Chiche P et al.; Overexpression of the glutathione S-transferase P1 (GSTP1) gene is related to drug resistance in human cancer cells . However, the mechanisms of the transcriptional activation of this gene remain unclear . In this study, we examined the molecular mechanisms underlying phorbol ester mediated gene regulation using human K562 leukemia cells as a model . Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor site was crucial for 12-O-tetradecanoyl phorbol 13-acetate (TPA)-mediated GSTP1 gene transcription . Electrophoretic mobility shift assays and transient transfection analysis demonstrated that both DNA binding and transactivation activities of AP-1 were induced by TPA . By supershift analysis, we identified transcription factors c-jun and fra-1 as well as NF-E2p45 as components of the induced binding complex . These results show for the first time that the phorbol ester TPA is involved in the molecular mechanism(s) mediating the activation of the GSTP1 promoter in a human leukemia model.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2550 - 4
Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression; Zaman Z et al.; The yeast transcriptional repressor Tup1, tethered to DNA, represses to strikingly different degrees transcription elicited by members of two classes of activators . Repression in both cases is virtually eliminated by mutation of either member of the cyclin-kinase pair Srb10/11 . In contrast, telomeric chromatin affects both classes of activators equally, and in neither case is that repression affected by mutation of Srb10/11 . In vitro, Tup1 interacts with RNA polymerase II holoenzyme bearing Srb10 as well as with the separated Srb10 . These and other findings indicate that at least one aspect of Tup1's action involves interaction with the RNA polymerase II holoenzyme.

EMBO J, 2001 Jan 15, 20(1-2), 250 - 61
PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER; Maeda Y et al.; Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins . Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences . In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI-MT-I) have different substrate specificities and are targets of species- specific inhibitors of GPI biosynthesis . GPI-MT-I, however, has not been molecularly characterized . Characterization of GPI-MT-I would also help to clarify the topology of GPI biosynthesis . Here, we report a human cell line defective in GPI-MT-I and the gene responsible, PIG-M . PIG-M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains . PIG-M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane.

EMBO J, 2001 Jan 15, 20(1-2), 118 - 27
Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1; Holm M et al.; Arabidopsis COP1 is a photomorphogenesis repressor capable of directly interacting with the photomorphogenesis-promoting factor HY5 . This interaction between HY5 and COP1 results in targeted deg radation of HY5 by the 26S proteasome . Here we characterized the WD40 repeat domain-mediated interactions of COP1 with HY5 and two new proteins . Mutational analysis of those interactive partners revealed a conserved motif responsible for the interaction with the WD40 domain . This novel motif, with the core sequence V-P-E/D-φ-G (φ = hydrophobic residue) in conjunction with an upstream stretch of 4-5 negatively charged residues, interacts with a defined surface area of the ss-propeller assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic interactions . Several residues in the COP1 WD40 domain that are critical for the interaction with this motif have been revealed . The fact that point mutations either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation in transgenic plants validates the biological significance of this defined interaction.

Fresenius J Anal Chem, 2000 Feb, 366(3), 303 - 6
Application of magdala red as a fluorescence probe in the determination of nucleic acids; Yang HH et al.; A fluorescence quenching method was developed for the rapid determination of DNA and RNA using magdala red as fluorescence probe . In weakly acidic medium, the fluorescence of magdala red (lambdaex/lambdaem = 540/555 nm) can be largely quenched by DNA or RNA . The calibration graphs are linear over the range 0.01-1.2 microg/mL for both calf thymus DNA (CT DNA) and salmon DNA (SM DNA), and 0.015-1.0 microg/mL for yeast RNA, respectively . The corresponding detection limits are 6.0 ng/mL for CT DNA, 7.0 ng/mL for SM DNA and 15.0 ng/mL for yeast RNA, respectively . CT DNA could be determined in the presence of 20% (w/w) yeast RNA, and the relative standard deviation of six replicate measurements is 3.18% for 400 ng/mL of CT DNA . Interference from coexisting substances in the determination of DNA was also examined . Real samples were determined with satisfactory results.

Cancer Res, 2001 Feb 1, 61(3), 943 - 9
Phorbol esters modulate the Ras exchange factor RasGRP3; Lorenzo PS et al.; RasGRP represents the prototype of a new class of guanine nucleotide exchange factors that activate small GTPases . The guanyl nucleotide-releasing protein (GRP) family members contain catalytic domains related to CDC25, the Ras exchange factor of Saccharomyces cerevisiae . They also contain a motif resembling a pair of calcium-binding EF-hands and a C1 domain similar to the diacylglycerol interaction domain of protein kinase C . The sequence of KIAA0846, identified in a human brain cDNA library, encodes a member of the GRP family that we refer to as RasGRP3 . We show here that RasGRP3 bound phorbol esters with high affinity . This binding depended on anionic phospholipids, which is characteristic of phorbol ester binding to C1 domain proteins . In addition, phorbol esters also caused activation of the RasGRP3 exchange activity in intact cells, as determined by an increase in RasGTP and phosphorylation of the extracellular-regulated kinases . Finally, both phorbol 12-myristate 13-acetate and the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol induced redistribution of RasGRP3 to the plasma membrane and/or perinuclear area in HEK-293 cells, as demonstrated using a green fluorescent fusion protein . We conclude that RasGRP3 serves as a PKC-independent pathway to link the tumor-promoting phorbol esters with activation of Ras GTPases.

Cancer Res, 2001 Feb 1, 61(3), 854 - 8
A mammalian two-hybrid system for adenomatous polyposis coli-mutated colon cancer therapeutics; Wakita K et al.; Colon cancer cells frequently lose expression of the tumor suppressor adenomatous polyposis coli (APC) . As result, beta-catenin accumulates and activates transcription of Tcf-responsive genes . Here we describe a novel mammalian two-hybrid system that selectively kills APC-mutated cells . This system consists of GAL4/beta-catenin, VP16/Tcf4, and a gene that is transcribed when GAL4 and VP16 associate . In APC-mutated human colon cancer cells, such as SW480, GAL4/beta-catenin accumulates, and in the presence of VP16/Tcf4, induces high levels of expression of the reporter gene . Expression of wild-type APC reduced GAL4/beta-catenin and intact beta-catenin levels and inhibited reporter gene expression . In colon cancer cells such as SW48 that have wild-type APC, GAL4/beta-catenin was degraded, and expression levels of the output gene were low . Replacement of the reporter gene with a suicide gene resulted in selective killing of SW480 cells . This system may be applicable for broader use of gene therapy by targeting diseases that involve protein degradation.

Cancer Res, 2001 Feb 1, 61(3), 1095 - 9
Characterization of the major histocompatibility complex class I deficiencies in B16 melanoma cells; Seliger B et al.; The murine B16 melanoma system represents an important in vivo model for the evaluation of T cell-based immunization and vaccination strategies, although deficient MHC class I surface expression has been identified in these cells . We postulate here that the MHC class I-deficient phenotype of B16 melanoma cells is attributable to down-regulation or the loss of the expression and function of multiple components of the MHC class I antigen-processing pathway, including the peptide transporter associated with antigen processing, the proteasome subunits LMP2, LMP7, and LMP10, PA28alpha and -beta, and the chaperone tapasin . In contrast, calnexin, calreticulin, ER60, and protein disulfide isomerase expression are unaltered or only marginally suppressed in these cells . The level of down-regulation of the components of the antigen-processing pathway is either transcriptionally or posttranscriptionally controlled and could be corrected in all cases by IFN-y treatment, which also reconstituted MHC class I surface expression . Thus, B16 melanoma cells can be used as a model for the characterization of the mechanisms underlying the coordinated dysregulation of the antigen-processing components, which should provide new insights into the development of tumors and the factors controlling this process.

Nat Struct Biol, 2001 Mar, 8(3), 258 - 64
Vam3p structure reveals conserved and divergent properties of syntaxins; Dulubova I et al.; Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion . All syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region . The N-terminal region of neuronal syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding . We have examined the generality of the structural properties of syntaxins by NMR analysis of Vam3p, a yeast syntaxin essential for vacuolar fusion . Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with syntaxin 1A in this region . However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by syntaxins to govern different types of membrane fusion.

Yeast, 2001 Mar 15, 18(4), 323 - 4
YJL159w does encode Pir2/Hsp150; Moukadiri I et al.; In this paper we compare the sequence of the gene HSP150/PIR2, independently determined by two different groups, with that present in the yeast database as YJL159w, determined within the Yeast Sequencing Project . Although YJL159w is believed to encode Hsp150/Pir2, there are important differences between the amino acid sequence coded by this ORF and that of HSP150/PIR2 . To find out if this divergence is due to strain polymorphism or to a possible sequencing error, we have analysed the diverging zone of this ORF in three strains and have found it entirely consistent with the sequence reported as HSP150/PIR2, concluding that the divergence is probably due to a sequencing error in YJL159w .

J Comp Neurol, 2001 Mar 19, 431(4), 481 - 91
Innervation of the ring gland of Drosophila melanogaster; Siegmund T et al.; In insects, peptidergic neurons of the central nervous system regulate the synthesis of the main developmental hormones . Neuropeptides involved in this neuroendocrine cascade have been identified in lepidopterans and dictyopterans . Since these organisms are not suitable for genetic research, we identified peptidergic brain neurons innervating the ring gland in Drosophila melanogaster . In larvae of Drosophila, ecdysteroids and juvenile hormones are produced by the ring gland, which is composed of the prothoracic gland, the corpus allatum, and the corpora cardiaca . Using the GAL4 enhancer trap system, we mapped those neurons of the central nervous system that innervate the ring gland . Eleven groups of neurosecretory neurons and their target tissues were identified . Five neurons of the lateral protocerebrum directly innervate the prothoracic gland or corpus allatum cells of the ring gland and are believed to regulate ecdysteroid and juvenile hormone titers . Axons of the circadian pacemaker neurons project onto dendritic fields of these five neurons . This connection might be the neuronal substrate of the circadian rhythms of molting and metamorphosis in Drosophila . Most of the neurons presented here have not been described before . The enhancer trap lines labeling them will be valuable tools for the analysis of neuronal as well as genetic regulation in insect development .

Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 459 - 61
Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases; Blanchard H et al.; Forkhead-associated (FHA) domains are modular protein-protein interaction domains of approximately 130 amino acids present in numerous signalling proteins . FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules . FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized . Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 127.3, c = 386.3 A; diffraction data have been collected to 3.1 A resolution on a synchrotron source . Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 A resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 A.

Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 393 - 400
Crystallographic phasing of myristoyl-CoA-protein N-myristoyltransferase using an iodinated analog of myristoyl-CoA; Futterer K et al.; Myristoyl-CoA-protein N-myristoyltransferase (Nmt; E.C . 2.1.3.97) catalyzes the covalent attachment of myristate to the N-terminal glycine amine of many eukaryotic and viral proteins . The molecular structure of the ternary complex of Saccharomyces cerevisiae Nmt1p with a bound non-hydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, and a competitive peptidomimetic inhibitor, SC-58272, was solved to 2.9 A resolution by X-ray crystallography . The structure determination utilized diffraction data from an iodinated ternary complex in which a newly designed and synthesized compound, S-(13-iodo-2-oxotridecyl)-CoA, was substituted for S-(2-oxopentadecyl)-CoA . Replacing the two terminal fatty acid C atoms of myristate by iodine produced, under the same crystallization conditions, heavy-atom-derivatized crystals of defined site occupancy that were isomorphous to the native complex . This approach for obtaining experimental phase information can be extended to other crystal structures of protein-fatty acyl complexes . The synthesis of S-(13-iodo-2-oxotridecyl)-CoA and the phasing procedure are described.

Gene, 2001 Jan 24, 263(1-2), 31 - 8
Characterization of a MEN1 ortholog from Drosophila melanogaster; Guru SC et al.; Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized by tumors of the parathyroid, entero-pancreatic neuroendocrine and pituitary tissues and caused by inactivating mutations in the MEN1 gene . Menin, the 610-amino acid nuclear protein encoded by MEN1, binds to the transcription factor JunD and can repress JunD-induced transcription . We report here the identification of a MEN1 ortholog in Drosophila melanogaster, Menin1, that encodes a 763 amino acid protein sharing 46% identity with human menin . Additionally, 69% of the missense mutations and in-frame deletions reported in MEN1 patients appear in amino acid residues that are identical in the Drosophila and human protein, suggesting the importance of the conserved regions . Drosophila Menin1 gene transcripts use alternative polyadenylation sites resulting in 4.3 and 5-kb messages . The 4.3-kb transcript appears to be largely maternal, while the 5-kb transcript appears mainly zygotic . The binding of Drosophila menin to human JunD or Drosophila Jun could not be demonstrated by the yeast two-hybrid analysis . The identification of the MEN1 ortholog from Drosophila melanogaster will provide an opportunity to utilize Drosophila genetics to enhance our understanding of the function of human menin.

FEBS Lett, 2001 Feb 16, 490(3), 179 - 89
Regulation of the G1 to S transition by the ubiquitin pathway; DeSalle LM et al.; This year the most prestigious prize in medical sciences, the Lasker Award, has been presented to the three scientists who discovered the ubiquitin pathway: Aaron Ciechanover, Avram Hershko, and Alexander Varshavsky {Nature Med . 6 (2000) 1073-1081} . During a time when the scientific community was focused on understanding how proteins were synthesized, they intently pursued the novel idea that cells were programmed to selectively destroy proteins . Their work led to the identification of an elaborate system of protein degradation targeting a myriad of cellular substrates . A small protein called ubiquitin is at the center of this process . Although the ubiquitin pathway was first described in the early 1980s, it has only more recently advanced to the forefront of basic research as a significant regulatory network within the cell . The field continues to grow as new ubiquitination enzymes and novel functions of this system are identified . Scientists are focused on elucidating the mechanisms by which cells deploy the ubiquitin pathway to control levels of selected proteins, such as cell cycle regulatory proteins, transcription factors and signaling molecules . Accelerated or decelerated rates of degradation of particular substrates participate in the genesis of many human diseases . Thus, understanding the mechanisms that confer specificity to the ubiquitin system will allow the development of novel therapeutic approaches to target aberrations in this pathway underlying tumorigenesis and other human pathologies.

Insect Biochem Mol Biol, 2001 Mar 15, 31(4-5), 401 - 5
Thiafatty acids as tracers to investigate biosynthetic pathways of lepidopteran sex pheromones; Pinilla A et al.; In order to investigate the potential utility of thiafatty acids as tracers for biosynthetic studies of moth sex pheromones, a series of thiatetradecanoic acids, namely 8-, 9-, 10-, 11-, 12- and 13-thiatetradecanoic, were prepared and their metabolism was investigated in pheromone glands of Spodoptera littoralis . Analysis by gas chromatography coupled to mass spectrometry of extracts from pheromone glands treated with the above acids showed that only 8-thiatetradecanoic acid and 13-thiatetradecanoic acid were metabolized by desaturation and were incorporated into the sex pheromone biosynthetic pathway . 13-Thiatetradecanoic acid was converted into (E)- and (Z)-13-thiatetradec-11-enoic acids, (Z,E)-13-thiatetradeca-9,11-dienoic acid, 11-thiadodecanoic acid, (E)- and (Z)-11-thiadodec-9-enoic acids and 15-thiahexadecanoic acid . 8-Thiatetradecanoic acid gave rise to two monoenoic thiafatty acids and two dienoic thiafatty acids, which were assigned to (Z)- and (E)-8-thiatetradec-11-enoic acids, (Z,E)-8-thiatetradeca-9,11-dienoic acid and (E,E)-8-thiatetradeca-10,12-dienoic acid . The other thiafatty acids tested, 9-, 10-, 11- and 12-thiatetradecanoic acids, were not metabolized by desaturation, although the corresponding products of beta-oxidation and chain elongation were detected . The occurrence of sulfoxides was not detected in this case, in disagreement with results on the metabolism of some thiaacids previously reported by other authors in yeast, Saccharomyces cerevisiae.

J Virol, 2001 Mar, 75(6), 2627 - 33
Antiviral response in cells containing Stat1 with heterologous transactivation domains; Shen Y et al.; The STATs (signal transducers and activators of transcription), latent cytoplasmic transcription factors, are activated by binding of extracellular polypeptides to cell surface receptors . Dimerization, accumulation in the nucleus, and transcriptional inductions of specific genes then occur . The COOH terminus of the STATs acts as a transcriptional activation domain (TAD) . Stat1, one of seven mammalian STAT genes, forms a homodimer after activation by gamma interferon and induces transcription of a number of genes . These induced genes in turn produce the antiviral state . In the present experiments we used a Stat1-deficient cell line complemented with Stat1 or various fusion constructs in which the wild-type Stat1 TAD was replaced by other TADs to test the possibility that a specific activating domain was necessary for the induction of the antiviral response . We found that a wide variety of TADs with different activation potential appended to the Stat1 COOH terminus could substitute for the wild-type protein in inducing the antiviral state.

Blood, 2001 Mar 1, 97(5), 1266 - 73
Initiation of polyoma virus origin-dependent DNA replication through STAT5 activation by human granulocyte-macrophage colony-stimulating factor; Watanabe S et al.; Several lines of evidence indicate that transcriptional activation is coupled with DNA replication initiation, but the nature of initiation of DNA replication in mammalian cells is unclear . Polyoma virus replicon is an excellent system to analyze the initiation of DNA replication in murine cells because its replication requires an enhancer, and all components of replication machinery, except for DNA helicase large T antigen, are supplied by host cells . This system was used to examine the role of signal transducer and activator of transcription (STAT5) in replication initiation of polyoma replicon in the mouse lymphoid cell line BA/F3 . The plasmid with tandem repeats of consensus STAT5 binding sites followed by polyoma replication origin was replicated by stimulation with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in the presence of polyoma large T antigen in BA/F3 cells . Mutation analysis of the hGM-CSF receptor beta subunit revealed that only the box1 region is essential, and the C-terminal tyrosine residues are dispensable for the activity . Addition of the tyrosine kinase inhibitor genistein suppressed this replication without affecting transcriptional activation of STAT5 . Because deletion analysis of STAT5 indicates the importance of the C-terminal transcriptional activation domain of STAT5 for the initiation of replication, the role of this region in the activation of replication was examined with a GAL4-STAT5 fusion protein . GAL4-STAT5 activated replication of the plasmid containing tandem repeats of GAL4 binding sites and polyoma replication origin in BA/F3 cells . Mutation analysis of GAL4-STAT5 indicated that multiple serine residues coordinately have a role in activating replication . This is the first direct evidence indicating the potential involvement of STAT5 in replication.

Bioinformatics, 2001 Jan, 17(1), 44 - 57
Functional and structural genomics using PEDANT; Frishman D et al.; MOTIVATION: Enormous demand for fast and accurate analysis of biological sequences is fuelled by the pace of genome analysis efforts . There is also an acute need in reliable up-to-date genomic databases integrating both functional and structural information . Here we describe the current status of the PEDANT software system for high-throughput analysis of large biological sequence sets and the genome analysis server associated with it . RESULTS: The principal features of PEDANT are: (i) completely automatic processing of data using a wide range of bioinformatics methods, (ii) manual refinement of annotation, (iii) automatic and manual assignment of gene products to a number of functional and structural categories, (iv) extensive hyperlinked protein reports, and (v) advanced DNA and protein viewers . The system is easily extensible and allows to include custom methods, databases, and categories with minimal or no programming effort . PEDANT is actively used as a collaborative environment to support several on-going genome sequencing projects . The main purpose of the PEDANT genome database is to quickly disseminate well-organized information on completely sequenced and unfinished genomes . It currently includes 80 genomic sequences and in many cases serves as the only source of exhaustive information on a given genome . The database also acts as a vehicle for a number of research projects in bioinformatics . Using SQL queries, it is possible to correlate a large variety of pre-computed properties of gene products encoded in complete genomes with each other and compare them with data sets of special scientific interest . In particular, the availability of structural predictions for over 300 000 genomic proteins makes PEDANT the most extensive structural genomics resource available on the web.

J Autoimmun, 2001 Feb, 16(1), 59 - 69
Monoclonal antibodies derived from BALB/c mice immunized with apoptotic Jurkat T cells recognize known autoantigens; Gensler TJ et al.; It has been postulated that post-translational modifications and relocalization of proteins during apoptosis may lead to presentation of these molecules to the immune system in such a way that normal mechanisms of tolerance are bypassed . In the present study, Jurkat cells were induced to undergo apoptosis by treatment with the chemotherapeutic agent Ara-C . BALB/c mice were then immunized with the apoptotic cells and hybridomas were generated . Using an indirect immunofluorescence assay, the monoclonal antibodies produced were screened by flow cytometry for those monoclonal antibodies demonstrating reactivity with permeabilized apoptotic Jurkat cells but not with non-permeabilized normal or apoptotic Jurkat cells . Of 281 monoclonal antibodies, 20 monoclonal antibodies with these properties were selected for further analysis . Using 32P- or 35S-metabolically labelled Jurkat cells, these selected monoclonal antibodies were screened for their ability to recognize autoantigens by immunoprecipitation and Western blotting . Well characterized autoimmune sera were then used to confirm the identity of autoantigens by immunoblotting . We demonstrate that immunization of normal mice with apoptotic Jurkat cells results in the formation of antibodies targeting multiple autoantigens or autoantigen complexes, including Ku, rRNPs, snRNPs and vimentin . These findings are consistent with the hypothesis that apoptosis can contribute to the development of autoimmunity.

Biol Pharm Bull, 2001 Feb, 24(2), 144 - 50
Identification of zinc finger proteins bound to a silencer region in the rat glutathione transferase P gene; Tanabe A et al.; The rat glutathione transferase P (GST-P) gene is strongly induced during chemical hepatocarcinogenesis, whereas mRNA of this gene is rarely expressed in normal rat liver . We previously identified a silencer region in the promoter of this gene . This silencer has several DNA binding sites and at least three proteins (Silencer factor A, -B, and -C (SF-A, SF-B, and SF-C)) bind to these sites . We previously cloned and characterized the Nuclear Factor 1 (NF1) family and the CCAAT/enhancer-binding protein (C/EBP) family as SF-A and SF-B, respectively . However, SF-C which binds to GST-P silencer 2 (GPS2) remains to be cloned . By screening using yeast one-hybrid system, several zinc finger proteins were identified as a candidate of SF-C . The gel-mobility shift analyses showed that BTEB2, EZF, LKLF, TFIIIA, TIEG1, and novel zinc finger protein MZFP bound to GPS2 with different affinities . Several proteins of these are known to be transcriptional activators or repressors, suggesting that zinc finger proteins bind to GPS2 and regulate GST-P expression in the rat liver.

Arzneimittelforschung, 2001 Jan, 51(1), 72 - 5
Antifungal activity of some mono, bis and quaternary Mannich bases derived from acetophenone; Gul HI et al.; The development of resistance to current antifungal therapeutics drives search for new effective agents . Some Mannich bases have antifungal activity, but no information is available regarding the antifungal activity of acetophenone derived Mannich bases . Mono Mannich bases of acetophenone 1-3 were synthesized and converted into their corresponding bis derivatives, 5-7 . Representative quaternary derivatives 4 and 8 were also synthesized . Antifungal activities of the compounds were evaluated using some yeasts and dermatophytes in vitro . Mono Mannich base 3 and quaternary compounds 4 and 8 were found to be 2-16 times more potent than the reference compound amphotericin B against dermatophytes: Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum canis . Compounds 4 and 8 were also found to be 2 times more effective compared with amphotericin B against the yeast Saccharomyces cerevisiae . Quaternization procedure improved the biological activity dramatically, whereas conversion of mono Mannich bases to corresponding bis derivatives generally did not affect antifungal activity . Our results suggest that acetophenone derived mono Mannich base 3 and quaternary derivatives 4 and 8 may serve as leading compounds for further studies to develop new antifungal agents with their highly potent antifungal activity.

RNA, 2001 Jan, 7(1), 94 - 105
The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop; Shukla GC et al.; The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome . U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants . To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay . Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo . In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing . Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity . However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function . These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.

RNA, 2001 Jan, 7(1), 5 - 15
Evidence that phosphorylation of human Upfl protein varies with intracellular location and is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway; Pal M et al.; Human Upf1 protein (p), a group 1 RNA helicase, has recently been shown to function in nonsense-mediated mRNA decay (NMD) in mammalian cells . Here, we demonstrate that the estimated 3 x 10(6) copies of hUpf1 p per exponentially growing HeLa cell are essentially equally distributed among polysomal, subpolysomal, and ribosome-free fractions . We also demonstrate that hUpf1p binds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonine . hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free . We find that serum-induced phosphorylation of hUpf1p is inhibited by wortmannin at a concentration that selectively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin . These and other data suggest that phosphorylation is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway . Comparisons are made of hUpf1p to Upf1p and SMG-2, which are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively.

RNA, 2001 Jan, 7(1), 29 - 43
A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation; Tuschl T et al.; In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated . One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing . The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine . It differs in that the leaving group is diphosphate rather than a 5' exon . The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation.

Antioxid Redox Signal, 2000 Winter, 2(4), 801 - 10
The mitochondrial thioredoxin system; Miranda-Vizuete A et al.; Eukaryotic organisms from yeast to human possess a mitochondrial thioredoxin system composed of thioredoxin and thioredoxin reductase, similar to the cytosolic thioredoxin system that exists in the same cells . Yeast and mammalian mitochondrial thioredoxins are monomers of approximately 12 kDa and contain the typical conserved active site WCGPC . However, there are important differences between yeast and mammalian mitochondrial thioredoxin reductases that resemble the differences between their cytosolic counterparts . Mammalian mitochondrial thioredoxin reductase is a selenoprotein that forms a homodimer of 55 kDa/subunit; while yeast mitochondrial thioredoxin reductase is a homodimer of 37 kDa/subunit and does not contain selenocysteine . A function of the mitochondrial thioredoxin system is as electron donor for a mitochondrial peroxiredoxin, an enzyme that detoxifies the hydrogen peroxide generated by the mitochondrial metabolism . Experiments with yeast mutants lacking both the mitochondrial thioredoxin system as well as the mitochondrial peroxiredoxin system suggest an important role for mitochondrial thioredoxin, thioredoxin reductase, and peroxiredoxin in the protection against oxidative stress.

J Environ Pathol Toxicol Oncol, 2000, 19(4), 401 - 13
Antimutagenesis studies of magnesium and calcium salts; Bronzetti G et al.; Magnesium is a microelement that is essential for biological functions and particularly for cellular metabolism . It has a central role in protein, lipid, carbohydrate, and nucleic acid synthesis, and it is important for muscular physiology and nerve excitability . Magnesium has an important role in the stability of biological membranes, it controls immune phenomena, and it activates over 300 enzymes . However, the mechanism of action of magnesium salts has not been well investigated and, in particular, its antimutagenesis properties and its effects in the detoxification of free radicals need further study . We investigated the effect of magnesium chloride, sulphate, carbonate, and oxide on the yeast Saccharomyces cerevisiae D7 strain, to evaluate their ability to protect against genotoxic damage . We found that magnesium salts induced antimutagenic effects in the cells harvested in the logarithmic phase by decreasing the induction of hydrogen peroxide . This, however, did not occur in the stationary phase . We also studied calcium salts of the type corresponding to those of magnesium and their protective role against the oxidative damage of free radicals and enzymatic activities, such as catalase, glutathione peroxidase, and superoxide dismutase, which are involved in antioxidative defenses.

Cell Mol Life Sci, 1999 Oct 15, 56(3-4), 233 - 42
Myosin-V: head to tail; Provance DW et al.; The myosin-V family is the most extensively studied of the unconventional myosin families . Most organisms examined have at least one member of the myosin-V family: many have multiple members . The wide range of species in which myosin-V has been identified suggests that myosin-V is a fundamental component of organelle transport in all higher eukaryotes . Possible cargoes for myosin-V range from melanosomes and synaptic vesicles in mammals to vacuoles and messenger RNA in yeast . In this review, we discuss the current state of research on the cellular function of myosin-V as described by the actions of the head, neck and tail domains.

Cell Mol Life Sci, 1999 Dec, 56(11-12), 894 - 907
Cathepsin A/protective protein: an unusual lysosomal multifunctional protein; Hiraiwa M; Cathepsin A/protective protein {3.4.16.5}, carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y . Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases . Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities . Cathepsin A was recently identified in human platelets as deamidase . In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions . Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function . The protective function of cathepsin A is distinct from its catalytic function . Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein' . Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase . Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A . The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis . Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A . Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis.

Methods Enzymol, 2001, 329, 431 - 8
Purification and properties of rat liver Sec23-Sec24 complex; Weissman JT et al.; We have demonstrated a protocol for purifying functional Sec23-24 complex from rat liver cytosol . Because the rat liver Sec23-24 complex is highly susceptible to proteolysis, we have noted several modifications which have allowed us to overcome the problem of degradation . If care is taken to prevent proteolysis, this procedure typically yields 2 mg of functional complex . The Sec23-24 complex can then be used to study Sar1 GTP hydrolysis in the Sec23 GTPase activation assay . Additionally, Sec23-24 can reconstitute ER vesicle formation in the presence of Sar1 and Sec13-31, allowing for the identification of novel proteins or compounds that affect cargo export.

Protein Sci, 2000 Dec, 9(12), 2470 - 6
Thirty-plus functional families from a single motif; Yu L et al.; It is now possible to identify over 30 functional subfamilies among the WD-repeat-containing proteins found in the completed genomes . The majority of these subfamilies have at least one member for which experimental data allow assignment to a cellular pathway or process . Half of the 63 WD-repeat-containing proteins in Saccharomyces cerevisiae, half of the 70 in Caenorhabditis elegans, and a third of the 100 plus predicted in Drosophila can be assigned to 23 of these functional subfamilies . Perhaps indicative of the future, 33 WD-repeat-containing proteins from the partial genome of Arabidopsis thaliana can now be assigned to 18 of these subfamilies . These assignments have been made possible by combining traditional sequence similarity with an implied common beta propeller structural context to obtain measures of protein-protein surface similarity . The beta propeller structural context is represented in the form of a Hidden Markov Model . The procedure is completely automated.

Protein Sci, 2000 Dec, 9(12), 2457 - 69
The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis; Rath A et al.; We have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a Tm of 60 degrees C and a deltaGu of 3.08 kcal/mol . Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild-type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment . Three of the eight mutants constructed in this way displayed increases in Tm ranging from 8 to 15 degrees C with concomitant increases in deltaGu of up to 1.4 kcal/mol . The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a Tm greater than 90 degrees C and a deltaGu more than double that of the wild-type domain . The folding rate of this hyperstable mutant was 10-fold faster than wild-type, while its unfolding rate was fivefold slower . All of the stabilized mutants were still able to bind a target peptide with wild-type affinity . We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies . In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site.

Philos Trans R Soc Lond B Biol Sci, 2001 Jan 29, 356(1405), 67 - 72
Evolution and the molecular basis of somatic hypermutation of antigen receptor genes; Diaz M et al.; Somatic hypermutation of immunoglobulin genes occurs in many vertebrates including sharks, frogs, camels, humans and mice . Similarities among species reveal a common mechanism and these include the AGC/T sequence hot spot, preponderance of base substitutions, a bias towards transitions and strand bias . There are some differences among species, however, that may unveil layers of the mechanism . These include a G:C bias in frog and shark IgM but not in nurse shark antigen receptor (NAR), a high frequency of doublets in NAR hypermutation, and the co-occurrence of somatic hypermutation with gene conversion in some species . Here we argue that some of the similarities and differences among species are best explained by error-prone DNA synthesis by the translesion synthesis DNA polymerase zeta (Pol zeta) and, as suggested by others, induction of DNA synthesis by DNA breaks in antigen receptor variable genes . Finally, targeting of the variable genes is probably obtained via transcription-related elements, and it is the targeting phase of somatic hypermutation that is the most likely to reveal molecules unique to adaptive immunity.

Philos Trans R Soc Lond B Biol Sci, 2001 Jan 29, 356(1405), 41 - 6
Mutagenesis in eukaryotes dependent on DNA polymerase zeta and Rev1p; Lawrence CW et al.; DNA polymerase zeta (Pol zeta) and Rev1p carry out translesion replication in budding yeast, Saccharomyces cerevisiae, and are jointly responsible for almost all base pair substitution and frameshift mutations induced by DNA damage in this organism . In addition, Pol zeta is responsible for the majority of spontaneous mutations in yeast and has been proposed as the enzyme responsible for somatic hypermutability . Pol zeta, a non-processive enzyme that lacks a 3' to 5' exonuclease proofreading activity, is composed of Rev3p, the catalytic subunit, and a second subunit encoded by REV7 . In keeping with its role, extension by Pol zeta is relatively tolerant of abnormal DNA structure at the primer terminus and is much more capable of extension from terminal mismatches than yeast DNA polymerase alpha (Pol alpha) . Rev1p is a bifunctional enzyme that possesses a deoxycytidyl transferase activity that incorporates deoxycytidyl opposite abasic sites in the template and a second, at present poorly defined, activity that is required for the bypass of a variety of lesions as well as abasic sites . Human homologues of the yeast REV1 and REV3 have been identified and, based on the phenotype of cells producing antisense RNA to one or other of these genes, their products appear also to be employed in translation replication and spontaneous mutagenesis . We suggest that Pol zeta is best regarded as a replication enzyme, albeit one that is used only intermittently, that promotes extension at forks the progress of which is blocked for any reason, whether the presence of an unedited terminal mismatch or unrepaired DNA lesion.

Arch Androl, 2001 Jan-Feb, 46(1), 29 - 35
Evidence for the binding of a human sperm component with diaphanous protein; Zhang SM et al.; The YWK-II component of human sperm membrane is related to the betaA4-amyloid precursor protein (APP) of Alzheimer's disease . A yeast 2-hybrid system was used to screen a mouse testis cDNA expression library for potential ligands capable of interacting with the extracellular domain of the YWK-II component . One of the bound proteins was identified as hDIA1, which has 96% identity with p140mDia . These proteins are members of the formin homology family and participate in cytokinesis and organization of the actin cytoskeleton . By interacting with these diaphanous proteins, the YWK-II component may be involved in germ cell differentiation and in the structural formation of the acrosome.

Nature, 2001 Jan 18, 409(6818), 346 - 9
Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake; Curie C et al.; Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality . Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups . The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron . The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface . The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter . Here we show that ys1 is a membrane protein that mediates iron uptake . Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media . Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots . Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants.

Plant Mol Biol, 2000 Nov, 44(5), 687 - 97
Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization; Gear ML et al.; A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries . Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members . The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa . The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers . The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily . Expression of the three genes in different organs of tomato was investigated by quantitative PCR . LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips . LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers . LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae . LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H+/hexose symporter . The Km of the symporter for glucose is 45 microM.

Faraday Discuss, 2000, (116), 119 - 34; discussion 171-90
Biomaterial engineered electrodes for bioelectronics; Pardo-Yissar V et al.; A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode . With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed . This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface . Biasing of the electrode at a negative potential (-0.3 V vs . SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface . Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps . While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1 . The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c . The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant . At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed . Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water . Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units.

Genes Immun, 2000, 1(4), 237 - 50
Ku autoantigen (DNA helicase) is required for interleukins-13/-4-induction of 15-lipoxygenase-1 gene expression in human epithelial cells; Kelavkar UP et al.; As reported previously in human monocytes, a human lung epithelial cell line, A549, showed de novo induction of 15-Lipoxygenase-1 (15-LO-1) in response to interleukins-13 (IL-13) and -4 (IL-4) . In this cell line, 15-LO-1 expression, by RT-PCR and western blotting, was observed following 6 and 24 h of exposure to human IL-13 (ED50 5 ng/ml) and IL-4 (ED50 0.2 ng/ml) . We have previously shown that no cis-acting regulatory elements exist within the 15-LO-1 promoter region . To define IL-13 and IL-4 responsive trans-acting elements, we identified a region (DP2: -353 to -304 bp site) within the 15-LO-1 promoter (by footprinting experiments) to which IL-13-responsive elements (or factors) bind specifically (Kelavkar et al, 1998, Mol Biol Rep 25, 173-182) . To further delineate this region, we constructed (by site-directed mutagenesis) several deletion mutants in the 'LOPB5' region containing the 29 bp within the -353 to -304 bp of the DP2 core element . These were: DP3 (site totally deleted), DP4 (5 bp deleted at the center of the site), DP5 (8 bp at the 5'-end of the site) and DP6 (13 bp at the 3'-end of the site) . Cotransfection of these deletion constructs (driving luciferase reporter genes) was associated with 90% (DP4, DP5 and DP6) or 100% (DP3) abrogation of promoter activity at 24 h . Purification of nuclear protein extracts from IL-13 and IL-4-stimulated A549 cells, using a DP2 core containing affinity column, identified a 150 kDa protein under non-denaturing conditions, and two, 70 and 85 kDa proteins under denaturing conditions . These were not detectable by Coomassie blue staining in control nuclear protein extracts . Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of the tryptic digests of these proteins, identified one as the 86 kDA Lupus KU autoantigen protein P86 and the second as the 70 kDa Lupus KU autoantigen protein P70 . Gel shift and supershift experiments using monoclonal antibodies toward Ku antigen and its individual subunits, and utilizing DP2 and other mutant oligonucleotides with purified nuclear protein extracts from control and cytokine-treated A549 cells, confirmed our findings . Furthermore, electroporation of neutralizing anti-Ku70, Ku 80 and Ku70/80 antibodies into A549 cells totally suppressed IL-13 and IL-4-stimulated 15-LO-1 induction in these cells . Further, immunoprecipitation experiments data suggests that IL-4 and IL-13 activate Ku antigens and 15-LO-1 expression through distinct signaling events . In summary, in A549 cells, Ku antigen is induced in response to the cytokines, IL-13 and -4, and a 29 bp region within the -353 to -304 bp region of the 15-LO-1 promoter is required for its binding and subsequent induction of 15-LO-1 gene expression . The findings may provide an important link between the established dysregulated function of Ku antigen in auto-immune diseases, such as systemic lupus erythematosus and thyroiditis, and the increasingly recognized 'anti-inflammatory' role of 15-LO-1.

Cancer Res, 2001 Jan 1, 61(1), 53 - 8
Topoisomerase I-mediated cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine: trapping of topoisomerase I by the O6-methylguanine; Pourquier P et al.; Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are known to covalently link alkyl groups at the position 6 of guanines (O6MG) in DNA . O6-alkylguanine-DNA alkyltransferase (AGT) specifically removes the methyl group of the O6MG . Using purified human topoisomerase I (Top1), we found an 8-10-fold enhancement of Top1 cleavage complexes when O6MG is incorporated in oligonucleotides at the +1 position relative to a unique Top1 cleavage site . Top1 poisoning by O6MG is attributable to a decrease of the Top1-mediated DNA religation as well as an increase in the enzyme cleavage step . Increased cleavage is probably linked to a change in the hydrogen bonding pattern, such as in the case of the 8-oxoguanine, whereas inhibition of religation could be attributed to altered base pairing, such as abasic sites or base mismatches, because incorporation of a 6-thioguanine did not affect Top1 activity . Top1-DNA covalent complexes are also induced in MNNG-treated CHO cells constitutively lacking the AGT enzyme . Conversely, no increase could be detected in CHO cells transfected with the wild-type human AGT . Moreover, we show that yeasts overexpressing the human Top1 are more sensitive to MNNG, whereas knock-out Top1 strain cells display some resistance to the drug . Altogether, these results suggest a role for Top1 poisoning by alkylated bases in the antiproliferative activity of alkylating agents as well as in the DNA lesions resulting from endogenous and carcinogenic DNA modifications.

Cancer Res, 2001 Jan 1, 61(1), 50 - 2
Highly elevated ultraviolet-induced mutation frequency in isolated Chinese hamster cell lines defective in nucleotide excision repair and mismatch repair proteins; Nara K et al.; We have isolated N-methyl-N'-nitro-N-nitrosoguanidine-resistant cell lines from 43-3B Chinese hamster ovary cells, which are deficient in the ERCC1 gene involved in nucleotide excision repair . By Western blotting analysis, we found cell lines that are deficient or decreased in the amount of MSH6, or PMS2, or MSH2 proteins . Cell extracts of these cell lines show reduced efficiency of G:T mismatch repair activity . Compared with 43-3B, these cell lines exhibit highly elevated UV-induced mutation rates, indicating that mammalian mismatch repair can suppress UV-induced mutagenesis and may play a role in the fidelity of DNA replication at the sites of UV damage.

Cancer Res, 2001 Jan 1, 61(1), 23 - 6
Alterations of the double-strand break repair gene MRE11 in cancer; Fukuda T et al.; MRE11 plays a role in DNA double-strand break repair . Hypomorphic mutations of MRE11 have been demonstrated in ataxia-telangiectasia (AT)-like disorder . ATM mutations play a causal role in AT and have been demonstrated in lymphoid malignancies in patients without AT histories . By analogy with the relationship of ATM to lymphoid malignancies, it is probable that alterations of MRE11 are associated with tumor formation . We performed a mutation analysis of MRE11 in 159 unselected primary tumors . Three missense mutations at conserved positions were found in breast and lymphoid tumors . Additionally, an aberrant transcript without genomic mutation was found in a breast tumor . These findings suggest an occasional role for MRE11 alterations in the development of primary tumors.

Mol Cell Biochem, 2000 Nov, 214(1-2), 121 - 9
Protein nitration; Kuo WN et al.; Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reducing agent in the step of SDS-PAGE . Histones II-S and VIII-S, IgG, cAMP-dependent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibited strong immunoreactive bands . Histone VI-S, glycogen synthase, lactate dehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate immunoreactivity . Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity . Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity . A variation of immunoreactivity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts . Additionally, nitrotyrosine immunoreactivity was observed in non-mammalian organisms including Eschericia coli, Saccharomyces cerevisiae and Triticum vulgaris . Upon the treatment of 15 microM peroxynitrite (PN), strong oxidant derived from nitric oxide (NO), the apparent Km of PKA for cAMP increased from approximately 10(-8) to 10(-6) M . The results imply that the varied nitration of tyrosine residues in proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal transduction cascade.

Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2490 - 2
Molecular cloning and characterization of a cDNA for a rice Sec31p homolog; Kato T et al.; A cDNA for a putative Sec31p in rice has been cloned and sequenced . In yeast, Sec31p is a component of a protein-coated vesicle, COPII, which functions in the transport of cargo proteins from the endoplasmic reticulum to the cis-Golgi network . Structural similarities between yeast Sec31p and the rice putative homolog are discussed.

Medicina (B Aires), 2000, 60 Suppl 2, 21 - 6
Mammalian histone acetyltransferase complexes; Ogryzko V; Over the last decade, great progress has been made in elucidating how the human genome operates in the chromatin context . This paper describes our work on two human acetyltransferases, PCAF and TIP60, and their interaction partners . This study provides new clues on the function of these enzymes . In a striking parallel with the general transcription factor TFIID, PCAF complex contains proteins that have histone-like domains . We speculate that these subunits can presumably form a nucleosome-like structure on DNA, which would allow PCAF to contribute to the maintenance of an active state of chromatin . On the other hand, TIP60 complex contains two eukaryotic homologs of bacterial RuvB helicase/ATPse, involved in recombination and repair . Accordingly, expression of a dominant negative mutant of TIP60 in living cells interferes with their ability to repair DNA damage, which points out, for the first time, a role for a histone acetyltransferase in a process other than transcription . We also have evidence implicating TIP60 in the apoptotic response to DNA damage.

IUBMB Life, 2000 Aug, 50(2), 125 - 9
An RNA polymerase II complex containing capping enzymes and viral oncoproteins; Cabrejos ME et al.; The aim of this work was to identify proteins from Adenovirus 2-infected HeLa cell extracts that interact with the carboxyl-terminal domain of the largest subunit of RNA polymerase II . First, a mammalian RNA polymerase II complex was isolated from Adenovirus 2-infected HeLa cell extracts by affinity chromatography against the carboxyl-terminal domain of the largest subunit of RNA polymerase II, followed by chromatography on a Mono S fast protein liquid chromatographic column . Second, the isolated complex was further characterized by Western blot analysis, the formation of a GMP-protein complex, and transcriptional activity . The isolated complex contains general transcription factors, chromatin-remodeling factors, histone acetyltransferases, Srbs, capping enzymes, and E1A viral oncoproteins . The RNA polymerase II complex is active in transcription when supplemented with recombinant transcription factor IIB.

J Bacteriol, 2001 Feb, 183(3), 1101 - 5
Experimental evolution of enzyme temperature activity profile: selection in vivo and characterization of low-temperature-adapted mutants of Pyrococcus furiosus ornithine carbamoyltransferase; Roovers M et al.; We have obtained mutants of Pyrococcus furiosus ornithine carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis . The mutants were double ones, still complementing at 15 degrees C, a temperature already in the psychrophilic range . Their kinetic analysis is reported.

Traffic, 2000 Mar, 1(3), 270 - 81
A new functional domain of guanine nucleotide dissociation inhibitor (alpha-GDI) involved in Rab recycling; Luan P et al.; Guanine nucleotide dissociation inhibitor (GDI) is a 55-kDa protein that functions in vesicular membrane transport to recycle Rab GTPases . We have now determined the crystal structure of bovine alpha-GDI at ultra-high resolution (1.04 A) . Refinement at this resolution highlighted a region with high mobility of its main-chain residues . This corresponded to a surface loop in the primarily alpha-helical domain II at the base of alpha-GDI containing the previously uncharacterized sequence-conserved region (SCR) 3A . Site-directed mutagenesis showed that this mobile loop plays a crucial role in binding of GDI to membranes and extraction of membrane-bound Rab . This domain, referred to as the mobile effector loop, in combination with Rab-binding residues found in the multi-sheet domain I at the apex of alpha-GDI may provide flexibility for recycling of diverse Rab GTPases . We propose that conserved residues in domains I and II synergize to form the functional face of GDI, and that domain II mediates a critical step in Rab recycling during vesicle fusion.

Traffic, 2000 Mar, 1(3), 195 - 202
Lipid metabolism and regulation of membrane trafficking; Huijbregts RP et al.; The past 20 years have witnessed tremendous progress in our understanding of the molecular machinery that controls protein and membrane transport between organelles (Scheckman R, Orci L . Coat proteins and vesicle budding . Science 1996;271: 1526-1533 and Rothman JE . Mechanisms of intracellular protein transport . Nature 1994;372: 55-63.) The research efforts responsible for these impressive advances have largely focused on the identification and characterization of protein factors that participate in membrane trafficking events . The role of membranes and their lipid constituents has received considerably less attention . Indeed, until rather recently, popular models for mechanisms of membrane trafficking had relegated membrane lipids to the status of a passive platform, subject to deformation by the action of coat proteins whose polymerization and depolymerization govern vesicle budding and fusion reactions . The 1990s, and particularly its last half, has brought fundamental reappraisals of the interface of lipids and lipid metabolism in regulating intracellular membrane trafficking events . Some of the emerging themes are reviewed here.

Cell, 2001 Jan 26, 104(2), 313 - 20
Protein sorting upon exit from the endoplasmic reticulum; Muniz M et al.; It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations . However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway . Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles . Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway . These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.

Genome Biol . 2001;2(2):REVIEWS1007 . Epub 2001 Feb 07.
Functional proteomics: large-scale analysis of protein kinase activity; Lawrence DS; Proteome-wide sampling of function can be used to shed light on complex biological systems . Protein microarrays have now been used to investigate the substrate specificities of essentially all the protein kinases encoded by the yeast genome.

Chem Biol, 2001 Jan, 8(1), 71 - 80
Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase; Salomon AR et al.; BACKGROUND: Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells . The goal of this study was to identify the molecular target of this newly discovered natural product . RESULTS: Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action . Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9 . PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line . Together these results suggested that apoptolidin might target a mitochondrial protein . Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F0F1-ATP synthase . The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations . The K(i) for apoptolidin was 4-5 microM . The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase . SIGNIFICANCE: Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated . The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways . The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.

J Exp Bot, 2001 Jan, 52(354), 181 - 2
The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C; Vranova E et al.; The AKT3 potassium channel protein was identified as a strongly interacting partner of the Arabidopsis thaliana protein phosphatase 2C (AtPP2CA) in a yeast two-hybrid screen . A deletion analysis indicated that the catalytic domain of AtPP2CA was essential for the interaction with AKT3 . Furthermore, the related PP2C phosphatase ABI1 did not interact with AKT3 in yeast.

J Cell Sci, 2001 Mar, 114(Pt 5), 975 - 86
Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii; Alberti-Segui C et al.; We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii . For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip . We could also show that mitotic division proceeds at a constant rate of 0 . 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae . Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration . For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e . generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae . This phenotype supported the view that dynein is important for the movement of nuclei towards the tip . In A . gossypii the opposite seems to be the case . A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches . Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules . The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.

Yeast, 2001 Feb, 18(3), 273 - 82
Disruption and functional analysis of six ORFs of chromosome IV: YDL103c (QRI1), YDL105w (QRI2), YDL112w (TRM3), YDL113c, YDL116w (NUP84) and YDL167c (NRP1); Reynaud A et al.; We have disrupted six ORFs (YDL103c, YDL105w, YDL112w, YDL113c, YDL116w and YDL167c) located on the left arm of chromosome IV . Except for YDL112w, the short flanking homology strategy was used to construct disruption cassettes using the KanMX4 marker . For YDL112w, a disruption cassette including the LEU2 gene was made . YDL103c and YDL105w are essential genes for vegetative growth . Disruption of YDL112w, YDL113c and YDL167c does not result in any detectable phenotype with the tests we used, while disruption of YDL116w confers slow growth, cryosensitivity and thermosensitivity, and the disrupted diploid homozygotes for the disruption failed to sporulate.

Int J Oncol, 2001 Mar, 18(3), 487 - 91
Interleukin 10 expression is correlated with thrombospondin expression and decreased vascular involvement in colon cancer; Kawakami T et al.; Interleukin 10 (IL-10) is an immuno-suppressive cytokine produced by T-lymphocytes, and a regulatory molecule for angiogenesis in various cancers . We examined IL-10 gene expression in 53 colon cancer patients who underwent surgical resection . IL-10 gene expression was correlated with TSP1 and TSP2 gene expression (P=0.0049, P=0.0285) . Colon cancer with IL-10 gene expression (19/53) showed significantly decreased venous involvement (P=0.0433) . The mean vessel counts in the colon cancers with IL-10 gene expression were significantly lower than those without IL-10 gene expression (P<0.001) . These results suggested that IL-10 stimulates angiostatic factor gene expression, and results in suppression of venous involvement.

Mol Biol Cell, 2001 Feb, 12(2), 475 - 85
Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles; Deloche O et al.; A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae . We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules . Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome . However, Vps10p sorting is independent of Apl2p . Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin . Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats . The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.

Mol Biol Cell, 2001 Feb, 12(2), 421 - 35
Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis; Dunn R et al.; Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins . Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis . Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity . In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex . We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast alpha-factor receptor, Ste2p . Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature . Deletion of the entire C2 domain had no effect on alpha-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization . These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo . In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.

Mol Biol Cell, 2001 Feb, 12(2), 309 - 21
Division of mitochondria requires a novel DMN1-interacting protein, Net2p; Cerveny KL et al.; Mitochondria are dynamic organelles that undergo frequent division and fusion, but the molecular mechanisms of these two events are not well understood . Dnm1p, a mitochondria-associated, dynamin-related GTPase was previously shown to mediate mitochondrial fission . Recently, a genome-wide yeast two-hybrid screen identified an uncharacterized protein that interacts with Dnm1p . Cells disrupted in this new gene, which we call NET2, contain a single mitochondrion that consists of a network formed by interconnected tubules, similar to the phenotype of dnm1 Delta cells . NET2 encodes a mitochondria-associated protein with a predicted coiled-coil region and six WD-40 repeats . Immunofluorescence microscopy indicates that Net2p is located in distinct, dot-like structures along the mitochondrial surface, many of which colocalize with the Dnm1 protein . Fluorescence and immunoelectron microscopy shows that Dnm1p and Net2p preferentially colocalize at constriction sites along mitochondrial tubules . Our results suggest that Net2p is a new component of the mitochondrial division machinery.

Mol Biol Cell, 2001 Feb, 12(2), 297 - 308
Genome-wide responses to mitochondrial dysfunction; Epstein CB et al.; Mitochondrial dysfunction can lead to diverse cellular and organismal responses . We used DNA microarrays to characterize the transcriptional responses to different mitochondrial perturbations in Saccharomyces cerevisiae . We examined respiratory-deficient petite cells and respiratory-competent wild-type cells treated with the inhibitors of oxidative phosphorylation antimycin, carbonyl cyanide m-chlorophenylhydrazone, or oligomycin . We show that respiratory deficiency, but not inhibition of mitochondrial ATP synthesis per se, induces a suite of genes associated with both peroxisomal activities and metabolite-restoration (anaplerotic) pathways that would mitigate the loss of a complete tricarboxylic acid cycle . The array data suggested, and direct microscopic observation of cells expressing a derivative of green fluorescent protein with a peroxisomal matrix-targeting signal confirmed, that respiratory deficiency dramatically induces peroxisome biogenesis . Transcript profiling of cells harboring null alleles of RTG1, RTG2, or RTG3, genes known to control signaling from mitochondria to the nucleus, suggests that there are multiple pathways of cross-talk between these organelles in yeast.

Infect Immun, 2001 Mar, 69(3), 1536 - 46
Efficacy of two alternate vaccines based on Plasmodium falciparum merozoite surface protein 1 in an Aotus challenge trial; Stowers AW et al.; In an attempt to produce a more defined, clinical-grade version of a vaccine based on Plasmodium falciparum merozoite surface protein 1 (MSP1), we evaluated the efficacy of two recombinant forms of MSP1 in an Aotus nancymai challenge model system . One recombinant vaccine, bvMSP1(42), based on the 42-kDa C-terminal portion of MSP1, was expressed as a secreted protein in baculovirus-infected insect cells . A highly pure baculovirus product could be reproducibly expressed and purified at yields in excess of 8 mg of pure protein per liter of culture . This protein, when tested for efficacy in the Aotus challenge model, gave significant protection, with only one of seven monkeys requiring treatment for uncontrolled parasitemia after challenge with P . falciparum . The second recombinant protein, P30P2MSP1(19), has been used in previous studies and is based on the smaller, C-terminal 19-kDa portion of MSP1 expressed in Saccharomyces cerevisiae . Substantial changes were made in its production process to optimize expression . The optimum form of this vaccine antigen (as judged by in vitro and in vivo indicators) was then evaluated, along with bvMSP1(42), for efficacy in the A . nancymai system . The new formulation of P30P3MSP1(19) performed significantly worse than bvMSP1(42) and appeared to be less efficacious than we have found in the past, with four of seven monkeys in the vaccinated group requiring treatment for uncontrolled parasitemia . With both antigens, protection was seen only when high antibody levels were obtained by formulation of the vaccines in Freund's adjuvant . Vaccine formulation in an alternate adjuvant, MF59, resulted in significantly lower antibody titers and no protection.

EMBO J, 2001 Feb 15, 20(4), 891 - 904
Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in PRT1 required for eIF3 integrity and ribosome binding; Valasek L et al.; eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met-tRNAiMet and mRNA to the pre-initiation complex . Saccharomyces cerevisiae contains an ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are not well defined . We found that HCR1 has a dual function in translation initiation: it binds to, and stabilizes, the eIF3-eIF5- eIF1-eIF2 multifactor complex and is required for the normal level of 40S ribosomes . The RNA recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1 and with an internal domain of eIF3 subunit TIF32 that has sequence and functional similarity to HCR1 . PRT1, HCR1 and TIF32 were also functionally linked by genetic suppressor analysis . We propose that HCR1 stabilizes or modulates interaction between TIF32 and the PRT1 RRM . Removal of the PRT1 RRM resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and destroyed 40S ribosome binding by the residual PRT1-TIF34-TIF35 subcomplex . Hence, the PRT1 RRM is crucial for the integrity and ribosome-binding activity of eIF3.

EMBO J, 2001 Feb 15, 20(4), 841 - 51
Gal80-Gal80 interaction on adjacent Gal4p binding sites is required for complete GAL gene repression; Melcher K et al.; Regulation of the GAL genes of Saccharomyces cerevisiae is determined by the interplay of the transcriptional activator Gal4p and the repressor Gal80p, which binds and masks the activation domain of Gal4p under non-inducing conditions . Here we demonstrate that Gal80p dimerizes with high affinity and that this dimerization appears to stabilize the Gal4p-Gal80p interaction and also, indirectly, the Gal4p-DNA interaction in a (Gal4p)2(Gal80p)2DNA complex . In addition, Gal80 dimers transiently interact with each other to form higher order multimers . We provide evidence that adjacent Gal4p binding sites, when correctly spaced, greatly stabilize Gal80p dimer-dimer interactions and that this stabilization results in the complete repression of GAL genes with multiple Gal4p binding sites . In contrast, GAL genes under the control of a single Gal4p binding site do not stabilize Gal80p multimers, resulting in significant and biologically important transcriptional leakage . Cooperative binding experiments indicate that Gal80p dimer-dimer interaction probably does not lead to a stronger Gal4p-Gal80p interaction, but most likely to a more complete shielding of the Gal4p activation domain.

J Mol Biol, 2001 Feb 23, 306(3), 539 - 53
Are trigger sequences essential in the folding of two-stranded alpha-helical coiled-coils?
Lee DL, Lavigne P, Hodges RS.
The amino acid residues comprising the interface between strands of the coiled-coil motif are usually hydrophobic and make a major contribution to coiled-coil folding and stability . However, in some cases the presence of excellent hydrophobic residues at the coiled-coil interface is insufficient for folding . It has been proposed that a "consensus trigger sequence" exists that is necessary within the coiled-coil domains of various proteins to trigger folding . Therefore, in this study we designed a 31-residue hybrid sequence based on sequences from the two-stranded parallel coiled-coil domains of the yeast transcriptional activator GCN4 and the actin-bundling protein Dictyostelium discoideum cortexillin I . The hybrid and its analogs were studied by CD spectroscopy and analytical ultracentrifugation . The hybrid had stable residues in the core "a" and "d" positions in the 3-4 hydrophobic repeat, denoted (abcdefg)n, but did not have a consensus trigger sequence and did not possess appreciable secondary structure as determined by CD spectroscopy . The substitutions in the parent peptide were introduced at positions other than "a" and "d", altering a variety of interactions including alpha-helical propensity, interchain and intrachain electrostatics, and hydrophobicity . Although the substitutions did not bring the overall sequence in closer agreement to the consensus trigger sequence, they increased coiled-coil folding and stability . Therefore, our results suggest that the combination of stabilizing effects along a protein sequence is a more general indicator of protein folding in coiled-coils than the identification of a specific trigger sequence . We propose that surpassing a critical threshold stability value using any type or combination of stabilizing effects will allow coiled-coils to fold, in the absence of a specific trigger sequence per se.

Arthritis Res, 2001, 3(2), 102 - 6 Epub 2000 Dec 20.
The human exosome: an autoantigenic complex of exoribonucleases in myositis and scleroderma; Brouwer R et al.; The anti-PM/Scl autoantibodies are known to characterize a subset of autoimmune patients with myositis, scleroderma (Scl), and the PM/Scl overlap syndrome . The major autoantigens that are recognized by anti-PM/Scl autoantibodies are designated PM/Scl-100 and PM/Scl-75 . These autoantigens have been reported to associate into a large complex consisting of 11 to 16 proteins and to play a role in ribosome synthesis . Recently, it was discovered that the PM/Scl complex is the human counterpart of the yeast (Saccharomyces cerevisiae) exosome, which is an RNA-processing complex consisting of 11 3' --> 5' exoribonucleases . To date, 10 human exosome components have been identified, although only some of these were studied in more detail . In this review, we discuss some recent advances in the characterization of the PM/Scl complex.

Nat Struct Biol, 2001 Feb, 8(2), 176 - 83
The structure of Sky1p reveals a novel mechanism for constitutive activity; Nolen B et al.; Sky1p is the only member of the SR protein kinase (SRPK) family in Saccharomyces cerevisiae . SRPKs are constitutively active kinases that display remarkable substrate specificity and have been implicated in RNA processing . Here we present the three-dimensional structure of a fully active truncated Sky1p . Analysis of the structure and structure-based functional studies reveal that the C-terminal tail, an unusual Glu residue located in the P+1 loop, and a unique mechanism for the positioning of helix alpha C act together to render Sky1p constitutively active . We have modeled a substrate peptide bound to Sky1p . The modeled complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase and suggest a mechanism by which SRPKs catalyze a sequential phosphorylation reaction of the consecutive RS dipeptide repeats characteristic of mammalian SRPK substrates.

Nat Struct Biol, 2001 Feb, 8(2), 135 - 40
A collapsed state functions to self-chaperone RNA folding into a native ribonucleoprotein complex; Webb AE et al.; Most large RNAs achieve their active, native structures only as complexes with one or more cofactor proteins . By varying the Mg(2+) concentration, the catalytic core of the bI5 group I intron RNA can be manipulated into one of three states, expanded, collapsed or native, or into balanced equilibria between these states . Under near-physiological conditions, the bI5 RNA folds rapidly to a collapsed but non-native state . Hydroxyl radical footprinting demonstrates that assembly with the CBP2 protein cofactor chases the RNA from the collapsed state to the native state . In contrast, CBP2 also binds to the RNA in the expanded state to form many non-native interactions . This structural picture is reinforced by functional splicing experiments showing that RNA in an expanded state forms a non-productive, kinetically trapped complex with CBP2 . Thus, rapid folding to the collapsed state functions to self-chaperone bI5 RNA folding by preventing premature interaction with its protein cofactor . This productive, self-chaperoning role for RNA collapsed states may be especially important to avert misassembly of large multi-component RNA-protein machines in the cell.

Nat Genet, 2001 Feb, 27(2), 167 - 71
Regulatory element detection using correlation with expression; Bussemaker HJ et al.; We present here a new computational method for discovering cis-regulatory elements that circumvents the need to cluster genes based on their expression profiles . Based on a model in which upstream motifs contribute additively to the log-expression level of a gene, this method requires a single genome-wide set of expression ratios and the upstream sequence for each gene, and outputs statistically significant motifs . Analysis of publicly available expression data for Saccharomyces cerevisiae reveals several new putative regulatory elements, some of which plausibly control the early, transient induction of genes during sporulation . Known motifs generally have high statistical significance.

Cell Death Differ, 2000 Dec, 7(12), 1210 - 7
Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death; Low P et al.; The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins . We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6' (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD) . Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres . S6' is present in degraded material only outside intact fibres . S7 can be detected in nuclei, cytoplasm and also in degraded material . S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD . 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes . These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD . Cell Death and Differentiation (2000) 7, 1210 - 1217.

J Biochem (Tokyo), 2001 Feb, 129(2), 185 - 91
SPT genes: key players in the regulation of transcription, chromatin structure and other cellular processes; Yamaguchi Y et al.; Suppressor of Ty (SPT) genes were originally identified through a genetic screen for mutations in the yeast Saccharomyces cerevisiae that restore gene expression disrupted by the insertion of the transposon Ty . Classic members of the SPT gene family, SPT11, SPT12, and SPT15, encode for the histones H2A and H2B, and for TATA-binding protein (TBP), respectively . Over the past few years, molecular complexes and cellular functions in which other SPT gene products involve have been discovered through genetic and biochemical studies in yeast and several other organisms: Key regulators of transcription and chromatin structure, such as DSIF, SAGA, and FACT, all contain SPT gene products as essential subunits . In addition, accumulating evidence suggests that SPT gene products play more diverse roles, including roles in DNA replication, DNA recombination and developmental regulation . Here we review the current understanding of the functions and roles of the SPT genes, with special emphasis on the role of SPT5 in transcript elongation and in neuronal development in vertebrates.

Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 254 - 9
Dynamic light-scattering analysis of full-length human RPA14/32 dimer: purification, crystallization and self-association; Habel JE et al.; Replication protein A (RPA) is a single-stranded DNA-binding protein involved in all aspects of eukaryotic DNA metabolism . A soluble heterodimeric form of RPA is composed of 14 and 32 kDa subunits (RPA14/32) . Dynamic light-scattering (DLS) analysis was used to improve the purification, stabilization and crystallization of RPA14/32 . Increasing the concentration of reducing agent in the last stage of purification diminished the size of a secondary peak in the anion-exchange chromatograph and promoted a single species in solution . This resulted in decreased polydispersity in the purified protein and enhanced the crystallization time from 9-12 months to 6 d . With this homogeneous preparation, the reversible association of RPA14/32 into a dimer of dimers was demonstrated by DLS . Four different crystal forms of RPA14/32 were obtained for structure determination and complete diffraction data were collected using synchrotron radiation for three of them . Data to 2.4 A resolution was collected from hexagonal crystals (P3(2) or P3(1); a = b = 63.0, c = 272.6 A) and to 2.2 and 1.9 A resolution from two orthorhombic crystal forms (both P2(1)2(1)2(1); form I, a = 61.4, b = 75.2, c = 131.6 A; form II, a = 81.8, b = 140.4, c = 173.1 A).

Trends Plant Sci, 2001 Feb, 6(2), 59 - 65
Histone acetylation: lessons from the plant kingdom; Lusser A et al.; Post-translational acetylation of core histones is an enigmatic process . The identification of histone acetyltransferases and deacetylases as co-regulators of transcription in yeast and vertebrates has advanced our understanding of the biological role of histone acetylation and also improved our general insight into the molecular network of gene regulation . Basic features of histone acetylation in plants resemble those of other eukaryotes but there are differences, which are reflected in novel classes of histone deacetylase . Investigating histone acetylation in higher plants might reveal regulatory pathways distinct from animals, yet of essential importance for gene expression in plants.

FEBS Lett, 2001 Feb 9, 490(1-2), 49 - 53
Multiple PIP2 binding sites in Kir2.1 inwardly rectifying potassium channels; Soom M et al.; Inwardly rectifying potassium channels require binding of phosphatidylinositol-4,5-bisphosphate (PIP2) for channel activity . Three independent sites (aa 175-206, aa 207-246, aa 324-365) were located in the C-terminal domain of Kir2.1 channels by assaying the binding of overlapping fragments to PIP2 containing liposomes . Mutations in the first site, which abolished channel activity, reduced PIP2 binding of this fragment but not of the complete C-terminus . Point mutations in the third site also reduced both, channel activity and PIP2 binding of this segment . The relevance of the third PIP2 binding site provides a basis for the understanding of constitutively active Kir2 channels.

Mol Cell, 2001 Jan, 7(1), 89 - 95
RSC unravels the nucleosome; Lorch Y et al.; RSC and SWI/SNF chromatin-remodeling complexes were previously reported to generate a stably altered nucleosome . We now describe the formation of hybrids between nucleosomes of different sizes, showing that the stably altered structure is a noncovalent dimer . A basis for dimer formation is suggested by an effect of RSC on the supercoiling of closed, circular arrays of nucleosomes . The effect may be explained by the interaction of RSC with DNA at the ends of the nucleosome, which could lead to the release 60--80 bp or more from the ends . DNA released in this way may be trapped in the stable dimer or lead to alternative fates such as histone octamer transfer to another DNA or sliding along the same DNA molecule.

Mol Cell, 2001 Jan, 7(1), 13 - 20
Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asf1; Emili A et al.; The evolutionarily conserved yeast checkpoint protein kinase Rad53 regulates cell cycle progression, transcription, and DNA repair in response to DNA damage . To uncover potential regulatory targets of Rad53, we identified proteins physically associated with it in vivo using protein affinity purification and tandem mass spectrometry . Here we report that Rad53 interacts in a dynamic functional manner with Asf1, a chromatin assembly factor recently shown to mediate deposition of acetylated histones H3 and H4 onto newly replicated DNA . Biochemical and molecular genetic studies suggest that Asf1 is an important target of the Rad53-dependent DNA damage response and that Rad53 may directly regulate chromatin assembly during DNA replication and repair.

Curr Med Chem, 2001 Feb, 8(2), 211 - 35
Histone deacetylase: a target for antiproliferative and antiprotozoal agents; Meinke PT et al.; Histone deacetylase (HDAC) and histone acetyltransferase (HAT) are enzymes that influence transcription by selectively deacetylating or acetylating the eta-amino groups of lysines located near the amino termini of core histone proteins . It is well-established that in transcriptionally active chromatin, histones generally are hyperacetylated and, conversely, hypoacetylated histones are coincident with silenced chromatin . Revived interest in these enzymatic pathways and how they modulate eukaryotic transcription has led to the identification of multiple cofactors whose complex interplay with HDAC affects gene expression . Concurrent with these discoveries, screening of natural product sources yielded new small molecules that were subsequently identified as potent inhibitors of HDAC . While predominantly identified using antiproliferative assays, the biological activity of these new HDAC inhibitors also encompasses significant antiprotozoal, antifungal, phytotoxic and antiviral applications . These newly discovered HDAC inhibitors served as lead structures for the development of improved derivatives including related reagents with considerable potential as tools to further elucidate the mechanism of transcriptional regulation.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1817 - 22
p53 associates with and targets Delta Np63 into a protein degradation pathway; Ratovitski EA et al.; A human p53 homologue, p63 (p40/p51/p73L/CUSP) that maps to the chromosomal region 3q27-29 was found to produce a variety of transcripts that encode DNA-binding proteins with and without a trans-activation domain (TA- or Delta N-, respectively) . The p63 gene locus was found to be amplified in squamous cell carcinoma, and overexpression of Delta Np63 (p40) led to increased growth of transformed cells in vitro and in vivo . Moreover, p63-null mice displayed abnormal epithelial development and germ-line human mutations were found to cause ectodermal dysplasia . We now demonstrate that certain p63 isotypes form complexes with p53 . p53 mutations R175H or R248W abolish the association of p53 with p63, whereas V143A or R273H has no effect . Deletion studies suggest that the DNA-binding domains of both p53 and p63 mediate the association . Overexpression of wild type but not mutant (R175H) p53 results in the caspase-dependent degradation of certain Delta Np63 proteins (p40 and Delta Np63 alpha) . The association between p53 and Delta Np63 supports a previously unrecognized role for p53 in regulation of Delta Np63 stability . The ability of p53 to mediate Delta Np63 degradation may balance the capacity of Delta Np63 to accelerate tumorigenesis or to induce epithelial proliferation.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1711 - 6
RAD50 function is essential for telomere maintenance in Arabidopsis; Gallego ME et al.; We have identified and characterized an Arabidopsis thaliana rad50 mutant plant containing a T-DNA insertion in the AtRAD50 gene and showing both meiotic and DNA repair defects . We report here that rad50/rad50 mutant cells show a progressive shortening of telomeric DNA relative to heterozygous rad50/RAD50 controls and that the mutant cell population rapidly enters a crisis, with the majority of the cells dying . Surviving rad50 mutant cells have longer telomeres than wild-type cells, indicating the existence in plants of an alternative RAD50-independent mechanism for telomere maintenance . These results report the role of a protein essential for double-strand break repair in telomere maintenance in higher eukaryotes.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1495 - 500
Ash1p is a site-specific DNA-binding protein that actively represses transcription; Maxon ME et al.; ASH1 encodes a protein that is localized specifically to the daughter cell nucleus, where it has been proposed to repress transcription of the HO gene . Using Ash1p purified from baculovirus-infected insect cells, we have shown that Ash1p binds specific DNA sequences in the HO promoter . DNase I protection analyses showed that Ash1p recognizes a consensus sequence, YTGAT . Mutation of this consensus abolishes Ash1p DNA binding in vitro . We have shown that Ash1p requires an intact zinc-binding domain in its C terminus for repression of HO in vivo and that this domain may be involved in DNA binding . A heterologous DNA-binding domain fused to an N-terminal segment of Ash1p functions as an active repressor of transcription . Our studies indicate that Ash1p is a DNA-binding protein of the GATA family with a separable transcriptional repression domain.

J Cell Sci, 2001 Feb, 114(Pt 4), 643 - 51
Where it all starts: eukaryotic origins of DNA replication; Bielinsky AK et al.; Chromosomal origins of DNA replication in eukaryotic cells not only are crucial for understanding the basic process of DNA duplication but also provide a tool to analyze how cell cycle regulators are linked to the replication machinery . During the past decade much progress has been made in identifying replication origins in eukaryotic genomes . More recently, replication initiation point (RIP) mapping has allowed us to detect start sites for DNA synthesis at the nucleotide level and thus to monitor replication initiation events at the origin very precisely . Beyond giving us the precise positions of start sites, the application of RIP mapping in yeast and human cells has revealed a single, defined start point at which replication initiates, a scenario very reminiscent of transcription initiation . More importantly, studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site . Therefore, in our pursuit of identifying ORC-binding sites in higher eukaryotes, RIP mapping may lead the way.

Biochem Soc Trans, 2000 Dec, 28(6), 658 - 60
Polyunsaturated fatty acid-specific elongation enzymes; Das T et al.; We have isolated a novel gene (GLELO) from Mortierella alpina and its homologue (CEELO1) from Caenorhabditis elegans and demonstrate the involvement of their encoded proteins in the elongation of C(18) polyunsaturated fatty acids.

Biochem Soc Trans, 2000 Dec, 28(6), 654 - 8
Cloning and functional expression of the first plant fatty acid elongase specific for Delta(6)-polyunsaturated fatty acids; Zank TK et al.; In order to elucidate the biosynthesis of long-chain polyunsaturated fatty acids (PUFAs) in plants we searched for a cDNA encoding a Delta(6)-specific PUFA elongase from Physcomitrella patens, which is known to contain high proportions of arachidonic acid (20:4 Delta(5,8,11,14)) . An EST clone from P . patens was identified by its low homology to the yeast gene ELO1, which is required for the elongation of medium-chain fatty acids . We functionally characterized this cDNA by heterologous expression in Saccharomyces cerevisiae grown in the presence of several fatty acids . Analysis of the fatty acid profile of the transgenic yeast revealed that the cDNA encodes a protein that leads to the elongation of the C(18) Delta(6)-polyunsaturated fatty acids gamma-linolenic acid (18:3 Delta(6,9,12)) and stearidonic acid (18:4 Delta(6,9,12,15)), which were recovered to 45-51% as their elongation products . In contrast, linoleic and alpha-linolenic acids were hardly elongated and we could not measure any elongation of saturated and mono-unsaturated fatty acids (including 18:1 Delta(6)), indicating that the elongase is highly specific for the polyunsaturated nature of the fatty acid acting as substrate.

Biochem Soc Trans, 2000 Dec, 28(6), 638 - 41
Further characterization of Delta(8)-sphingolipid desaturases from higher plants; Sperling P et al.; A previously cloned cDNA from Helianthus annuus codes for a fusion protein composed of an N-terminal cytochrome b(5) and a C-terminal desaturase domain . For a functional identification, this cDNA was expressed in Saccharomyces cerevisiae and the structures of sphingolipid long-chain bases were analysed . The expression of this sunflower enzyme resulted in the formation of new Delta(8)-trans/cis-phytosphingenine from C(18)- and C(20)-phytosphinganine present in wild-type yeast cells . To elucidate the substrate specificity, the recently cloned Delta(8)-sphingolipid desaturases from Arabidopsis thaliana and Brassica napus were expressed in the yeast mutant sur2Delta that lacked the sphinganine C(4)-hydroxylase and was thus unable to form phytosphinganine . Long-chain base analysis of the transformed mutant cells did not show any conversion of C(18)- or C(20)-sphinganine into Delta(8)-sphingenine, whereas exogenously added C(18)-phytosphinganine was desaturated to Delta(8)-trans/cis-phytosphingenine . Furthermore, GLC-MS analysis did not reveal the presence of any Delta(9)-regioisomers as reported before . These results show that the sunflower gene codes for a Delta(8)-sphingolipid desaturase which accepts C(18)- and C(20)-phytosphinganine . The absence of Delta(8)-sphingenine as desaturation product in the transformed mutant suggests that C(4)-hydroxylation of sphinganine precedes Delta(8)-desaturation . Therefore, in yeast, the substrate for the plant Delta(8)-sphingolipid desaturase seems to be the phytosphinganine residue.

Biochem Soc Trans, 2000 Dec, 28(6), 636 - 8
Mutagenesis of the borage Delta(6) fatty acid desaturase; Sayanova O et al.; The consensus sequence of the third histidine box of a range of Delta(5), Delta(6), Delta(8) and sphingolipid desaturases differs from that of the membrane-bound non-fusion Delta(12) and Delta(15) desaturases in the presence of glutamine instead of histidine . We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b(5) fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Delta(6) desaturase.

Biochem Soc Trans, 2000 Dec, 28(6), 625 - 7
Molecular properties of the oleoyl-phosphatidylcholine desaturase from Arachis hypogaea L; Powell GL et al.; Deficiencies in the activity of the microsomal oleoyl phosphatidylcholine (oleoyl-PC) desaturase from peanuts are the basis of the high oleate oil . Mutation of aspartate-150 to asparagine and the attendant decrease in activity, together with the loss in expression of the higher activity transcript, was the molecular basis of the high oleate trait . The ability of oleoyl-PC desaturase to desaturate palmitoleate, oleate and 10(Z) nonadecenoate to methylene-interrupted products was not consistent with description of this activity as a Delta(12) or omega-6 desaturase . Electrospray MS was used to examine the intact phospholipid products of desaturation by the oleoyl-PC desaturase . PC and phosphatidylinositol containing unsaturated moieties could be desaturated . The enzyme can act on either sn-1 or sn-2 moieties . Phosphatidylethanolamine was a poor substrate.

Biochem Soc Trans, 2000 Dec, 28(6), 615 - 6
Protein interactions of fatty acid synthase II; Honeyman G et al.; We have used a yeast two-hybrid approach to detect direct protein interactions between fatty acid synthase components . Enoyl-acyl carrier protein (ACP) reductase was found to interact with stearoyl-ACP desaturase and acyl-ACP thioesterase, but none of these proteins interacted with ACP in the yeast nucleus.

Biochem J, 2001 Feb 1, 353(Pt 3), 689 - 99
Cholesterol biosynthesis from lanosterol: molecular cloning, chromosomal localization, functional expression and liver-specific gene regulation of rat sterol delta8-isomerase, a cholesterogenic enzyme with multiple functions; Bae S et al.; Sterol Delta(8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil binding protein or sigma receptor, catalyses the conversion of the 8-ene isomer into the 7-ene isomer in the cholesterol biosynthetic pathway in mammals . Recently, mutations of SI have been found to be associated with Conradi-Hunermann syndrome in humans . To investigate the in vitro and in vivo modes of molecular regulation of SI and its role in cholesterol biosynthesis in mammals, we isolated a full-length cDNA encoding rat SI . The deduced amino-acid sequence of rat SI predicts a 230-residue protein (26737 Da) with 87% and 80% amino-acid identity to mouse and human counterparts . The rat SI gene was mapped to chromosome 12q1.2 using fluorescence in situ hybridization (FISH) . The biological function of the cloned rat SI cDNA was verified by overexpressing recombinant Myc-SI in Saccharomyces cerevisiae . It showed a characteristic pattern of inhibition on exposure to trans-2-{4-(1,2-diphenylbuten-1-yl)phenoxy}-N,N-dimethylethylamine (tamoxifen; IC(50)=11.2 microM) and 3beta-{2-(diethylamino)ethoxy}androst-5-en-17-one (U18666A; IC(50)=4.2 microM), two well known potent inhibitors of SI . Northern-blot analysis of 3-week-old rats compared with 2-year-old rats showed that SI mRNA expression in both age groups was restricted to liver, where a 70% reduction in mRNA levels was observed in 2-year-old rats . The FISH studies revealed ubiquitous expression of SI mRNA in rat hepatocytes . The in vitro studies showed that the SI mRNA was highly suppressed by 25-hydroxycholesterol in H4IIE cells . Treatment of H4IIE cells grown in medium supplemented with fetal bovine serum with tamoxifen for 24 h resulted in a dose-dependent induction of SI mRNA, with a concomitant suppression of sterol regulatory element binding protein-1 mRNA . Interestingly, this effect was not seen in emopamil-treated cells . The in vivo experiments also indicate that both mRNA expression and enzymic activity of SI in liver were induced approx . 3-fold in rats fed 5% (w/w) cholestyramine plus 0.1% (w/w) lovastatin in normal chow for 2 weeks . With this newly cloned rat SI cDNA, it becomes possible to gain molecular understanding of previously unknown and tamoxifen-mediated gene regulation of SI that is involved in cholesterol metabolism, ischaemia and genetic diseases.

Fungal Genet Biol, 2000 Nov, 31(2), 69 - 78
The rpl16a gene for ribosomal protein L16A identified from expressed sequence tags is differentially expressed during sexual development of Aspergillus nidulans; Jeong HY et al.; We obtained 305 expressed sequence tags (ESTs), which are from the poly(A) site to the most proximal MboI site, from mycelia at the early sexual developmental (ESD) stage of Aspergillus nidulans . By comparison of these ESTs with those obtained previously from the vegetative stage and from the late sexual developmental stage followed by Northern blot analyses, genes of 17 ESTs were identified as being expressed more abundantly at the ESD stage than at the vegetative stage . Five of 17 genes were expressed more abundantly in the presence of the veA gene or the nsdD gene, suggesting that these 5 genes may be involved in sexual development . In a gene of one EST, appearing three times among 305 ESTs and identified by GenBank, polyadenylation seemed to occur at two sites . Nucleotide sequences of the gene having the EST and its cDNA revealed that the gene can code for a 202-amino-acid polypeptide with an estimated molecular mass of 23 kDa . The deduced amino acid showed 73% identity to Saccharomyces cerevisiae ribosomal protein L16A (RPL16A), and therefore the gene was named rpl16a . A . nidulans RPL16A had a putative leucine zipper motif and a basic leucine zipper motif like those of other organisms . The expression level of the rpl16a gene, present as a single copy in this organism, reached a maximum after 2 h, decreased thereafter, and increased again 30 to 50 h after the end of induction of sexual development . These results clearly indicated that the rpl16a gene is expressed differentially during sexual development.

J Nat Prod, 2001 Jan, 64(1), 2 - 5
Isolation, synthesis, and structure-activity relationships of bioactive benzoquinones from Miconia lepidota from the Suriname rainforest; Gunatilaka AA et al.; Bioactivity-directed fractionation of an EtOAc extract from the leaves of Miconia lepidota afforded the two benzoquinones 2-methoxy-6-heptyl-1,4-benzoquinone (1) and 2-methoxy-6-pentyl-1,4-benzoquinone (primin) (2) . This is the first reported isolation of 1 . Both quinones 1 and 2 exhibited activity toward mutant yeast strains based on Saccharomyces cerevisiae, indicative of their cytotoxicity and potential anticancer activity . A number of previously synthesized and new analogues were prepared and tested in the same strains . Compounds 1, 2, 2-methoxy-6-butyl-1,4-benzoquinone (5), and 2-methoxy-6-decyl-1,4-benzoquinone (6) were tested in two cytotoxicity assays . In the M109 tumor cell lines, quinones 1, 2, and 6 had an IC(50) value of 10 microg/mL . In the A2780 cell line, compounds 1, 2 and 5 had IC(50) values of 7.9, 2.9, and 3.2 microg/mL, respectively.

Biochemistry, 2001 Jan 30, 40(4), 994 - 1001
Probing the relative timing of hydrogen abstraction steps in the flavocytochrome b2 reaction with primary and solvent deuterium isotope effects and mutant enzymes; Sobrado P et al.; Flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate . Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate O-H and C-H bonds by the wild-type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme . The (D)V(max) and (D)(V/K(lactate)) values are both 3.0 with the wild-type enzyme at pH 7.5 and 25 degrees C, increasing to about 3.6 with the flavin domain and increasing further to about 4.5 with the D282N enzyme . Under these conditions, the (D20)V(max) values are 1.38, 1.18, and 0.98 for the wild-type enzyme, the flavin domain, and the D282N enzyme, respectively; the (D20)(V/K(lactate)) values are 0.9, 0.44, and 1.0, respectively . The (D)k(red) value is 5.4 for the wild-type enzyme and 3.5 for the flavin domain, whereas the solvent isotope effect on this kinetic parameter is 1.0 for both enzymes . The V(max) values for the wild-type enzyme and the flavin domain are 32 and 15% limited by viscosity, respectively . In contrast, the V/K(lactate) value for the flavin domain increases about 2-fold at moderate concentrations of glycerol . The data are consistent with a minimal chemical mechanism in which the lactate hydroxyl proton is not in flight in the transition state for C-H bond cleavage and there is an internal equilibrium involving the lactate-bound enzyme prior to C-H bond cleavage which is sensitive to solution conditions . Removal of the hydroxyl proton may occur in this pre-equilibrium.

Biochemistry, 2001 Jan 23, 40(3), 719 - 31
Mapping the energy surface for the folding reaction of the coiled-coil peptide GCN4-p1; Ibarra-Molero B et al.; The energy surface for the folding/unfolding reactions of the homodimeric coiled-coil peptide M2V GCN4-p1, a 33-residue segment comprising the leucine zipper domain of the transcriptional activator GCN4, was mapped by equilibrium and kinetic methods . Circular dichroism (CD) spectroscopy was used to monitor the urea-induced unfolding reaction at a series of temperatures and temperature-induced unfolding at a series of urea concentrations . A global analysis of the urea- and temperature-induced equilibrium unfolding data provides strong support for a two-state mechanism . The absence of a detectable population of intermediate states is also consistent with differential scanning calorimetry and thermal CD melts as a function of peptide concentration . Furthermore, a global analysis of stopped-flow CD kinetic data is consistent with a kinetic two-state mechanism as well . The urea dependence of the apparent folding and unfolding rate constants at a series of temperatures reveals that the activation enthalpy and entropy for unfolding in the absence of denaturant are both significantly greater than those for the refolding reaction . Although the unfolding barrier is dominated by the activation enthalpy, the activation entropy dominates the refolding barrier . The relative magnitudes of the urea dependence of the unfolding and refolding rate constants indicate that 55-65% of the surface area is buried in the transition state . The activation parameters imply a partially organized transition state and are consistent with a previous model in which the pair of C-terminal heptad repeats are docked in a coiled-coil-like motif {Zitzewitz et al . (2000) J . Mol . Biol . 296, 1105-1116}.

Biochemistry, 2001 Jan 23, 40(3), 712 - 8
DNA topoisomerase II as the target for the anticancer drug TOP-53: mechanistic basis for drug action; Byl JA et al.; TOP-53 is a promising anticancer agent that displays high activity against non-small cell lung cancer in animal tumor models {Utsugi, T., et al . (1996) Cancer Res . 56, 2809-2814} . Compared to its parent compound, etoposide, TOP-53 is considerably more toxic to non-small cell lung cancer cells, is more active at generating chromosomal breaks, and displays improved cellular uptake and pharmacokinetics in animal lung tissues . Despite the preclinical success of TOP-53, several questions remain regarding its cytotoxic mechanism . Therefore, this study characterized the basis for drug action . Results indicate that topoisomerase II is the primary cytotoxic target for TOP-53 . Furthermore, the drug kills cells by acting as a topoisomerase II poison . TOP-53 exhibits a DNA cleavage site specificity that is identical to that of etoposide . Like its parent compound, the drug increases the number of enzyme-mediated DNA breaks by interfering with the DNA religation activity of the enzyme . TOP-53 is considerably more efficient than etoposide at enhancing topoisomerase II-mediated DNA cleavage and exhibits high activity against human topoisomerase IIalpha and IIbeta in vitro and in cultured cells . Therefore, at least in part, the enhanced cytotoxic activity of TOP-53 can be attributed to an enhanced activity against topoisomerase II . Finally, TOP-53 displays nearly wild-type activity against a mutant yeast type II enzyme that is highly resistant to etoposide . This finding suggests that TOP-53 can retain activity against systems that have developed resistance to etoposide, and indicates that substituents on the etoposide C-ring are important for topoisomerase II-drug interactions.

Bioconjug Chem, 2001 Jan-Feb, 12(1), 35 - 43
Solid-phase synthesis of a radiolabeled, biotinylated, and farnesylated Ca(1)a(2)X peptide substrate for Ras- and a-mating factor converting enzyme; Dolence EK et al.; Eukaryotic proteins with carboxyl-terminal Ca(1)a(2) motifs undergo three posttranslational processing reactions--prenylation, endoproteolysis, and carboxymethylation . Two genes in yeast encoding Ca(1)a(2)X endoproteases, AFC1 and RCE1, have been identified . Rce1p is solely responsible for proteolysis of yeast Ras proteins . When proteolysis is blocked, localization of Ras2p to the outer membrane is impaired . The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics . A biotinylated, farnesylated Ca(1)a(2)X peptide {(1-N-biotinyl-(13-N-succinimidyl-(S-(E,E-farnesyl)-L-cysteinyl)-L-valinyl-L-isoleucinyl-L-alanine))-4,7,10-trioxatridecanediamine} 1 containing a poly(ethylene glycol) linker was prepared by solid-phase synthesis for use in an assay for Ca(1)a(2)X endoprotease activity that relies on the strong affinity of avidin for biotin . The peptide was radiolabeled in the penultimate step of the synthesis by cleavage of the biotinylated, farnesylated Ca(1)a(2) precursor from Kaiser's oxime resin with {(14)C}-L-alanine methyl ester . {(14)C}1 was a good substrate for yRce1p with K(M) = 1.3 +/- 0.3 microM . Analysis of the carboxyl terminal products by reverse phase HPLC confirmed that VIA was the only radioactive fragment released upon incubation of {(14)C}1 with a yeast membrane preparation of recombinant yRce1p . The solid-phase methodology developed using Kaiser's benzophenone oxime resin to synthesize {(14)C}1 should be generally applicable for peptides containing sensitive side chains . In addition, introduction of the radiolabeled unit at the end of the synthesis mostly circumvents problems associated with handling radioactive materials.

Plant J, 2001 Jan, 25(1), 43 - 53
The jasmonate-inducible AP2/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element; van der Fits L et al.; The AP2/ERF-domain transcription factor ORCA3 is a master regulator of primary and secondary metabolism in Catharanthus roseus (periwinkle) . Here we demonstrate that ORCA3 specifically binds to and activates gene expression via a previously characterized jasmonate- and elicitor-responsive element (JERE) in the promoter of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str) . Functional characterization of different domains in the ORCA3 protein in yeast and plant cells revealed the presence of an N-terminal acidic activation domain and a serine-rich C-terminal domain with a negative regulatory function . Orca3 mRNA accumulation was rapidly induced by the plant stress hormone methyljasmonate with biphasic kinetics . A precursor and an intermediate of the jasmonate biosynthetic pathway also induced Orca3 gene expression, further substantiating the role for ORCA3 in jasmonate signaling . The protein synthesis inhibitor cycloheximide did not inhibit jasmonate-responsive expression of Orca3, nor of its target genes Str and Tryptophan decarboxylase (Tdc) . In conclusion, ORCA3 regulates jasmonate-responsive expression of the Str gene via direct interaction with the JERE . The activating activities of ORCA proteins do not seem to depend on jasmonate-induced de novo protein synthesis, but presumably occur via modification of pre-existing ORCA protein.

Plant J, 2001 Jan, 25(1), 31 - 41
Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity; Gallego ME et al.; The Rad50 protein is involved in the cellular response to DNA-double strand breaks (DSBs), including the detection of damage, activation of cell-cycle checkpoints, and DSB repair via recombination . It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice . Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene . A single copy of this gene is present in the Arabidopsis genome, located on chromosome II . Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells . The predicted protein presents strong conservation with the other known Rad50 homologues of the amino- and carboxy-terminal regions . Mutant plants present a sterility phenotype which co-segregates with the T-DNA insertion . Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T-DNA insertion . An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA-damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double-strand break repair in plant cells . This is the first report of a plant mutated in a protein of the Rad50-Mre11-Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.

Genes Cells, 2000 Dec, 5(12), 1039 - 48
Sed4p functions as a positive regulator of Sar1p probably through inhibition of the GTPase activation by Sec23p; Saito-Nakano Y et al.; BACKGROUND: Sar1p belongs to a unique subfamily of small GTPases and is essential for formation of the transport vesicles from the endoplasmic reticulum (ER) that are destined to the Golgi apparatus . To understand how the GTPase cycle of Sar1p is regulated, we screened for multicopy suppressors of sar1 ts mutants and identified SED4 . RESULTS: Although deletion of sed4 alone shows no growth defect, sar1 delta(sed4) double mutant cells are inviable . In the delta(sed4) background, the suppression activity of SAR1 towards sec12 and sec16 is lost . These observations suggest that SED4 is a very close partner of SAR1 and imply that Sed4p may act to increase the active Sar1p in the cell . Over-expression of SEC12 does not remedy the lethality of sar1 delta(sed4) . The purified cytoplasmic domain of Sed4p does not show a guanine nucleotide exchange (GEF) activity toward Sar1p nor increase the GEF activity of Sec12p . On the contrary, over-expression of SED4 aggravates the ts growth of sec23 cells . The cytoplasmic domain of Sed4p weakly inhibits the GTPase-activating (GAP) activity of Sec23p toward Sar1p . In a microsome-based COPII binding assay, the binding of the GDP-form mutant Sar1p (D32G) is lower on the delta(sed4) microsomes than on the wild-type membranes . CONCLUSION: We propose a model that Sed4p counteracts the GAP action of Sec23p on to Sar1p.

Gene, 2000 Dec 31, 261(2), 305 - 10
Investigating 42 candidate orthologous protein groups by molecular evolutionary analysis on genome scale; Xie T et al.; It is one of key problems for comparative genomics to accurately identify orthologous genes/proteins . Here 42 quartettes of human, yeast Saccharomyces cerevisiae, nematode Caenorhabditis elegans, and fruit fly Drosophila melanogaster candidate orthologs, defined by using similarity-based highest hit criteria (Mushegian et al., 1998 Genome Res . 8: 590-598), were reconsidered according to molecular evolutionary analysis . We found that only 14 of the 42 candidate orthologous groups can be identified to have truly one-to-one orthologous relationships, whereas other groups were characterized by one (many)-to-many orthologous relationships or even more complex scenarios involving gene duplications and/or gene losses . The result could imply that the classical one-to-one orthology might be not as common as typically accepted and automated similarity-based methods should be used with caution when accurate orthology/paralogy discrimination is required.

Mol Biochem Parasitol, 2001 Jan 15, 112(1), 39 - 49
Identification of a spliced leader RNA binding protein from Trypanosoma cruzi; Xu P et al.; Nuclear mRNAs in trypanosomatids are generated by trans-splicing . Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified . Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA . XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae . Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T . cruzi protein binds the SL RNA in vitro . The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system . Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T . cruzi extract.

Trends Cell Biol, 2001 Feb, 11(2), 60 - 5
The Pif1p subfamily of helicases: region-specific DNA helicases?
Bessler JB, Torredagger JZ, Zakian VA.
DNA helicases are required for DNA replication, recombination and repair . Despite a common enzymatic function - the ability to unwind duplex DNA - most helicases share only limited amino acid sequence similarity . Helicases that have significant sequence similarity define a subfamily . It remains unclear, however, how this sequence similarity relates to helicase function . The Saccharomyces cerevisiae Pif1p helicase is the prototype member of a helicase subfamily that is conserved from yeasts to humans . As the two Pif1p subfamily members studied to date affect the same DNA sequences, the amino acid similarity that defines this subfamily might reflect common substrates.

Mech Dev, 2001 Feb, 100(2), 335 - 8
Identification of SorCS2, a novel member of the VPS10 domain containing receptor family, prominently expressed in the developing mouse brain; Rezgaoui M et al.; We report the identification of a fourth member of the VPS10 domain containing receptor family, SorCS2, highly expressed in the developing and mature murine central nervous system . During early central nervous system development its main site of expression is the floor plate . In addition, high transcript levels were detected transiently in a variety of brain regions including the dopaminergic midbrain nuclei and the dorsal thalamus . Outside the nervous system expression is detected in lung and heart and transiently in a variety of mesodermally derived tissues.

Mol Cell Endocrinol, 2001 Jan 22, 171(1-2), 205 - 10
Evolution of 17beta-HSD type 4, a multifunctional protein of beta-oxidation; Breitling R et al.; 17beta-Hydroxysteroid dehydrogenase type 4 (17beta-HSD4) is the most unusual among human 17beta-HSDs . It is characterized by a multidomain structure, in which the dehydrogenase domain is fused to a hydratase and a lipid transfer domain . 17beta-HSD4 not only inactivates estradiol by conversion to estrone but its three protein domains also participate in successive steps of peroxisomal beta-oxidation of long- and branched-chain fatty acids . We have compared the genomic structure of human 17beta-HSD4 and several homologous genes from lower animals and fungi . Our data suggest an evolutionary scenario for the three protein domains and indicate a highly dynamic history of the enzyme but also a very high conservation of multifunctionality . This suggests that the main function of human 17beta-HSD4 is still its involvement in fatty-acid metabolism, while steroid conversion is only a secondary and possibly minor activity in vivo.

Plant Sci, 2001 Jan 5, 160(2), 209 - 218
Cloning and characterization of a cDNA encoding hexokinase from tomato; Menu T et al.; Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato (Lycopersicon esculentum) by RT-PCR using degenerate primers . Southern blot analysis suggested the existence of two divergent HXK genes . A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit . The 1770 bp cDNA of LeHXK2 contained an open reading frame encoding a 496 amino acid protein that has 69% identity with the two Arabidopsis HXKs, 83 and 85% identity with potato StHXK1 and tobacco NtHXK, respectively . However, this clone had 97% amino acid identity with potato StHXK2 and, therefore, was named LeHXK2 . LeHXK2 cDNA was expressed in a triple mutant yeast (Saccharomyces cerevisiae) strain which lacked the ability to phosphorylate glucose and fructose and, therefore, was unable to grow on these sugars as carbon sources . Mutant cells expressing LeHXK2 grew on both glucose and fructose with shorter doubling time on glucose . The kinetic properties of LeHXK2 expressed in yeast were determined after the purification of LeHXK2 by HPLC-ion exchange chromatography, confirming the identity of LeHXK2 as hexokinase with higher affinity to glucose . LeHXK2 mRNA was detected by RT-PCR expression analysis in all organs and tissues and at all stages of fruit development . However, semi-quantitative RT-PCR analysis showed that LeHXK2 was most highly expressed in flowers.

Mech Ageing Dev, 2000 Dec 20, 121(1-3), 5 - 19
Modulation of X-ray-induced damage recognition and repair in ageing human peripheral blood mononuclear cells by an interleukin-6-type cytokine; Frasca D et al.; We have investigated the effects of an interleukin (IL)-6-type cytokine on the DNA-binding activity of ku and on unscheduled DNA repair in X-ray-treated peripheral blood mononuclear cells (PBMC) from human subjects of different ages . The cytokine used, called K-7/D-6, is an IL-6 variant with increased in vivo and in vitro biological activity compared to the wild type molecule . Ku is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK) . It binds the ends of various types of DNA discontinuity and is involved in the repair of DNA breaks caused by V(D)J recombination, isotype switching, physiological oxidation reactions, ionizing radiation and some chemotherapeutic drugs . The ku-dependent repair process, called non-homologous end joining, is the main DNA double strand break repair mechanism in irradiated mammalian cells . Results show that K-7/D-6 significantly increases DNA-binding activity of ku in irradiated PBMC from young but not from elderly subjects . However, K-7/D-6 is able to induce unscheduled DNA repair in irradiated PBMC from both young and elderly subjects . These effects of K-7/D-6 are relevant to the mechanisms of the cellular response to DNA damage.

FEBS Lett, 2001 Jan 12, 488(1-2), 91 - 4
Prolactin activation of IRF-1 transcription involves changes in histone acetylation; Book McAlexander M et al.; In response to prolactin (PRL) signaling, transcription of the interferon regulatory factor-1 gene (IRF-1) is rapidly induced during early G(1), declines in mid G(1), and rises again over the G(1)/S transition phase of the cell cycle in Nb2 T cells . Using chromatin immunoprecipitation assays, we show that histone H4 acetylation increases over a 1.7 kb region of the IRF-1 promoter in early G(1) and again at the G(1)/S transition in response to PRL stimulation . These results demonstrate a correlation between histone H4 hyperacetylation at the IRF-1 promoter and biphasic transcription of IRF-1 in response to PRL signaling in vivo.

Int J Radiat Oncol Biol Phys, 2001 Jan 1, 49(1), 161 - 7
Expression of genes involved in repair of DNA double-strand breaks in normal and tumor tissues; Sakata K et al.; BACKGROUND: DNA double-strand breaks (DSB) are the major lethal lesions induced by ionizing radiation . The capability for DNA DSB repair is crucial for inherent radiosensitivity of tumor and normal cells . DNA-PKcs, Ku 70, Ku 85, Xrcc4, and Nbs1 play a critical role in DNA DSB repair . METHODS: We immunohistochemically investigated the expression of DNA-PKcs, Ku 70, Ku85, Xrcc4, and Nbs1 in 134 specimens from various normal and tumor tissues with different radiosensitivity . RESULTS AND CONCLUSION: Immunopositivity to Ku70, Ku85, DNA-PKcs, Xrcc4, and Nbs1 was found in all tumor tissues examined . The staining for Ku70, Ku85, and DNA-PKcs was nuclear; but, for Xrcc4 and Nbs1, it was nuclear and cytoplasmic . There were no apparent differences in the expression of these five proteins among cancerous tissues and the corresponding normal tissues . No apparent differences in nuclear staining intensity were detected in the expression of these five proteins among tumor tissues with different radiosensitivity, although non-Hodgkins' lymphoma (B or T cell) tended to show a lower expression than the others . The stromal cells generally expressed these five proteins at much lower frequency than either tumor or epithelial cells in both tumor and normal tissues.

Mol Biochem Parasitol, 2000 Dec, 111(2), 275 - 82
The CYC3 gene of trypanosoma brucei encodes a cyclin with a short half-life; Van Hellemond JJ et al.; Recently, we identified two Trpanosoma brucei cyclin genes, CYC2 and CYC3, by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G1 cyclin function . CYC3 has a low level of sequence identity to mitotic B-type cyclins from a variety of organisms . In order to examine whether CYC3 associates in vivo with a trypanosome cdc2-related kinase (CRK), the CYC3 gene was fused with the TY-epitope tag, integrated into the trypanosome genome and expressed under inducible control . CYC3ty was demonstrated to associate with the CRK-binding factor p12cks1 and histone H1 kinase activity could be detected in CYC3ty immune precipitated fractions, which demonstrates that CYC3ty associates in vivo with an active trypanosome CRK . Both CYC3ty and CYC2ty were shown to have a half-life of less than one cell cycle, which was significantly elongated by specific proteasome inhibitors, strongly suggesting that CYC3ty and CYC2ty are substrates for proteasome degradation . This is consistent with the presence in CYC3 of a putative destruction box motif that defines proteins for degradation via the ubiquitin degradation pathway . These results are consistant with proteolysis by the proteasome being involved in regulation of the cellular cyclin concentration in trypanosomes.

Cell, 2001 Jan 12, 104(1), 131 - 42
FASCIATA genes for chromatin assembly factor-1 in arabidopsis maintain the cellular organization of apical meristems; Kaya H et al.; Postembryonic development of plants depends on the activity of apical meristems established during embryogenesis . The shoot apical meristem (SAM) and the root apical meristem (RAM) have similar but distinct cellular organization . Arabidopsis FASCIATA1 (FAS1) and FAS2 genes maintain the cellular and functional organization of both SAM and RAM, and FAS gene products are subunits of the Arabidopsis counterpart of chromatin assembly factor-1 (CAF-1) . fas mutants are defective in maintenance of the expression states of WUSCHEL (WUS) in SAM and SCARECROW (SCR) in RAM . We suggest that CAF-1 plays a critical role in the organization of SAM and RAM during postembryonic development by facilitating stable maintenance of gene expression states.

Cell, 2001 Jan 12, 104(1), 119 - 30
Regulation of histone acetylation and transcription by INHAT, a human cellular complex containing the set oncoprotein; Seo SB et al.; Acetylation of histones by p300/CBP and PCAF is considered to be a critical step in transcriptional regulation . In order to understand the role of cellular activities that modulate histone acetylation and transcription, we have purified and characterized a multiprotein cellular complex that potently inhibits the histone acetyltransferase activity of p300/CBP and PCAF . We have mapped a novel acetyltransferase-inhibitory domain of this INHAT (inhibitor of acetyltransferases) complex that binds to histones and masks them from being acetyltransferase substrates . Endogenous INHAT subunits, which include the Set/TAF-Ibeta oncoprotein, associate with chromatin in vivo and can block coactivatormediated transcription when transfected in cells . We propose that histone masking by INHAT plays a regulatory role in chromatin modification and serves as a novel mechanism of transcriptional regulation.

Mol Cell, 2000 Dec, 6(6), 1473 - 84
Structure of the AAA ATPase p97; Zhang X et al.; p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion . It is thought to disassemble SNARE complexes formed during the process of membrane fusion . Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution . Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible . A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis . Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.

Mol Cell, 2000 Dec, 6(6), 1309 - 20
Gcn4 activator targets Gcn5 histone acetyltransferase to specific promoters independently of transcription; Kuo MH et al.; Histone acetylation correlates well with transcriptional activity, and histone acetyltransferases (HATs) selectively regulate subsets of target genes by mechanisms that remain unclear . Here, we provide in vivo evidence that the yeast transcriptional activator Gcn4 recruits Gcn5 HAT complexes to selective promoters positioned in natural or ectopic locations, thereby creating local domains of histone H3 hyperacetylation and subsequent transcriptional activation . A significant portion of the Gcn4-targeted histone acetylation by Gcn5 is independent of transcriptional activity . These observations provide strong evidence for promoter-selective, targeted histone acetylation by Gcn5 that facilitates transcription in a causal fashion . In addition, Gcn5 also functions in an untargeted manner to acetylate H3 on a genome-wide scale.

Cell, 2000 Dec 22, 103(7), 1121 - 31
Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon; Lykke-Andersen J et al.; Nonsense-mediated decay (NMD) rids eukaryotic cells of aberrant mRNAs containing premature termination codons . These are discriminated from true termination codons by downstream cis-elements, such as exon-exon junctions . We describe three novel human proteins involved in NMD, hUpf2, hUpf3a, and hUpf3b . While in HeLa cell extracts these proteins are complexed with hUpf1, in intact cells hUpf3a and hUpf3b are nucleocytoplasmic shuttling proteins, hUpf2 is perinuclear, and hUpf1 cytoplasmic . hUpf3a and hUpf3b associate selectively with spliced beta-globin mRNA in vivo, and tethering of any hUpf protein to the 3'UTR of beta-globin mRNA elicits NMD . These data suggest that assembly of a dynamic hUpf complex initiates in the nucleus at mRNA exon-exon junctions and triggers NMD in the cytoplasm when recognized downstream of a translation termination site.

Curr Opin Genet Dev, 2001 Feb, 11(1), 71 - 7
ATM and ATR: networking cellular responses to DNA damage; Shiloh Y; Maintenance of genome stability depends on the appropriate response to DNA damage . This response is based on complex networks of signaling pathways that activate numerous processes and lead ultimately to damage repair and cellular survival - or apoptosis . The protein kinases ATM and ATR are master controllers of some of these networks, acting either in concert or separately to orchestrate the responses to specific types of DNA damage or stalled replication . Understanding their mode of action is essential to our understanding of how cells cope with genotoxic stress.

Biochem Biophys Res Commun, 2001 Feb 2, 280(4), 1135 - 41
cDNA cloning and expression of a novel cytochrome p450 (cyp4f12) from human small intestine; Hashizume T et al.; A cDNA encoding a novel human CYP4F enzyme (designated CYP4F12) was cloned by PCR from a human small intestine cDNA library . RT-PCR analysis demonstrated that CYP4F12 is expressed in human small intestine and liver . This cDNA contains an entire coding region of a 524-amino-acid protein that is 81.7, 78.3, and 78.2% identical to CYP4F2, CYP4F3, and CYP4F8, respectively . When expressed in Saccharomyces cerevisiae, the P450 catalyzes leukotriene B(4) omega-hydroxylation and arachidonic acid omega-hydroxylation, typical reactions of CYP4F isoforms . Their activity levels are, however, much lower than those of CYP4F2 . Interestingly, CYP4F12 catalyzes the hydroxylation of the antihistamine ebastine with significantly higher catalytic activity relative to CYP4F2 (385 vs 5 pmol/min/nmol P450) . These results indicate that CYP4F12 has a different profile of substrate specificity from other CYP4F isoforms, enzymes responsible for metabolizing endogenous autacoids, therefore suggesting that it may play an important role in xenobiotic biotransformation in the human small intestine .

Biochem Biophys Res Commun, 2001 Jan 26, 280(3), 845 - 7
One-substrate transketolase-catalyzed reaction; Bykova IA et al.; Apart from catalyzing the common two-substrate reaction with ketose as donor substrate and aldose as acceptor substrate, transketolase is also able to catalyze a one-substrate reaction utilizing only ketose (xylulose 5-phosphate) as substrate . The products of this one-substrate reaction were glyceraldehyde 3-phosphate and erythrulose . No free glycolaldehyde (a product of xylulose 5-phosphate splitting in the transketolase reaction) was revealed .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 407 - 14
Murine and human SDF2L1 is an endoplasmic reticulum stress-inducible gene and encodes a new member of the Pmt/rt protein family; Fukuda S et al.; We isolated murine and human cDNAs for SDF2L1 (stromal cell-derived factor 2-like1) and characterized the genomic structures . Northern blot analysis of the gene expression in various tissues revealed that both murine Sdf2l1 and human SDF2L1 genes are expressed ubiquitously, with particularly high expression in the testis . The SDF2L1 protein has an endoplasmic reticulum (ER)-retention-like motif, HDEL, at the carboxy (C)-terminus . Interestingly, SDF2L1 protein also shows significant similarity to the central hydrophilic part of protein O-mannosyltransferase (Pmt) proteins of Saccharomyces cerevisiae, the human homologues of Pmt (POMT1 and POMT2) and Drosophila melanogaster rotated abdomen (rt) protein . In a murine hepatocellular carcinoma cell line, Sdf2l1 was strongly induced by tunicamycin and a calcium ionophore, A23187, and weakly induced by heat stress but was not induced by cycloheximide . In conclusion, SDF2L1 protein is a new member of Pmt/rt protein family and Sdf2l1 is a new ER stress-inducible gene .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 280 - 5
Repression of the HSP70B promoter by NFIL6, Ku70, and MAPK involves three complementary mechanisms; Tang D et al.; We have studied mechanisms of HSP70 gene regulation at 37 degrees C by the cellular factors NF-IL6 and Ku70 . As both factors repress HSF1, we first examined whether phosphorylation on serine 303 and 307 of HSF1 by MAPK and GSK3, which has known to inhibit HSF1, was involved in the repression . However, repression by NF-IL6 was found using HSF1 mutants S303G and S307G refractory to the effects of MAPK and GSK3 . We then examined whether NF-IL6 repressed HSP70B by a mechanism resembling Ku proteins . However, in Ku-deficient cells, NF-IL6 was still able to displace HSF1 from heat shock element (HSE) and repressed HSF1 activation . In addition, activation of the HSP 70B promoter by wild type, S303G, or S307G HSF1 was observed to be much more pronounced in Ku-deficient cells . In vitro translated Ku70 interacted with HSF1 by binding to and displacing it from HSE . These data indicate that the repression of the HSP70B promoter by NF-IL6, Ku70, and MAPK occurs independently of each other and involves three complementary mechanisms .

Biochem Biophys Res Commun, 2000 Dec 29, 279(3), 779 - 85
Chimeras of Delta6-fatty acid and Delta8-sphingolipid desaturases; Libisch B et al.; The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species . In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast . Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids . However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6 . These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site . This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets.

J Mol Biol, 2001 Jan 26, 305(4), 927 - 38
Detection of altered protein conformations in living cells; Raquet X et al.; The maturation, conformational stability, and the rate of in vivo degradation are specific for each protein and depend on both the intrinsic features of the protein and those of the surrounding cellular environment . While synthesis and degradation can be measured in living cells, stability and maturation of proteins are more difficult to quantify . We developed the split-ubiquitin method into a tool for detecting and analyzing changes in protein conformation . The biophysical parameter that forms the basis of these measurements is the time-averaged distance between the N terminus and C terminus of a protein . Starting from three proteins of known structure, we demonstrate the feasibility of this approach, and employ it to elucidate the effect of a previously described mutation in the protein Sec62p on its conformation in living cells .

Genomics, 2000 Dec 15, 70(3), 315 - 26
The transcription factor-like nuclear regulator (TFNR) contains a novel 55-amino-acid motif repeated nine times and maps closely to SMN1; Kelter AR et al.; The transcription factor-like nuclear regulator (TFNR) is a novel human gene that maps on 5q13, distal to the duplicated region that includes SMN1, the spinal muscular atrophy (SMA) determining gene . The location of TFNR allowed us to design an evolutionary model of the SMA region . The 9.5-kb TFNR transcript is highly expressed in cerebellum and weakly in all other tissues tested . TFNR encodes a protein of 2254 amino acids (aa) and contains nine repeats of a novel 55-aa motif, of yet unknown function . The coding region is organized in 32 exons . Alternative splicing of exon 15 results in a truncated protein of 796 aa . TFNR comprises a series of polypeptides that range from 55 to 250 kDa . Immunocytological studies showed that the TFNR protein is present exclusively in the nucleus, where it is concentrated in several nuclear structures . Amino acids 155-474 show significant homology to TFC5, a subunit of the yeast transcription factor TFIIIB, suggesting that TFNR is a putative transcription factor . Based on its proximity to SMN1 and its expression pattern, TFNR may be a candidate gene for atypical forms of SMA with cerebral atrophy and axonal neuropathy that have been shown to carry large deletions in the SMA region.

Dev Biol, 2001 Feb 15, 230(2), 161 - 76
Structure-specific abnormalities associated with mutations in a DNA replication accessory factor in Drosophila; Jaffe AB et al.; We have phenotypically and molecularly analyzed the cutlet locus in Drosophila . Homozygous cutlet flies exhibit abnormal development of a subset of adult tissues, including the eye, wing, and ovary . We show that abnormal development of these tissues is due to a defect in normal cell growth . Surprisingly, cell growth is affected in all developing precursor tissues in cutlet mutant animals, including those that give rise to phenotypically wild-type adult structures . The cutlet gene encodes a Drosophila homologue of yeast CHL12 and has similarity to mammalian replication factor C . In addition, cutlet genetically interacts with multiple subunits of Drosophila replication factor C . Our results suggest that the cutlet gene product acts as an accessory factor for DNA replication and has different requirements for the formation of various adult structures during Drosophila development .

Plant Physiol, 2001 Feb, 125(2), 1086 - 93
Trehalose and trehalase in Arabidopsis; Muller J et al.; Trehalase is ubiquitous in higher plants . So far, indications concerning its function are scarce, although it has been implicated in the detoxification of exogenous trehalose . A putative trehalase gene, T19F6.15, has been identified in the genome sequencing effort in Arabidopsis . Here we show that this gene encodes a functional trehalase when its cDNA is expressed in yeast, and that it is expressed in various plant organs . Furthermore, we present results on the distribution and activity of trehalase in Arabidopsis and we describe how inhibition of trehalase by validamycin A affects the plants response to exogenous trehalose (alpha-D-glucopyranosyl-{1, 1}-alpha-D-glucopyranoside) . Trehalase activity was highest in floral organs, particularly in the anthers (approximately 700 nkat g(-1) protein) and maturing siliques (approximately 250 nkat g(-1) protein) and much lower in leaves, stems, and roots (less than 50 nkat g(-1) protein) . Inhibition of trehalase in vivo by validamycin A led to the accumulation of an endogenous substance that had all the properties of trehalose, and to a strong reduction in sucrose and starch contents in flowers, leaves, and stems . Thus, trehalose appears to be an endogenous substance in Arabidopsis, and trehalose and trehalase may play a role in regulating the carbohydrate allocation in plants.

Plant Physiol, 2001 Feb, 125(2), 847 - 55
Identification and analysis of a gene from Calendula officinalis encoding a fatty acid conjugase; Qiu X et al.; Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase chain reaction-based cloning strategy . Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes . In C . officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds . Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a Delta12 desaturase that introduces a double bond at the 12 position of 16:1(9Z) and 18:1(9Z) . Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a fatty acid conjugase converting 18:2(9Z, 12Z) to calendic acid 18:3(8E, 10E, 12Z) . The enzyme also has weak activity on the mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes.

Nucleic Acids Res . 2001 Feb 15;29(4):E24.
A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex; Honey S et al.; A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed . It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope . This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity . Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2 . Associated proteins were identified using mass spectrometry . These included the known associated proteins Cdc28, Sic1 and Cks1 . Several other proteins were found including the 70 kDa chaperone, Ssa1.

Nucleic Acids Res . 2001 Feb 15;29(4):E18.
A novel approach for the identification of protein-protein interaction with integral membrane proteins; Hubsman M et al.; Protein-protein interaction plays a major role in all biological processes . The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins . Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein . The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant . Protein-protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast . We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein-protein interaction . The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits . The system serves as an attractive approach to explore novel protein-protein interactions with high specificity and selectivity, where other methods fail.

Nucleic Acids Res, 2001 Feb 1, 29(3), 629 - 37
Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1; Ahmad A et al.; We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1, chHAT-1, comprising 408 amino acids including a putative initiation Met . It exhibits 80.4% identity to the human homolog and possesses a typical leucine zipper motif . The glutathione S:-transferase (GST) pull-down assay, involving truncated and missense mutants of the chicken chromatin assembly factor-1 (chCAF-1)p48, revealed not only that a region (comprising amino acids 376-405 of chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction . The GST pull-down assay, involving truncated and missense chHAT-1 mutants, established that a region, comprising amino acids 380-408 of chHAT-1 and containing the leucine zipper motif, is required for its in vitro interaction with chCAF-1p48 . In addition, mutation of each of four Leu residues in the leucine zipper motif prevents the in vitro interaction . The yeast two-hybrid assay revealed that all four Leu residues within the leucine zipper motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48 . These results indicate not only that the proper leucine zipper motif of chHAT-1 is essential for its interaction with chCAF-1p48, but also that the propeller structure of chCAF-1p48 expected to act as a platform for protein-protein interactions may not be necessary for this interaction of chHAT-1.

Mol Biol Cell, 2001 Jan, 12(1), 201 - 19
The Sda1 protein is required for passage through start; Zimmerman ZA et al.; We have used affinity chromatography to identify proteins that interact with Nap1, a protein previously shown to play a role in mitosis . Our studies demonstrate that a highly conserved protein called Sda1 binds to Nap1 both in vitro and in vivo . Loss of Sda1 function causes cells to arrest uniformly as unbudded cells that do not increase significantly in size . Cells arrested by loss of Sda1 function have a 1N DNA content, fail to produce the G1 cyclin Cln2, and remain responsive to mating pheromone, indicating that they arrest in G1 before Start . Expression of CLN2 from a heterologous promoter in temperature-sensitive sda1 cells induces bud emergence and polarization of the actin cytoskeleton, but does not induce cell division, indicating that the sda1 cell cycle arrest phenotype is not due simply to a failure to produce the G1 cyclins . The Sda1 protein is absent from cells arrested in G0 and is expressed before Start when cells reenter the cell cycle, further suggesting that Sda1 functions before Start . Taken together, these findings reveal that Sda1 plays a critical role in G1 events . In addition, these findings suggest that Nap1 is likely to function during G1 . Consistent with this, we have found that Nap1 is required for viability in cells lacking the redundant G1 cyclins Cln1 and Cln2 . In contrast to a previous study, we have found no evidence that Sda1 is required for the assembly or function of the actin cytoskeleton . Further characterization of Sda1 is likely to provide important clues to the poorly understood mechanisms that control passage through G1.

Mol Biol Cell, 2001 Jan, 12(1), 53 - 62
Regulation of cell cycle progression by Swe1p and Hog1p following hypertonic stress; Alexander MR et al.; Exposure of yeast cells to an increase in external osmolarity induces a temporary growth arrest . Recovery from this stress is mediated by the accumulation of intracellular glycerol and the transcription of several stress response genes . Increased external osmolarity causes a transient accumulation of 1N and 2N cells and a concomitant depletion of S phase cells . Hypertonic stress triggers a cell cycle delay in G2 phase cells that appears distinct from the morphogenesis checkpoint, which operates in early S phase cells . Hypertonic stress causes a decrease in CLB2 mRNA, phosphorylation of Cdc28p, and inhibition of Clb2p-Cdc28p kinase activity, whereas Clb2 protein levels are unaffected . Like the morphogenesis checkpoint, the osmotic stress-induced G2 delay is dependent upon the kinase Swe1p, but is not tightly correlated with inhibition of Clb2p-Cdc28p kinase activity . Thus, deletion of SWE1 does not prevent the hypertonic stress-induced inhibition of Clb2p-Cdc28p kinase activity . Mutation of the Swe1p phosphorylation site on Cdc28p (Y19) does not fully eliminate the Swe1p-dependent cell cycle delay, suggesting that Swe1p may have functions independent of Cdc28p phosphorylation . Conversely, deletion of the mitogen-activated protein kinase HOG1 does prevent Clb2p-Cdc28p inhibition by hypertonic stress, but does not block Cdc28p phosphorylation or alleviate the cell cycle delay . However, Hog1p does contribute to proper nuclear segregation after hypertonic stress in cells that lack Swe1p . These results suggest a hypertonic stress-induced cell cycle delay in G2 phase that is mediated in a novel way by Swe1p in cooperation with Hog1p.

Mol Biol Cell, 2001 Jan, 12(1), 37 - 51
Vps41p function in the alkaline phosphatase pathway requires homo-oligomerization and interaction with AP-3 through two distinct domains; Darsow T et al.; Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p . However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin . Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway . These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes . Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways . The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex . Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain . We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p . These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

Mol Biol Cell, 2001 Jan, 12(1), 13 - 26
Ric1p and the Ypt6p GTPase function in a common pathway required for localization of trans-Golgi network membrane proteins; Bensen ES et al.; In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes . To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain . This screen yielded a mutant allele of RIC1 . Cells carrying a deletion of RIC1 (ric1Delta) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole . Delivery to the vacuole occurs in ric1Delta cells also harboring end3Delta to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes . SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Delta . Further comparison of ric1Delta and ypt6Delta cells demonstrated identical phenotypes . Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Delta and ypt6Delta cells . SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor . Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.

J Virol, 2001 Mar, 75(5), 2097 - 106
Mutation of host delta9 fatty acid desaturase inhibits brome mosaic virus RNA replication between template recognition and RNA synthesis; Lee WM et al.; All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes . Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae . BMV encodes RNA replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain . Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA replication complexes on the perinuclear ER . We show that BMV RNA replication is severely inhibited by a mutation in OLE1, an essential yeast chromosomal gene encoding delta9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis . OLE1 deletion and medium supplementation show that BMV RNA replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels . In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally . However, RNA replication is blocked prior to initiation of negative-strand RNA synthesis . The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA replication . The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy.

J Virol, 2001 Feb, 75(4), 1744 - 50
RNA triphosphatase component of the mRNA capping apparatus of Paramecium bursaria Chlorella virus 1; Ho CK et al.; Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host . The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed . PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5' diphosphate end of RNA to form a GpppN cap structure . Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the gamma-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end . We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo . The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses) . Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses . Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.

Glycobiology, 2000 Dec, 10(12), 1283 - 9
N-linked oligosaccharide processing enzyme glucosidase II produces 1,5-anhydrofructose as a side product; Hirano K et al.; alpha-1,4-Glucan lyase cleaves alpha-1,4-linkages of nonreducing termini of alpha-1,4-glucans to produce 1,5-anhydrofructose (1,5-AnFru) . The enzymes isolated from fungi and algae show high homology with glycoside hydrolase family 31 . Purification of alpha-1,4-glucan lyase from rat liver using DEAE Cellulose chromatography resulted in separation of two enzymatic active fractions, one was bound to the column and the other was in the flow-through . Partial amino acid sequence determined from the lyase, retained on the anion exchange column, were identical with that of the N:-linked oligosaccharide processing enzyme glucosidase II . The lyase showed similar enzymatic properties as the microsomal glucosidase such as inhibition by 1-deoxynojirimycin and castanospermine . On the other hand, glucosidase II purified from rat liver microsomes produced not only glucose but also a small amount of 1,5-AnFru using maltose as substrate . Furthermore, CHO cells overexpressing pig liver glucosidase II showed a 1.5- to 2-fold higher lyase activity compared to the nontransfected CHO cells . Conversely, no lyase activity was detectable either in PHAR2.7, the glucosidase II-deficient mutant from a mouse lymphoma cell line, or in Saccharomyces cerevisiae strain YG427 having the glucosidase II gene disrupted . These data demonstrate that glucosidase II possesses an additional enzymatic activity of releasing 1,5-AnFru from maltose.

Biophys J, 2001 Feb, 80(2), 939 - 51
Temperature dependence of the folding and unfolding kinetics of the GCN4 leucine zipper via 13C(alpha)-NMR; Holtzer ME et al.; Studies by one-dimensional NMR are reported on the interconversion of folded and unfolded forms of the GCN4 leucine zipper in neutral saline buffer . The peptide bears 99% 13C(alpha) labels at three sites: V9, L12, and G31 . Time-domain 13C(alpha)-NMR spectra are interpreted by global Bayesian lineshape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 degrees C . The data are well fit by the assumption that the same rate constants apply at each labeled site, confirming that only two conformational states need be considered . Results show that 1) both processes require a free energy of activation; 2) unfolding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages approximately 40% helical . The activation parameters for unfolding, derived from NMR data at the elevated temperatures where both conformations are populated, lead to estimates of the rate constant at low temperatures (5-15 degrees C) that agree with extant values determined by stopped-flow CD via dilution from denaturing media . However, the corresponding estimated values for the folding rate constant are larger by two to three orders of magnitude than those obtained by stopped flow . We propose that this apparent disagreement is caused by the necessity, in the stopped-flow experiment, for initiation of new helices as the highly denaturant-unfolded molecule adjusts to the newly created benign solvent conditions . This must reduce the success rate of collisions in producing the folded molecule . In the NMR determinations, however, the unfolded chains always have a small, but essential, helix content that makes such initiation unnecessary . Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-chain helical peptides and from demonstration that the folding rate constants for coiled coils, as obtained by stopped flow, are influenced by the nature of the denaturant used.

Bioinformatics, 2000 Dec, 16(12), 1073 - 81
Frequency-domain analysis of biomolecular sequences; Anastassiou D; MOTIVATION: Frequency-domain analysis of biomolecular sequences is hindered by their representation as strings of characters . If numerical values are assigned to each of these characters, then the resulting numerical sequences are readily amenable to digital signal processing . RESULTS: We introduce new computational and visual tools for biomolecular sequences analysis . In particular, we provide an optimization procedure improving upon traditional Fourier analysis performance in distinguishing coding from noncoding regions in DNA sequences . We also show that the phase of a properly defined Fourier transform is a powerful predictor of the reading frame of protein coding regions . Resulting color maps help in visually identifying not only the existence of protein coding areas for both DNA strands, but also the coding direction and the reading frame for each of the exons . Furthermore, we demonstrate that color spectrograms can visually provide, in the form of local 'texture', significant information about biomolecular sequences, thus facilitating understanding of local nature, structure and function.

Bioinformatics, 2000 Nov, 16(11), 1023 - 37
Biochemical systems analysis of genome-wide expression data; Voit EO et al.; MOTIVATION: Modern methods of genomics have produced an unprecedented amount of raw data . The interpretation and explanation of these data constitute a major, well-recognized challenge . RESULTS: Biochemical Systems Theory (BST) is the mathematical basis of a well-established methodological framework for analyzing networks of biochemical reactions . An existing BST model of yeast glycolysis is used here to explain and interpret the glycolytic gene expression pattern of heat shocked yeast . Our analysis demonstrates that the observed gene expression profile satisfies the primary goals of increased ATP, trehalose, and NADPH production, while maintaining intermediate metabolites at reasonable levels . Based on a systematic exploration of alternative, hypothetical expression profiles, we show that the observed profile outperforms other profiles . Conclusion: BST is a useful framework for combining DNA microarray data with enzymatic process information to yield new insights into metabolic pathway regulation . AVAILABILITY: All analyses were executed with the software PLAS(Copyright), which is freely available at for academic use . CONTACT: VoitEO@MUSC.edu

Science, 2001 Jan 26, 291(5504), 650 - 3
Establishment of transcriptional silencing in the absence of DNA replication; Li YC et al.; Transcriptional repression of the silent mating-type loci in Saccharomyces cerevisiae requires a cell cycle-dependent establishment step that is commonly assumed to involve DNA replication . Using site-specific recombination, we created a nonreplicating DNA ring in vivo to test directly the role of replication in establishment of silencing . Sir1 was tethered to the ring following excision from the chromosome to activate a dormant silencer . We show here that silencing can be established in DNA that does not replicate . The silenced ring adopted structural features characteristic of bona fide silent chromatin, including an altered level of DNA supercoiling and reduced histone acetylation . In addition, the process required silencing factors Sir2, Sir3, and Sir4 and progression between early S and M phases of the cell cycle . The results indicate that passage of a replication fork is not the cell-cycle event required for establishment of silencing in yeast.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 914 - 9 Epub 2001 Jan 23.
Subunit interactions influence the biochemical and biological properties of Hsp104; Schirmer EC et al.; Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo . In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization . We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing V(max) with little effect on K(m) . In a complementary fashion, the detergent 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering V(max) with little effect on K(m) . ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions . To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities . An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro . In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104 . Thus, interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological functions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo.

Plant Cell Physiol, 2001 Jan, 42(1), 37 - 45
Two types of putative nuclear factors that physically interact with histidine-containing phosphotransfer (Hpt) domains, signaling mediators in His-to-Asp phosphorelay, in Arabidopsis thaliana; Suzuki T et al.; The higher plant, Arabidopsis thaliana, has a large number of genes, each of which encodes a component of His-to-Asp phosphorelay signal transduction systems . One type of such signal transducers are the histidine-containing phosphotransmitters (termed AHPs), which presumably mediate His-to-Asp phosphorelay . Here we attempted to isolate a factor or factors that interact with AHP1, AHP2 and AHP3 by means of a yeast two-hybrid system . This allowed us to identify two types of nuclear-localizing proteins . They are the members of the type-B family of response regulators (specifically, ARR1, APP2 and ARR10), and a novel protein named TCP10 . The binding of ARR1 to AHP2 was also confirmed by in vitro binding assays . Moreover, dephosphorylation of AHP2 was observed in a manner dependent on ARR in vitro . A subset of AHPs appeared to also interact with a protein that contains a TCP domain, a recently proposed basic helix-loop-helix motif . Because several factors carrying the TCP domain have been implicated in the regulation of growth and development in lateral organs, the binding of TCP10 to this subset of AHPs suggests a possible linkage between the His-to-Asp phosphorelay systems and plant growth regulation.

Mol Endocrinol, 2001 Feb, 15(2), 219 - 27
Glucocorticoid repression of AP-1 is not mediated by competition for nuclear coactivators; De Bosscher K et al.; Interleukin-6 (IL-6) is a pleiotropic cytokine that is involved in many autoimmune and inflammatory diseases . Transcriptional control of IL-6 gene expression is exerted by various compounds, among which glucocorticoids are the most potent antiinflammatory and immunosuppressive agents currently in use . Glucocorticoids exert their transrepressive actions by negatively interfering with transcription factors, such as nuclear factor-kappaB (NF-kappaB) and AP-1 . Both factors make use of the coactivator cAMP response element-binding protein (CREB)-binding protein (CBP) to enhance their transcriptional activities, which led to the hypothesis that a mutual antagonism between p65 or c-Jun and activated glucocorticoid receptor (GR) results from a limited amount of CBP . Recently, we showed that glucocorticoid repression of NF-kappaB-driven gene expression occurs irrespective of the amount of coactivator levels in the cell . In the current study, we extend this observation and demonstrate that also AP-1-targeted gene repression by glucocorticoids is refractory to increased amounts of nuclear coactivators . From results with Gal4 chimeric proteins we conclude that glucocorticoid repression occurs by a promoter-independent mechanism involving a nuclear interplay between activated GR and AP-1, independently of CBP levels in the cell.

J Cell Biol, 2001 Feb 5, 152(3), 483 - 90
DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9; Ekert PG et al.; MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis . DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID . Here, we show that after UV radiation, MIHA prevented apoptosis by inhibiting caspase 9 and caspase 3 activation . Unlike Bcl-2, MIHA functioned after release of cytochrome c and DIABLO from the mitochondria and was able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms . Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.

Hum Mol Genet, 2001 Feb 15, 10(4), 423 - 9
Direct interactions of the five known Fanconi anaemia proteins suggest a common functional pathway; Medhurst AL et al.; Fanconi anaemia (FA) is an autosomal recessive inherited disorder associated with a progressive aplastic anaemia, diverse congenital abnormalities and cancer . The condition is genetically heterogeneous, with at least seven complementation groups (A-G) described . Cells from individuals who are homozygous for mutations in FA genes are characterized by chromosomal instability and hypersensitivity to DNA interstrand crosslinking agents . These features suggest a possible role for the encoded proteins in the recognition or repair of these lesions, but neither their function nor whether they operate in a concerted or discrete functional pathways is known . The recent cloning of the FANCF and FANCE genes has allowed us to investigate the interaction of the proteins encoded by five of the seven complementation groups of FA . We used the yeast two-hybrid system and co-immunoprecipitation analysis to test the 10 possible pairs of proteins for direct interaction . In addition to the previously described binding of FANCA to FANCG, we now demonstrate direct interaction of FANCF with FANCG, of FANCC with FANCE and a weaker interaction of FANCE with both FANCA and FANCG . These findings show that the newly identified FANCE protein is an integral part of the FA pathway, and support the concept of a functional link between all known proteins encoded by the genes that are mutated in this disorder . These proteins may act either as a multimeric complex or by sequential recruitment of subsets of the proteins in a common pathway that protects the genomic integrity of mammalian cells.

Hum Mol Genet, 2001 Feb 15, 10(4), 395 - 404
Fusion of the MORF and CBP genes in acute myeloid leukemia with the t(10;16)(q22;p13); Panagopoulos I et al.; The CBP gene at 16p13 fuses to MOZ and MLL as a result of the t(8;16)(p11;p13) in acute (myelo)monocytic leukemias (AML M4/M5) and the t(11;16)(q23;p13) in treatment-related AML, respectively . We show here that a novel t(10;16)(q22;p13) in a childhood AML M5a leads to a MORF-CBP chimera . RT-PCR using MORF forward and CBP reverse primers amplified a MORF-CBP fusion in which nucleotide 3103 of MORF was fused in-frame with nucleotide 284 of CBP . Nested RT-PCR with CBP forward and MORF reverse primers generated a CBP-MORF transcript in which nucleotide 283 of CBP was fused in-frame with nucleotide 3104 of MORF . Genomic analyses revealed that the breaks were close to Alu elements in intron 16 of MORF and intron 2 of CBP and that duplications had occurred near the breakpoints . A database search using MORF cDNA enabled us to construct an exon-intron map of the MORF gene . The MORF-CBP protein retains the zinc fingers, two nuclear localization signals, the histone acetyltransferase (HAT) domain, a portion of the acidic domain of MORF and the CBP protein downstream of codon 29 . Thus, the part of CBP encoding the RARA-binding domain, the CREB-binding domain, the three Cys/His-rich regions, the bromodomain, the HAT domain and the Glu-rich domains is present . In the reciprocal CBP-MORF, part of the acidic domain and the C-terminal Ser- and Met-rich regions of MORF are likely to be driven by the CBP promoter . Since both fusion transcripts were present, their exact role in the leukemogenic process remains to be elucidated.

Genome Res, 2001 Feb, 11(2), 218 - 29
Low-complexity regions in Plasmodium falciparum proteins; Pizzi E et al.; Full-sequence data available for Plasmodium falciparum chromosomes 2 and 3 are exploited to perform a statistical analysis of the long tracts of biased amino acid composition that characterize the vast majority of P . falciparum proteins and to make a comparison with similarly defined tracts from other simple eukaryotes . When the relatively minor subset of prevalently hydrophobic segments is discarded from the set of low-complexity segments identified by current segmentation methods in P . falciparum proteins, a good correspondence is found between prevalently hydrophilic low-complexity segments and the species-specific, rapidly diverging insertions detected by multiple-alignment procedures when sequences of bona fide homologs are available . Amino acid preferences are fairly uniform in the set of hydrophilic low-complexity segments identified in the two P . falciparum chromosomes sequenced, as well as in sequenced genes from Plasmodium berghei, but differ from those observed in Saccharomyces cerevisiae and Dictyostelium discoideum . In the two plasmodial species, amino acid frequencies do not correlate with properties such as hydrophilicity, small volume, or flexibility, which might be expected to characterize residues involved in nonglobular domains but do correlate with A-richness in codons . An effect of phenotypic selection versus neutral drift, however, is suggested by the predominance of asparagine over lysine.

Genes Dev, 2001 Jan 15, 15(2), 147 - 57
The Sir1 protein's association with a silenced chromosome domain; Gardner KA et al.; Silencing of the cryptic mating-type locus HMR requires recognition of a small DNA sequence element, the HMR-E silencer, by the Sir1p, one of four Sir proteins required for the assembly of silenced chromatin domains in Saccharomyces cerevisiae . The Sir1p recognizes the silencer through interactions with the origin recognition complex (ORC), a protein complex that binds the silencer DNA directly . Sir1p was physically associated with HMR in chromatin, and this association required a Sir1p-ORC interaction, suggesting that it reflected the Sir1p silencer-recognition function required for silencing . Sir1p was not associated with nonsilencer replication origins that bind the ORC, indicating that a Sir1p-ORC interaction is confined to silencers . Significantly, the other SIR genes were required for Sir1p's association with HMR . Thus, multiple protein contacts required for and unique to silent chromatin may confine a Sir1p-ORC interaction to silencers . The Sir1p was present at extremely low concentrations in yeast cells yet was associated with HMR at all stages of the cell cycle examined . These data provide insights into the mechanisms that establish and restrict the assembly of silenced chromatin to only a few discrete chromosomal domains.

Genes Dev, 2001 Jan 15, 15(2), 128 - 33
The proteasome regulates the UV-induced activation of the AP-1-like transcription factor Gcn4; Stitzel ML et al.; The proteasome is well known for its regulation of the cell cycle and degradation of mis-folded proteins, yet many of its functions are still unknown . We show that RPN11, a gene encoding a subunit of the regulatory cap of the proteasome, is required for UV-stimulated activation of Gcn4p target genes, but is dispensable for their activation by the general control pathway . We provide evidence that RPN11 functions downstream of RAS2, and show that mutation of two additional proteasome subunits results in identical phenotypes . Our analysis defines a novel function of the proteasome: regulation of the RAS- and AP-1 transcription factor-dependent UV resistance pathway.

EMBO J, 2001 Feb 1, 20(3), 589 - 600
AtSPO11-1 is necessary for efficient meiotic recombination in plants; Grelon M et al.; The Saccharomyces cerevisiae Spo11 protein catalyses DNA double-strand breaks (DSBs) that initiate meiotic recombination . The model plant Arabidopsis thaliana possesses at least three SPO11 homologues . T-DNA and ethyl-methane sulfonate mutagenesis allowed us to show that meiotic progression is altered in plants in which the AtSPO11-1 gene is disrupted . Both male and female meiocytes formed very few bivalents . Furthermore, no fully synapsed chromosomes were observed during prophase I . Later, in meiosis I, we observed that chromosomes segregated randomly, leading to the production of a large proportion of non-functional gametes . These meiotic aberrations were associated with a drastic reduction in meiotic recombination . Thus, our data show that initiation of meiotic recombination by SPO11- induced DSBs is a mechanism conserved in plants . Furthermore, unlike Drosophila and Caenorhabditis elegans, but like fungi, SPO11 is necessary for normal synapsis in plants.

EMBO J, 2001 Feb 1, 20(3), 532 - 40
Splicing and 3' end formation in the definition of nonsense-mediated decay-competent human beta-globin mRNPs; Neu-Yilik G et al.; Premature translation termination codons are common causes of genetic disorders . mRNAs with such mutations are degraded by a surveillance mechanism termed nonsense-mediated decay (NMD), which represents a phylogenetically widely conserved post-transcriptional mechanism for the quality control of gene expression . How NMD-competent mRNPs are formed and specified remains a central question . Here, we have used human beta-globin mRNA as a model system to address the role of splicing and polyadenylation for human NMD . We show that (i) splicing is an indispensable component of the human beta-globin NMD pathway, which cannot be compensated for by exonic beta-globin 'failsafe' sequences; (ii) the spatial requirements of human beta-globin NMD, as signified by the maximal distance of the nonsense mutation to the final exon-exon junction, are less constrained than in yeast; and (iii) non-polyadenylated mRNAs with a histone 3' end are NMD competent . Thus, the formation of NMD-competent mRNP particles critically depends on splicing but does not require the presence of a poly(A) tail.

Appl Environ Microbiol, 2001 Feb, 67(2), 865 - 71
Expression of cmg1, an exo-beta-1,3-glucanase gene from Coniothyrium minitans, increases during sclerotial parasitism; Giczey G et al.; During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains beta-1,3-glucan as its major component . A PCR-based strategy was used to clone a beta-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus . The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-beta-1,3-glucanase genes . The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73 . Saccharomyces cerevisiae INVSc1 expressing the cmg1 gene secreted a approximately 100-kDa beta-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium . N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site . The purified recombinant glucanase inhibited in vitro mycelial growth of S . sclerotiorum by 35 and 85% at concentrations of 300 and 600 microg x ml(-1), respectively . A single copy of the cmg1 gene is present in the genome of C . minitans . Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S . sclerotiorum; only slight repression was observed in the presence of 2% glucose . Expression of cmg1 increased during parasitic interaction with S . sclerotiorum.

J Virol, 2000 May, 74(9), 4310 - 8
Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a; Chen J et al.; Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes RNA replication proteins 1a and 2a . 1a contains a C-terminal helicase-like domain and an N-terminal domain implicated in viral RNA capping, and 2a contains a central polymerase-like domain . 1a and 2a colocalize in an endoplasmic reticulum (ER)-associated replication complex that is the site of BMV-specific RNA-dependent RNA synthesis in plant and yeast cells . 1a also localizes to the ER in the absence of 2a or viral RNA replication templates . To investigate the determinants of 2a localization, we fused 2a to the green fluorescent protein (GFP), creating a functional GFP-2a fusion that supported BMV RNA replication and subgenomic mRNA transcription . In the absence of 1a, the GFP-2a fusion was found to be diffused throughout the cytoplasm and in punctate spots not associated with any cytoplasmic organelle so far tested . Formation of these spots was dependent on the C-terminal half of 2a and may represent aggregation of a fraction of 2a . When coexpressed with 1a, GFP-2a colocalized with 1a and ER-resident protein Kar2p in a partial or complete ring around the nucleus . Consistent with these results, cell fractionation showed that both the GFP-2a fusion and wild-type (wt) 2a remained soluble when expressed alone, while in cells coexpressing 1a, most of the GFP-2a fusion or wt 2a cofractionated with 1a in the rapidly sedimenting membrane fraction . Deletion analysis showed that the N-terminal 120-amino-acid segment of 2a, containing one of two 2a regions previously shown to interact with 1a, was necessary and sufficient for 1a-directed localization of GFP-2a derivatives to the ER . These results suggest that 1a, which also interacts independently with the ER and viral RNA, is a key organizer of RNA replication complex assembly.

J Biol Chem, 2000 Apr 14, 275(15), 11521 - 8
Evidence for overlapping and distinct functions in protein transport of coat protein Sec24p family members; Peng R et al.; Budding of transport vesicles from the endoplasmic reticulum in yeast requires the formation, at the budding site, of a coat protein complex (COPII) that consists of two heterodimeric subcomplexes (Sec23p/Sec24p and Sec13p/Sec31p) and the Sar1 GTPase . Sec24p is an essential protein and involved in cargo selection . In addition to Sec24p, the yeast Saccharomyces cerevisiae expresses two non-essential Sec24p-related proteins, termed Sfb2p (product of YNL049c) and Sfb3p/Lst1p (product of YHR098c) . We here show that Sfb2p and, less efficiently, Sfb3p/Lst1p are able to bind, like Sec24p, the integral membrane cargo protein Sed5p . We also demonstrate that Sfb2p, like Sec24p and Sfb3p/Lst1p, forms a complex with Sec23p in vivo . Whereas the deletion of SFB2 did not affect transport kinetics of various proteins, the maturation of the glycolipid-anchored plasma membrane protein Gas1p was differentially impaired in sfb3 knock-out cells . We generated several conditional-lethal sec24 mutants that, combined with null alleles of SFB2 and SFB3/LST1, led to a complete block of transport between the endoplasmic reticulum and the Golgi (sec24-11/Deltasfb2) or to cell death (sec24-11/Deltasfb3) . Of the Sec24p family members, Sfb2p is the least abundant at steady state, but high intracellular concentrations of Sfb2p can rescue sec24 mutants under restrictive conditions . The data presented strongly suggest that the Sec24p-related proteins function as COPII components.

J Biol Chem, 2000 Apr 14, 275(15), 11141 - 6
Interaction in vivo and in vitro of the metastasis-inducing S100 protein, S100A4 (p9Ka) with S100A1; Wang G et al.; The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems . mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients . Recombinant S100A4 (p9Ka) has been reported to interact in vitro with cytoskeletal components and to form oligomers, particularly homodimers in vitro . Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected . Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions . The interaction between S100A4 and S100A1 was also observed in vitro using affinity column chromatography and gel overlay techniques . Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4.

J Biol Chem, 2000 Apr 14, 275(15), 10887 - 92
E2F family members are differentially regulated by reversible acetylation; Marzio G et al.; The six members of the E2F family of transcription factors play a key role in the control of cell cycle progression by regulating the expression of genes involved in DNA replication and cell proliferation . E2F-1, -2, and -3 belong to a structural and functional subfamily distinct from those of the other E2F family members . Here we report that E2F-1, -2, and -3, but not E2F-4, -5, and -6, associate with and are acetylated by p300 and cAMP-response element-binding protein acetyltransferases . Acetylation occurs at three conserved lysine residues located at the N-terminal boundary of their DNA binding domains . Acetylation of E2F-1 in vitro and in vivo markedly increases its binding affinity for a consensus E2F DNA-binding site, which is paralleled by enhanced transactivation of an E2F-responsive promoter . Acetylation of E2F-1 can be reversed by histone deacetylase-1, indicating that reversible acetylation is a mechanism for regulation also of non-histone proteins.

Dev Biol, 2000 Apr 15, 220(2), 412 - 23
The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase transition from late embryo development to germination; Nambara E et al.; Arabidopsis abi3 and fus3 mutants are defective in late embryo development and their embryos show precocious growth . To understand the function and role of ABI3 and FUS3, we analyzed expression patterns of genes which were normally activated during late embryo development and germination in these mutants . Using the differential display method, both upregulated and downregulated genes were observed in immature siliques of the abi3 fus3 double mutant . Four clones having more abundant expression in the abi3 fus3 double mutant than in wild type were isolated . These genes were activated during wild-type germination, suggesting that some genes that are activated during wild-type germination are precociously activated in the abi3 fus3 mutant during late embryo development . Also, genes that were activated during wild-type germination were isolated and their expression patterns during late embryo development in the wild type and in abi3, fus3, and abi3 fus3 mutants were analyzed . Sixteen such clones were found, and 11 of these showed derepression or precocious activation of gene expression in the mutants . These results indicate that ABI3 and FUS3 negatively regulate a particular set of genes during late embryo development . We also showed that immature fus3 siliques accumulated one-third of the wild-type level of abscisic acid (ABA), but mature fus3 siliques accumulated ABA at a level comparable to that in the wild type . The possible mechanisms of controlling developmental timing in late embryo development as well as collaborative and distinct roles of ABI3 and FUS3 are discussed .

Science, 2000 Apr 7, 288(5463), 136 - 40
Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons; Loots GG et al.; Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them . To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs . Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals . Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases.

J Biol Chem, 2001 Apr 6, 276(14), 11167 - 73 Epub 2001 Jan 10.
Dimerization and nuclear localization of ku proteins; Koike M et al.; Ku, a heterodimer of Ku70 and Ku80, plays a key role in multiple nuclear processes, e.g . DNA repair, chromosome maintenance, and transcription regulation . Heterodimerization is essential for Ku-dependent DNA repair in vivo, although its role is poorly understood . Some lines of evidence suggest that heterodimerization is required for the stabilization of Ku70 and Ku80 . Here we show that the heterodimerization of these Ku subunits is important for their nuclear entry . When transfected into Ku-deficient xrs-6 cells, exogenous Ku70 and Ku80 tagged with green fluorescent protein accumulated into the nucleus, whereas each nuclear localization signal (NLS)-dysfunctional mutant was undetectable in the nucleus, supporting the idea that each Ku can translocate to the nucleus through its own NLS . On the other hand, the nuclear accumulation of each NLS-dysfunctional mutant was markedly enhanced by the presence of an exogenous wild-type counterpart . In Ku-expressing HeLa cells, each NLS-dysfunctional mutant, as well as wild-type Ku70 and Ku80, was still detectable in the nucleus, whereas the double mutant of each Ku subunit with decreased functions of both nuclear targeting and dimerization was undetectable in the nucleus . Our results indicate that each Ku subunit can translocate to the nucleus not only through its own NLS but also through heterodimerization with each other.

J Biol Chem, 2001 Mar 30, 276(13), 9825 - 31 Epub 2001 Jan 04.
Evidence that proteolysis of Gal4 cannot explain the transcriptional effects of proteasome ATPase mutations; Russell SJ et al.; The Gal system of Saccharomyces cerevisiae is a paradigm for eukaryotic gene regulation . Expression of genes required for growth on galactose is regulated by the transcriptional activator Gal4 . The activation function of Gal4 has been localized to 34 amino acids near the C terminus of the protein . The gal4D allele of GAL4 encodes a truncated protein in which only 14 amino acids of the activation domain remain . Expression of GAL genes is dramatically reduced in gal4D strains and these strains are unable to grow on galactose as the sole carbon source . Overexpression of gal4D partially relieves the defect in GAL gene expression and allows growth on galactose . A search for extragenic suppressors of gal4D identified recessive mutations in the SUG1 and SUG2 genes, which encode ATPases of the 19S regulatory complex of the proteasome . The proteasome is responsible for the ATP-dependent degradation of proteins marked for destruction by the ubiquitin system . It has been commonly assumed that effects of SUG1 and SUG2 mutations on transcription are explained by alterations in the proteolysis of gal4D protein . We have investigated this assumption . Surprisingly, we find that SUG1 and SUG2 alleles that are unable to suppress gal4D cause a larger increase in gal4D protein levels than do suppressing alleles . In addition, mutations in genes encoding subunits of the proteolytic 20S sub-complex of the proteasome increase the levels of gal4D protein but do not rescue its transcriptional activity . Therefore, an alteration in the proteolysis of gal4D by the proteasome cannot explain the effects of mutations in SUG1 and SUG2 on expression of GAL genes . These findings suggest that the 19S regulatory complex may play a more direct role in transcription.

J Biol Chem, 2001 Apr 6, 276(14), 10646 - 54 Epub 2001 Jan 04.
The neural cell recognition molecule neurofascin interacts with syntenin-1 but not with syntenin-2, both of which reveal self-associating activity; Koroll M et al.; Neurofascin belongs to the L1 subgroup of the immunoglobulin superfamily of cell adhesion molecules and is implicated in axonal growth and fasciculation . We used yeast two-hybrid screening to identify proteins that interact with neurofascin intracellularly and therefore might link it to trafficking, spatial targeting, or signaling pathways . Here, we demonstrate that rat syntenin-1, previously published as syntenin, mda-9, or TACIP18 in human, is a neurofascin-binding protein that exhibits a wide-spread tissue expression pattern with a relative maximum in brain . Syntenin-1 was found not to interact with other vertebrate members of the L1 subgroup such as L1 itself or NrCAM . We confirmed the specificity of the neurofascin-syntenin-1 interaction by ligand-overlay assay, surface plasmon resonance analysis, and colocalization of both proteins in heterologous cells . The COOH terminus of neurofascin was mapped to interact with the second PDZ domain of syntenin-1 . Furthermore, we isolated syntenin-2 that may be expressed in two isoforms . Despite their high sequence similarity to syntenin-1, syntenin-2alpha, which interacts with neurexin I, and syntenin-2beta do not bind to neurofascin or several other transmembrane proteins that are binding partners of syntenin-1 . Finally, we report that syntenin-1 and -2 both form homodimers and can interact with each other.

J Biol Chem, 2001 Mar 30, 276(13), 9896 - 902 Epub 2001 Jan 04.
Requirements for the nucleolytic processing of DNA ends by the Werner syndrome protein-Ku70/80 complex; Li B et al.; Werner syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability . The WS gene encodes a protein with helicase and exonuclease activities . Our previous studies indicated that the Werner syndrome protein (WRN) interacts with Ku, a heterodimeric factor of 70- and 80-kDa subunits implicated in the repair of double strand DNA breaks . Moreover, we demonstrated that Ku70/80 strongly stimulates and alters WRN exonuclease activity . In this report, we investigate further the association between WRN and Ku70/80 . First, using various WRN deletion mutants we show that 50 amino acids at the amino terminus are required and sufficient to interact with Ku70/80 . In addition, our data indicate that the region of Ku80 between amino acids 215 and 276 is necessary for binding to WRN . Then, we show that the amino-terminal region of WRN from amino acid 1 to 388, which comprise the exonuclease domain, can be efficiently stimulated by Ku to degrade DNA substrates, indicating that the helicase domain and the carboxyl-terminal tail are not required for the stimulatory process . Finally, using gel shift assays, we demonstrate that Ku recruits WRN to DNA . Taken together, these results suggest that Ku-mediated activation of WRN exonuclease activity may play an important role in a cellular pathway that requires processing of DNA ends.

J Biol Chem, 2001 Apr 6, 276(14), 10663 - 9 Epub 2001 Jan 03.
Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation; Deak PM et al.; The endoplasmic reticulum contains a protein quality control system that discovers malfolded or unassembled secretory proteins and subjects them to degradation in the cytosol . This requires retrograde transport of the respective proteins from the endoplasmic reticulum back to the cytosol via the Sec61 translocon . In addition, a fully competent ubiquitination machinery and the 26 S proteasome are necessary for retrotranslocation and degradation . Ubiquitination of mutated and malfolded proteins of the endoplasmic reticulum is dependent mainly on the ubiquitin-conjugating enzyme Ubc7p . In addition, several new membrane components of the endoplasmic reticulum are required for degradation . Here we present the topology of the previously discovered RING-H2 finger protein Der3/Hrd1p, one of the new components of the endoplasmic reticulum membrane . The protein spans the membrane six times . The amino terminus and the carboxyl terminus containing the RING finger domain face the cytoplasm . Altogether, RING finger-dependent ubiquitination of malfolded carboxypeptidase yscY in vivo, as well as of Der3/Hrd1p itself in vitro and RING finger-dependent binding of Ubc7p, uncovers Der3/Hrd1p as the ubiquitin-protein ligase (E3) of the endoplasmic reticulum-associated protein degradation process.

J Biol Chem, 2001 Mar 30, 276(13), 9861 - 7 Epub 2001 Jan 02.
Energy-dependent flip of fluorescence-labeled phospholipids is regulated by nutrient starvation and transcription factors, PDR1 and PDR3; Hanson PK et al.; The yeast Saccharomyces cerevisiae readily accumulates short-chain, fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled phosphatidylcholine and phosphatidylethanolamine at the nuclear envelope/endoplasmic reticulum and mitochondria . The net intracellular accumulation reflects the sum of their inwardly and outwardly directed transbilayer translocation across the plasma membrane (flip and flop, respectively) . The rate of flop is negligible in energy-depleted cells as well as at low temperature (2 degrees C) . Although flip is reduced at 2 degrees C, it can still be measured by flow cytometry, allowing the rate of flip, independent of flop, to be characterized at this temperature . Flip requires the energy of the plasma membrane proton electrochemical gradient and is down-regulated as cells pass through the diauxic shift and enter stationary phase . Furthermore, drug-resistant, gain-of-function mutations in the transcription factors, PDR1 and PDR3, result in a dramatic down-regulation of flip in addition to their already established up-regulation of flop . These results imply that down-regulation of the NBD-phospholipid flip pathway is a physiological response to environmental stress.

J Biol Chem, 2001 Mar 30, 276(13), 10398 - 406 Epub 2000 Dec 29.
Biochemical analysis of fructose-1,6-bisphosphatase import into vacuole import and degradation vesicles reveals a role for UBC1 in vesicle biogenesis; Shieh HL et al.; When Saccharomyces cerevisiae are shifted from medium containing poor carbon sources to medium containing fresh glucose, the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is imported into Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation . Here, we show that FBPase import is independent of vacuole functions and proteasome degradation . However, FBPase import required the ubiquitin-conjugating enzyme Ubc1p . A strain containing a deletion of the UBC1 gene exhibited defective FBPase import . Furthermore, FBPase import was inhibited when cells overexpressed the K48R/K63R ubiquitin mutant that fails to form multiubiquitin chains . The defects in FBPase import seen for the Deltaubc1 and the K48R/K63R mutants were attributed to the Vid vesicle fraction . In the Deltaubc1 mutant, the level of the Vid vesicle-specific marker Vid24p was reduced in the vesicle fraction, suggesting that UBC1 is required for either Vid vesicle production or Vid24p binding to Vid vesicles . However, the K48R/K63R mutant did not prevent Vid24p binding to Vid vesicles, indicating that ubiquitin chain formation is dispensable for Vid24p binding to these structures . Our results support the findings that ubiquitin conjugation and ubiquitin chain formation play important roles in a number of cellular processes including organelle biogenesis.

J Biol Chem, 2001 Mar 30, 276(13), 10374 - 86 Epub 2000 Dec 28.
A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission; Bardwell AJ et al.; The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals . We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction . Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2 . Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs . Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro . In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal . This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised . Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo.

J Biol Chem, 2001 Apr 13, 276(15), 11735 - 42 Epub 2000 Dec 27.
TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes; Bishop N et al.; Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies . We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex . Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101 . TSG101 and hVPS28 are predominantly cytosolic . However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles . We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.

J Biol Chem, 2001 Apr 6, 276(14), 11189 - 98 Epub 2000 Dec 27.
Myomegalin is a novel protein of the golgi/centrosome that interacts with a cyclic nucleotide phosphodiesterase; Verde I et al.; Subcellular targeting of the components of the cAMP-dependent pathway is thought to be essential for intracellular signaling . Here we have identified a novel protein, named myomegalin, that interacts with the cyclic nucleotide phosphodiesterase PDE4D, thereby targeting it to particulate structures . Myomegalin is a large 2,324-amino acid protein mostly composed of alpha-helical and coiled-coil structures, with domains shared with microtubule-associated proteins, and a leucine zipper identical to that found in the Drosophila centrosomin . Transcripts of 7.5-8 kilobases were present in most tissues, whereas a short mRNA of 2.4 kilobases was detected only in rat testis . A third splicing variant was expressed predominantly in rat heart . Antibodies against the deduced sequence recognized particulate myomegalin proteins of 62 kDa in testis and 230-250 kDa in heart and skeletal muscle . Immunocytochemistry and transfection studies demonstrate colocalization of PDE4D and myomegalin in the Golgi/centrosomal area of cultured cells, and in sarcomeric structures of skeletal muscle . Myomegalin expressed in COS-7 cells coimmunoprecipitated with PDE4D3 and sequestered it to particulate structures . These findings indicate that myomegalin is a novel protein that functions as an anchor to localize components of the cAMP-dependent pathway to the Golgi/centrosomal region of the cell.

J Biol Chem, 2001 Apr 27, 276(17), 14139 - 52 Epub 2000 Dec 22.
A rice functional transcriptional activator, RISBZ1, responsible for endosperm-specific expression of storage protein genes through GCN4 motif; Onodera Y et al.; The GCN4 motif, a cis-element that is highly conserved in the promoters of cereal seed storage protein genes, plays a central role in controlling endosperm-specific expression . This motif is the recognition site for a basic leucine zipper transcriptional factor that belongs to the group of maize Opaque-2 (O2)-like proteins . Five different basic leucine zipper cDNA clones, designated RISBZ1-5, have been isolated from a rice seed cDNA library . The predicted gene products can be divided into two groups based on their amino acid sequences . Although all the RISBZ proteins are able to interact with the GCN4 motif, only RISBZ1 is capable of activating (more than 100-fold expression) the expression of a reporter gene under a minimal promoter fused to a pentamer of the GCN4 motif . Loss-of-function and gain-of-function experiments using the yeast GAL4 DNA binding domain revealed that the proline-rich N-terminal domain (27 amino acids in length) is responsible for transactivation . The RISBZ1 protein is capable of forming homodimers as well as heterodimers with other RISBZ subunit proteins . RISBZ1 gene expression is restricted to the seed, where it precedes the expression of storage protein genes . When the RISBZ1 promoter was transcriptionally fused to the beta-glucuronidase reporter gene and the chimeric gene was introduced into rice, the beta-glucuronidase gene is specifically expressed in aleurone and subaleurone layer of the developing endosperm . These findings suggest that the specific expression of transcriptional activator RISBZ1 gene may determine the endosperm specificity of the storage protein genes.

Genome Res, 2001 Jan, 11(1), 112 - 23
Assessing clusters and motifs from gene expression data; Jakt LM et al.; Large-scale gene expression studies and genomic sequencing projects are providing vast amounts of information that can be used to identify or predict cellular regulatory processes . Genes can be clustered on the basis of the similarity of their expression profiles or function and these clusters are likely to contain genes that are regulated by the same transcription factors . Searches for cis-regulatory elements can then be undertaken in the noncoding regions of the clustered genes . However, it is necessary to assess the efficiency of both the gene clustering and the postulated regulatory motifs, as there are many difficulties associated with clustering and determining the functional relevance of matches to sequence motifs . We have developed a method to assess the potential functional significance of clusters and motifs based on the probability of finding a certain number of matches to a motif in all of the gene clusters . To avoid problems with threshold scores for a match, the top matches to a motif are taken in several sample sizes . Genes from a sample are then counted by the cluster in which they appear . The probability of observing these counts by chance is calculated using the hypergeometric distribution . Because of the multiple sample sizes, strong and weak matching motifs can be detected and refined and significant matches to motifs across cluster boundaries are observed as all clusters are considered . By applying this method to many motifs and to a cluster set of yeast genes, we detected a similarity between Swi Five Factor and forkhead proteins and suggest that the currently unidentified Swi Five Factor is one of the yeast forkhead proteins.

Mol Cell Biol, 2001 Feb, 21(3), 966 - 76
Molecular dissection of interactions between Rad51 and members of the recombination-repair group; Krejci L et al.; Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation . In the yeast Saccharomyces cerevisiae, recombination requires products of the RAD52 epistasis group . The Rad51 protein associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic complex . We describe a new strategy to screen for mutations which cause specific disruption of the interaction between certain proteins in the complex, leaving other interactions intact . This approach defines distinct protein interaction domains and protein relationships within the Rad51 complex . Alignment of the mutations onto the constructed three-dimensional model of the Rad51 protein reveal possible partially overlapping interfaces for the Rad51-Rad52 and the Rad51-Rad54 interactions . Rad51-Rad55 and Rad51-Rad51 interactions are affected by the same spectrum of mutations, indicating similarity between the two modes of binding . Finally, the detection of a subset of mutations within Rad51 which disrupt the interaction with mutant Rad52 protein but activate the interaction with Rad54 suggests that dynamic changes within the Rad51 protein may contribute to an ordered reaction process.

Cell Signal, 2000 Dec, 12(11-12), 745 - 50
Human colon carcinoma cell-line HCT116 transfected by antisense cDNA as a tool to study the Ku86 involvement in cell proliferation; Sadji Z et al.; In this work, we used colon carcinoma cell-line HCT116 to study the involvement of the 86-kDa subunit (Ku86) of DNA-protein kinase (DNA-PK) in human tumoural cell proliferation . We transfected these cells with a 639-bp cDNA encoding a Ku86 portion inserted into pcDNA3.1 vector in the antisense orientation . After selection by neomycin, we obtained more than 300 resistant colonies . In the Y'A5 colony that we chose as total population, we showed by PCR and RT-PCR that pcDNA3/Ku86 antisense was integrated in genomic DNA and that transcript was present . After cloning, we selected two clones, A20 and A23, which contained significatively reduced level of Ku86 protein . These two clones displayed a reduced DNA-PK activity from 44% to 71% and a slower growth than control cells . These results suggest that the HCT116 cell-line is a useful tool to investigate the role of Ku86 in the regulation of human tumoural cell growth.

Physiol Rev, 2001 Jan, 81(1), 153 - 208
Small GTP-binding proteins; Takai Y et al.; Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members . This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families . They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions . They furthermore play key roles in not only temporal but also spatial determination of specific cell functions . The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization . Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated . Cascades and cross-talks of small G proteins have also been clarified . In this review, functions of small G proteins and their modes of activation and action are described.

Hum Mol Genet, 2001 Jan 15, 10(2), 99 - 105
Rent1, a trans-effector of nonsense-mediated mRNA decay, is essential for mammalian embryonic viability; Medghalchi SM et al.; The ability to detect and degrade transcripts that lack full coding potential is ubiquitous but non-essential in lower eukaryotes, leaving in question the evolutionary basis for complete maintenance of this function . One hypothesis holds that nonsense-mediated RNA decay (NMD) protects the organism by preventing the translation of truncated peptides with dominant negative or deleterious gain-of-function potential . All organisms studied to date that are competent for NMD express a structural homolog of Saccharomyces cerevisiae Upf1p . We have now explored the consequences of loss of NMD function in vertebrates through targeted disruption of the Rent1 gene in murine embryonic stem cells which encodes a mammalian ortholog of Upf1p . Mice heterozygous for the targeted allele showed no apparent phenotypic abnormalities but homozygosity was never observed, demonstrating that Rent1 is essential for embryonic viability . Homozygous targeted embryos show complete loss of NMD and are viable in the pre-implantation period, but resorb shortly after implantation . Furthermore, Rent1(-/-) blastocysts isolated at 3.5 days post-coitum undergo apoptosis in culture following a brief phase of cellular expansion . These data suggest that NMD is essential for mammalian cellular viability and support a critical role for the pathway in the regulated expression of selected physiologic transcripts.

J Mol Biol, 2001 Jan 19, 305(3), 441 - 50
Varying levels of positive and negative supercoiling differently affect the efficiency with which topoisomerase II catenates and decatenates DNA; Roca J; Type II DNA topoisomerases catalyze the transport of one DNA double helix through another . Here, by using a non-hydrolyzable analog of ATP, I examined the single-step DNA transport preferences of the yeast type II topoisomerase bound to positively and negatively supercoiled DNA rings . I found that negative supercoiling favors decatenation of DNA rings more than positive supercoiling . Conversely, positive supercoiling favors the catenation and knotting of DNA rings more than negative supercoiling . This vectorial effect of DNA supercoiling handedness supports a model in which type II topoisomerases can recognize three DNA segments, and highlights a novel influence of DNA supercoiling in global DNA topology .

J Virol, 2001 Feb, 75(3), 1557 - 60
Ten-kilodalton domain in Ty3 Gag3-Pol3p between PR and RT is dispensable for Ty3 transposition; Claypool JA et al.; Ty3 is a gypsy-type, retrovirus-like element found in the budding yeast Saccharomyces cerevisiae . In cells overexpressing Ty3 under the GAL1 upstream activation sequence, Ty3 RNA, proteins, and DNA are made . Elucidation of the molecular masses and amino-terminal sequences of protease and reverse transcriptase indicated the existence of an additional intervening domain, designated J, in the Ty3 Gag3-Pol3p polyprotein . A region analogous to J can be found in many retrotransposable elements closely related to Ty3; however, J does not correspond to any of the highly conserved retroviral protein domains . Ty3 mutants deleted for the J-coding region showed moderately reduced transposition frequency but greatly reduced levels of Ty3 DNA . These results show that under galactose regulation, the Ty3 J domain is not absolutely essential.

J Virol, 2001 Feb, 75(3), 1348 - 58
Hepatitis C virus 3'X region interacts with human ribosomal proteins; Wood J et al.; To identify proteins that can bind the 3' untranslated region (UTR) of hepatitis C virus (HCV) we screened human cDNA libraries using the Saccharomyces cerevisiae three-hybrid system . Screening with an RNA sequence derived from the 3'-terminal 98 nucleotides (3'X region) of an infectious clone of HCV (H77c) yielded clones of human ribosomal proteins L22, L3, S3, and mL3, a mitochondrial homologue of L3 . We performed preliminary characterization of the binding between the 3'X region and these proteins by a three-hybrid mating assay using mutant 3'X sequences . We have further characterized the interaction between 3'X and L22, since this protein is known to be associated with two small Epstein-Barr virus (EBV)-encoded RNA species (EBERs) which are abundantly produced in cells latently infected with EBV . The EBERs, which have similar predicted secondary structure to the HCV 3'X, assemble into ribonucleoprotein particles that include L22 and La protein . To confirm that L22 binds HCV 3'X we performed in vitro binding assays using recombinant L22 (expressed as a glutathione S-transferase {GST} fusion protein) together with a 3'X riboprobe . The 3'X region binds to the GST-L22 fusion protein (but not to GST alone), and this interaction is subject to competition with unlabeled 3'X RNA . To establish the functional role played by L22 in internal ribosome entry site (IRES)-mediated translation of HCV sequences we performed translational analysis in HuH-7 cells using monocistronic and bicistronic reporter constructs . The relative amount of core-chloramphenicol acetyltransferase reporter protein translated under the control of the HCV IRES was stimulated in the presence of L22 and La when these proteins were supplied in trans.

J Virol, 2001 Feb, 75(3), 1265 - 73
Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain; Taylor DR et al.; Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by interferon and activated upon autophosphorylation . We previously identified four autophosphorylated amino acids and elucidated their participation in PKR activation . Three of these sites are in the central region of the protein, and one is in the kinase domain . Here we describe the identification of four additional autophosphorylated amino acids in the spacer region that separates the two dsRNA-binding motifs in the RNA-binding domain . Eight amino acids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2 . This region of E2 is required for its inhibition of PKR although the mechanism of inhibition is not known . Replacement of all four of these residues in PKR with alanines did not dramatically affect kinase activity in vitro or in yeast Saccharomyces cerevisiae . However, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity . A synthetic peptide corresponding to this region of PKR was phosphorylated in vitro by PKR, but phosphorylation was strongly inhibited after PKR was preincubated with HCV E2 . Another synthetic peptide, corresponding to the central region of PKR and containing serine 242, was also phosphorylated by active PKR, but E2 did not inhibit this peptide as efficiently . Neither of the PKR peptides was able to disrupt the HCV E2-PKR interaction . Taken together, these results show that PKR is autophosphorylated on serine 83 and threonines 88, 89, and 90, that this autophosphorylation may enhance kinase activation, and that the inhibition of PKR by HCV E2 is not solely due to duplication of and competition with these autophosphorylation sites.

J Virol, 2001 Feb, 75(3), 1172 - 85
Herpes simplex virus virion host shutoff protein requires a mammalian factor for efficient in vitro endoribonuclease activity; Lu P et al.; The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system . Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity . As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae . Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source . The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions . The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff . Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity . However, activity was readily detected when such extracts were mixed with RRL . These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.

J Pept Res, 2000 Dec, 56(6), 427 - 37
Ion channel activation by SPC3, a peptide derived from the HIV-1 gp120 V3 loop; Carlier E et al.; SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence . This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains . However, the molecular mode of action of SPC3 remains unclear . Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection . To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors . Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration . This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3 . The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor . The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity . Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.

Chromosoma, 2000 Nov, 109(7), 453 - 9
A GAL4-HP1 fusion protein targeted near heterochromatin promotes gene silencing; Seum C et al.; We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator . The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences . These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain . We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin . These rearrangements induce variegation of both white and lacZ . Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3-7 and Su(var)2-5 . Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain . Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2-5, is shown to enhance variegation of Heidi and Tell . Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera . Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes.

FEBS Lett, 2000 Dec 29, 487(2), 161 - 5
The substitution of the C-terminus of bax by that of bcl-xL does not affect its subcellular localization but abrogates its pro-apoptotic properties; Oliver L et al.; The interaction of the anti-apoptotic members of the Bcl-2 family with mitochondria, through their hydrophobic C-terminus, has been proposed to play a crucial role in the execution phase of apoptosis . We report here that a substitution of the C-terminal end of pro-apoptotic bax by that of anti-apoptotic bcl-xL (baxCxL) does not modify its association with mitochondria in human and rat cells or in Saccharomyces cerevisiae . In addition, while bax sensitizes these cells to apoptotic stimuli, the construct baxCxL does not affect the apoptotic response in transfected cells . These results suggest that the C-terminus of bax plays an important role in apoptosis independently of its membrane addressing/targeting mechanism.

J Cell Biol, 2001 Jan 8, 152(1), 213 - 29
The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly; Aridor M et al.; Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes . We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro . We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection . These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro . By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures . Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

J Cell Biol, 2001 Jan 8, 152(1), 197 - 212
Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex; Cheeseman IM et al.; Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae . Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p . Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex . Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity . Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects . Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex . duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function . In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle.

J Cell Biol, 2001 Jan 8, 152(1), 51 - 64
Membrane recruitment of Aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires Aut1p, Aut2p, and the autophagy conjugation complex; Kim J et al.; Autophagy is a degradative pathway by which cells sequester nonessential, bulk cytosol into double-membrane vesicles (autophagosomes) and deliver them to the vacuole for recycling . Using this strategy, eukaryotic cells survive periods of nutritional starvation . Under nutrient-rich conditions, autophagy machinery is required for the delivery of a resident vacuolar hydrolase, aminopeptidase I, by the cytoplasm to vacuole targeting (Cvt) pathway . In both pathways, the vesicle formation process requires the function of the starvation-induced Aut7 protein, which is recruited from the cytosol to the forming Cvt vesicles and autophagosomes . The membrane binding of Aut7p represents an early step in vesicle formation . In this study, we identify several requirements for Aut7p membrane association . After synthesis in the cytosol, Aut7p is proteolytically cleaved in an Aut2p-dependent manner . While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additional physical interactions with Aut1p and the autophagy (Apg) conjugation complex before recruitment to the membrane . Lack of these interactions results in a cytosolic distribution of Aut7p rather than localization to forming Cvt vesicles and autophagosomes . This study assigns a functional role for the Apg conjugation system as a mediator of Aut7p membrane recruitment . Further, we demonstrate that Aut1p, which physically interacts with components of the Apg conjugation complex and Aut7p, constitutes an additional factor required for Aut7p membrane recruitment . These findings define a series of steps that results in the modification of Aut7p and its subsequent binding to the sequestering transport vesicles of the autophagy and cytoplasm to vacuole targeting pathways.

Plant Cell, 2000 Dec, 12(12), 2339 - 2350
Nuclear localization of NPR1 is required for activation of PR gene expression; Kinkema M et al.; Systemic acquired resistance (SAR) is a broad-spectrum resistance in plants that involves the upregulation of a battery of pathogenesis-related (PR) genes . NPR1 is a key regulator in the signal transduction pathway that leads to SAR . Mutations in NPR1 result in a failure to induce PR genes in systemic tissues and a heightened susceptibility to pathogen infection, whereas overexpression of the NPR1 protein leads to increased induction of the PR genes and enhanced disease resistance . We analyzed the subcellular localization of NPR1 to gain insight into the mechanism by which this protein regulates SAR . An NPR1-green fluorescent protein fusion protein, which functions the same as the endogenous NPR1 protein, was shown to accumulate in the nucleus in response to activators of SAR . To control the nuclear transport of NPR1, we made a fusion of NPR1 with the glucocorticoid receptor hormone binding domain . Using this steroid-inducible system, we clearly demonstrate that nuclear localization of NPR1 is essential for its activity in inducing PR genes.

J Cell Sci, 2001 Jan, 114(Pt 2), 247 - 55
Search, capture and signal: games microtubules and centrosomes play; Schuyler SC et al.; Accurate distribution of the chromosomes in dividing cells requires coupling of cellular polarity cues with both the orientation of the mitotic spindle and cell cycle progression . Work in budding yeast has demonstrated that cytoplasmic dynein and the kinesin Kip3p define redundant pathways that ensure proper spindle orientation . Furthermore, it has been shown that the Kip3p pathway components Kar9p and Bim1p (Yeb1p) form a complex that provides a molecular link between cortical polarity cues and spindle microtubules . Recently, other studies indicated that the cortical localization of Kar9p depends upon actin cables and Myo2p, a type V myosin . In addition, a BUB2-dependent cell cycle checkpoint has been described that inhibits the mitotic exit network and cytokinesis until proper centrosome position is achieved . Combined, these studies provide molecular insight into how cells link cellular polarity, spindle position and cell cycle progression.

Biochemistry, 2001 Jan 16, 40(2), 327 - 35
Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc1 complex affects the mobility of the {2Fe2S} domain; Ghosh M et al.; Mutating three conserved alanine residues in the tether region of the iron-sulfur protein of the yeast cytochrome bc(1) complex resulted in 22-56% decreases in enzymatic activity {Obungu et al . (2000) Biochim . Biophys . Acta 1457, 36-44} . The activity of the cytochrome bc(1) complex isolated from A86L was decreased 60% compared to the wild-type without loss of heme or protein and without changes in the 2Fe2S cluster or proton-pumping ability . The activity of the bc(1) complex from mutant A92R was identical to the wild-type, while loss of both heme and activity was observed in the bc(1) complex isolated from mutant A90I . Computer simulations indicated that neither mutation A86L nor mutation A92R affects the alpha-helical backbone in the tether region; however, the side chain of the leucine substituted for Ala-86 interacts with the side chain of Leu-89 . The Arrhenius plot for mutant A86L was apparently biphasic with a transition observed at 17-19 degrees C and an activation energy of 279.9 kJ/mol below 17 degrees C and 125.1 kJ/mol above 17 degrees C . The initial rate of cytochrome c(1) reduction was lowered 33% in mutant A86L; however, the initial rate of cytochrome b reduction was unaffected, suggesting that movement of the tether region of the iron-sulfur protein is necessary for maximum rates of enzymatic activity . Substituting a leucine for Ala-86 impedes the unwinding of the alpha-helix and hence movement of the tether.

Nat Cell Biol, 2001 Jan, 3(1), 38 - 42
Multi-step control of spindle pole body duplication by cyclin-dependent kinase; Haase SB et al.; Organelles called centrosomes in metazoans or spindle pole bodies (SPBs) in yeast direct the assembly of a bipolar spindle that is essential for faithful segregation of chromosomes during mitosis . Abnormal accumulation of multiple centrosomes leads to genome instability, and has been observed in both tumour cells and cells with targeted mutations in tumour-suppressor genes . The defects that lead to centrosome amplification are not understood . We have recapitulated the multiple-centrosome phenotype in budding yeast by disrupting the activity of specific cyclin-dependent kinase (CDK) complexes . Our observations are reminiscent of mechanisms that govern DNA replication, and show that specific cyclin/CDK activities function both to promote SPB duplication and to prevent SPB reduplication.

Nat Cell Biol, 2001 Jan, 3(1), 24 - 9
Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation; Bays NW et al.; In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins . ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation . Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination . Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD . Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s) . A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins . In this capacity, Hrd1p has an apparent preference for misfolded proteins . We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.

Genetica, 2000, 108(1), 9 - 17
Using the COG database to improve gene recognition in complete genomes; Natale DA et al.; A complete understanding of the biology of an organism necessarily starts with knowledge of its genetic makeup . Proteins encoded in a genome must be identified and characterized, and the presence or absence of specific sets of proteins must be noted in order to determine the possible biochemical pathways or functional systems utilized by that organism . The COG database presents a set of tools suited to these purposes, including the ability to select protein families (COGs) that contain proteins from a specified set of species . The selection is based upon a phylogenetic pattern, which is a shorthand representation of the presence or absence of a particular species in a COG . Here we present the use of phylogenetic patterns as a means to perform targeted searches for undetected protein-coding genes in complete genomes.

Biol Pharm Bull, 2000 Dec, 23(12), 1477 - 80
Anti-estrogenic activity of diesel exhaust particles; Taneda S et al.; Estrogenic and anti-estrogenic activities of diesel exhaust particles (DEP) were evaluated using yeast cells expressing the human estrogen receptor and the responsive element regulating the expression of the receptor gene for beta-galactosidase (Routledge and Sumpter, 1996) . It was found that a suspension of whole DEP suspension is not estrogenic but that this preparation possesses the ability to reduce the estrogen-dependent reporter activity . DEP were serially extracted with hexane, benzene, dichloromethane, methanol, and 1 M ammonia, and the estrogenic and anti-estrogenic activities of these preparations were determined . None of the extracts of DEP were estrogenic, but the extracts of benzene, dichloromethane and methanol possessed anti-estrogenic activity, and the activity of estrogen in the presence of hexane extract was slightly decreased . These results indicated that DEP contain heterologous compounds having anti-estrogenic activity . It is thought that those compounds in DEP can modulate the activity of estrogen, leading to the distruption of balance between estrogen and androgen . In this paper, the environmental effects of DEP in relation to the endocrine disrupting effect of organic compounds in DEP are discussed.

RNA, 2000 Dec, 6(12), 1882 - 94
The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity; Rho SB et al.; The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration . However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear . As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron . We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA-protein interactions to the large bI4 group I intron . We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners . In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity . These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.

RNA, 2000 Dec, 6(12), 1773 - 80
The odyssey of a regulated transcript; Vilardell J et al.; The transcript of the Saccharomyces cerevisiae gene, RPL30, is subject to regulated splicing and regulated translation, due to a structure that interacts with its own product, ribosomal protein L30 . We have followed the fate of the regulated RPL30 transcripts in vivo . Initially, these transcripts abortively enter the splicing pathway, forming an unusually stable association with U1 snRNP . A large proportion of the unspliced molecules, however, are found in the cytoplasm . Most of these are still bound by L30, as only a small fraction are engaged in translation . Eventually, the unspliced RPL30 transcripts escape the grasp of L30, associate with ribosomes, and fall prey to nonsense mediated decay.

RNA, 2000 Dec, 6(12), 1737 - 49
Pre-mRNA processing factors are required for nuclear export; Brodsky AS et al.; RNA export from the nucleus is thought to be linked to proper processing and packaging into ribonucleoprotein protein complexes . A system to observe mRNA nuclear export in living yeast cells was developed by fusing the U1A RNA-binding protein to the green fluorescent protein to follow specific mRNAs with U1A hairpins engineered into them . RNAs encoding Rpl25, Pgk1, and Ssa4 were examined for the effects of 3' UTRs, introns, RNA processing factors, nucleoporins, and transport factors on their export . All accumulated in the nucleus in mutants affecting components of the nuclear export machinery and certain nucleoporins . However, under conditions of stress, PGK1 and RPL25 transcripts accumulate in the nucleus whereas SSA4 RNA is exported . Moreover, when export is blocked, only RNAs containing the ASH1 3' UTR accumulated in the nucleolus . Mutations in the splicing machinery selectively blocked export of only intron-containing RNAs . Mutations in RNA14, RNA15, and PAP1, which encode factors important for 3' processing, also blocked export of all RNAs, including SSA4, thereby linking export to the process of polyadenlyation . Taken together, these data graphically display the connections between mRNA processing and nuclear export.

Biochemistry, 2001 Jan 9, 40(1), 256 - 67
Deuterated delta 7-cholestenol analogues as mechanistic probes for wild-type and mutated delta 7-sterol-C5(6)-desaturase; Rahier A; Deuterium-labeled 5alpha-cholest-7-en-3beta-ol (1) bearing one or two deuteriums at the C-5alpha and (or) C-6alpha positions was synthesized in high isotopic and chiral purity . These compounds were used as substrates with the microsomal wild-type Zea mays and recombinant Arabidopsis thaliana Delta(7)-sterol-C5(6)-desaturases (5-DES) to probe directly the stereochemistry and the mechanism of the enzymatic reaction . Clearly, in the conversion of 1 by both 5-DESs, the 6alpha-hydrogen is removed . {6alpha-(2)H}-5alpha-Cholest-7-en-3beta-ol shows an intermolecular deuterium kinetic isotope effect (DKIE) on V and V/K, (D6)V = 2.6+/-0.3, (D6)V/K = 2.4+/-0.1; and (D6)V = 2.3 +/-0.3, (D6)V/K = 2.3+/-0.2 for the Zea mays and A . thaliana wild-type 5-DES, respectively . In contrast, negligible or minor isotope effects, (D5)V = 0.99+/-0.04, (D5)V/K = 0.91+/-0.08; and (D5)V = 0.93 +/-0.06, (D5)V/K = 0.96+/-0.04, respectively, were observed with {5alpha-(2)H}-cholest-7-en-3beta-ol . The observed pattern of isotope effects strongly suggests that the plant 5-DES initiates oxidation by cleavage of the chemically activated C6alpha-H bond, a step which appears to be partially rate-limiting in the desaturation process . Cleavage of the C5-H bond has a negligible isotope effect, indicating that the desaturation involves asynchronous scission of the two C-H bonds at C5 and C6 . We showed previously {Taton, M., et al . (2000) Biochemistry 39, 701} that threonine 114 was not essential to maintaining desaturase activity, although V/K values for mutant T114I and T114S were respectively 10-fold lower and 4-fold higher than that of the native 5-DES . In this study, we combined variation in enzyme structure and DKIE studies and showed that (D6)V and (D6)V/K increased respectively to 3.8+/-0.3 and 3.8+/-0.4 in mutant T114I and decreased respectively to 1.6+/-0.4 and 1.7+/- 0.1 in mutant T114S . The data suggest that the conserved hydroxyl function at position 114 in the ERG3 family makes the abstraction of the 6alpha-hydrogen atom substantially less rate-limiting during the 5-DES reaction . Based on the data, a tentative mechanism for the desaturation of cholest-7-en-3beta-ol is proposed.

Nucleic Acids Res, 2001 Jan 15, 29(2), 527 - 35
Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors; Pinson B et al.; Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation . Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo . C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA . The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2 . Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents . Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e . a DNA-induced conformational change in the second repeat leads to formation of a full helix-turn-helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

Nucleic Acids Res, 2001 Jan 15, 29(2), 499 - 505
Solution structure and dynamics of GCN4 cognate DNA: NMR investigations; Khandelwal P et al.; A 12 bp long GCN4-binding, self-complementary duplex DNA d(CATGACGTCATG)(2) has been investigated by NMR spectroscopy to study the structure and dynamics of the molecule in aqueous solution . The NMR structure of the DNA obtained using simulated annealing and iterative relaxation matrix calculations compares quite closely with the X-ray structure of ATF/CREB DNA in complex with GCN4 protein (DNA-binding domain) . The DNA is also seen to be curved in the free state and this has a significant bearing on recognition by the protein . The dynamic characteristics of the molecule have been studied by (13)C relaxation measurements at natural abundance . A correlation has been observed between sequence-dependent dynamics and recognition by GCN4 protein.

Biochem J, 2001 Jan 15, 353(Pt 2), 199 - 205
Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis; Keresztessy Z et al.; The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae . The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein . Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme . Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program . Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile . The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad . The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment . A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase.

Exp Cell Res, 2001 Jan 15, 262(2), 75 - 83
The human histone deacetylase family; Gray SG et al.; Since the identification of the first histone deacetylase (Taunton et al., Science 272, 408-411), several new members have been isolated . They can loosely be separated into entities on the basis of their similarity to various yeast histone deacetylases . The first class is represented by its closeness to the yeast Rpd3-like proteins, and the second most recently discovered class has similarities to yeast Hda1-like proteins . However, due to the fact that several different research groups isolated the Hda1-like histone deacetylases independently, there have been various different nomenclatures used to describe the various members, which can lead to confusion in the interpretation of this family's functions and interactions . With the discovery of another novel murine histone deacetylase, homologous to yeast Sir2, the number of members of this family is set to increase, as 7 human homologues of this gene have been isolated . In the light of these recent discoveries, we have examined the literature data and conducted a database analysis of the isolated histone deacetylases and potential candidates . The results obtained suggest that the number of histone deacetylases within the human genome may be as high as 17 and are discussed in relation to their homology to the yeast histone deacetylases .

J Biol Chem, 2001 Apr 20, 276(16), 13145 - 52 Epub 2001 Jan 03.
Signal-binding specificity of the mu4 subunit of the adaptor protein complex AP-4; Aguilar RC et al.; The medium (mu) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXphi) sorting signals found within the cytoplasmic domains of integral membrane proteins . Here, we describe the signal-binding specificity and affinity of the medium subunit mu4 of the recently described adaptor protein complex AP-4 . To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using mu4 as a selector protein . Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi) . In addition, we examined the interaction of mu4 with naturally occurring YXXphi signals by both two-hybrid and in vitro binding analyses . These experiments showed that mu4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF . Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the mu4-YXXphi interaction to be in the micromolar range . To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a mu4-specific YXXphi signal . This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.

Toxicol Lett, 2001 Jan 3, 118(3), 147 - 55
Evaluation of in vitro methods for detecting the effects of various chemicals on the human progesterone receptor, with a focus on pyrethroid insecticides; Sumida K et al.; The progesterone receptor (PR) is associated with physiological events such as implantation and the maintenance of pregnancy . Recently, it has become a social concern that chemicals may exert agonistic or antagonistic effects on hormone receptors . Therefore, we examined the effects of various chemicals on the human PR, with a focus on pyrethroid insecticides, using three in vitro methods . Eight pyrethroid insecticides (fenvalerate, d-allethrin, d-phenothrin, prallethrin, empenthrin, permethrin, cypermethrin and imiprothrin), examples of environmental pollutants and positive control chemicals were subjected to a reporter gene assay (luciferase assay) using human breast cancer T-47D cells, a two-hybrid assay and a binding assay using the same whole cells or receptors (cell-free) . In none of these did the eight pyrethroid insecticides show any binding to the PR, agonistic or antagonistic effects.

Curr Biol, 2000 Dec 14-28, 10(24), R908 - 11
Checkpoints: it takes more than time to heal some wounds; Rhind N et al.; The S-phase DNA damage checkpoint seems to provide a twist on the checkpoint theme . Instead of delaying replication and allowing repair as a consequence, it may activate repair and delay replication as a consequence.

Curr Biol, 2000 Dec 14-28, 10(24), 1599 - 602
S-phase cyclins are required for a stable arrest at metaphase; Meyn MA 3rd et al.; A critical DNA damage checkpoint in Saccharomyces cerevisiae is an arrest at the metaphase stage of mitosis . Here we show that the S-phase cyclins Clb5 and Clb6 are required for this arrest . Strains lacking Clb5 and Clb6 are hypersensitive to DNA damage . Furthermore, in the presence of the DNA alkylating agent methyl methanesulfonate (MMS) over 50% of clb5 clb6 mutants by-passed the metaphase checkpoint and arrested instead with separated sister chromatids . Levels of Pds1, an inhibitor of anaphase that accumulates following DNA damage, were similar in the wild-type and mutant strains following MMS treatment . Furthermore, unlike wild-type cells, clb5 clb6 mutants undergo nuclear division despite the presence of nuclear non-degradable Pds1 . Our results suggest a novel role for the S-phase cyclins Clb5 and Clb6 in maintaining sister chromatid cohesion during a metaphase arrest, perhaps by regulating Pds1 activity.

Curr Biol, 2000 Dec 14-28, 10(24), 1574 - 81
Partitioning the transcriptional program induced by rapamycin among the effectors of the Tor proteins; Shamji AF et al.; BACKGROUND: In all organisms, nutrients are primary regulators of signaling pathways that control transcription . In Saccharomyces cerevisiae, the Tor proteins regulate the transcription of genes sensitive to the quality of available nitrogen and carbon sources . Formation of a ternary complex of the immunosuppressant rapamycin, its immunophilin receptor Fpr1p and Tor1p or Tor2p results in the nuclear import of several nutrient- and stress-responsive transcription factors . RESULTS: We show that treating yeast cells with rapamycin results in a broader modulation of functionally related gene sets than previously understood . Using chemical epistasis and vector-based global expression analyses, we partition the transcriptional program induced by rapamycin among five effectors (TAP42, MKS1, URE2, GLN3, GAT1) of the Tor proteins, and identify how the quality of carbon and nitrogen sources impinge upon components of the program . Biochemical data measuring Ure2p phosphorylation coupled with the partition analysis indicate that there are distinct signaling branches downstream of the Tor proteins . CONCLUSIONS: Whole-genome transcription profiling reveals a striking similarity between shifting to low-quality carbon or nitrogen sources and treatment with rapamycin . These data suggest that the Tor proteins are central sensors of the quality of carbon and nitrogen sources . Depending on which nutrient is limited in quality, the Tor proteins can modulate a given pathway differentially . Integrating the partition analysis of the transcriptional program of rapamycin with the biochemical data, we propose a novel architecture of Tor protein signaling and of the nutrient-response network, including the identification of carbon discrimination and nitrogen discrimination pathways.

Curr Biol, 2000 Dec 14-28, 10(24), 1557 - 64
Pds5 cooperates with cohesin in maintaining sister chromatid cohesion; Panizza S et al.; BACKGROUND: Sister chromatid cohesion depends on a complex called cohesin, which contains at least four subunits: Smc1, Smc3, Scc1 and Scc3 . Cohesion is established during DNA replication, is partially dismantled in many, but not all, organisms during prophase, and is finally destroyed at the metaphase-to-anaphase transition . A quite separate protein called Spo76 is required for sister chromatid cohesion during meiosis in the ascomycete Sordaria . Spo76-like proteins are highly conserved amongst eukaryotes and a homologue in Aspergillus nidulans, called BimD, is required for the completion of mitosis . The isolation of the cohesin subunit Smc3 as a suppressor of BimD mutations suggests that Spo76/BimD might function in the same process as cohesin . RESULTS: We show here that the yeast homologue of Spo76, called Pds5, is essential for establishing sister chromatid cohesion and maintaining it during metaphase . We also show that Pds5 co-localizes with cohesin on chromosomes, that the chromosomal association of Pds5 and cohesin is interdependent, that Scc1 recruits Pds5 to chromosomes in G1 and that its cleavage causes dissociation of Pds5 from chromosomes at the metaphase-to-anaphase transition . CONCLUSIONS: Our data show that Pds5 functions as part of the same process as cohesin . Sequence similarities and secondary structure predictions indicate that Pds5 consists of tandemly repeated HEAT repeats, and might therefore function as a protein-protein interaction scaffold, possibly in the cohesin-DNA complex assembly.

Proc Natl Acad Sci U S A, 2001 Jan 2, 98(1), 54 - 9
Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures; Lee JK et al.; The minichromosome maintenance (Mcm) proteins 2-7 are required for both the initiation and elongation steps of chromosomal DNA replication . Previous studies have shown that the Mcm complex consisting of the Mcm 4, 6, and 7 proteins contains 3' to 5' DNA helicase activity with limited processivity (displacing duplex DNA regions up to 30 nt) . In this report, we show that the presence of both 5' and 3' single-stranded tails in DNA helicase substrates is essential for the processive helicase activity of the Mcm complex . The presence of both 5' and 3' tails facilitated the formation of double heterohexameric complexes of Mcm4/6/7 on substrate DNA, which appeared to be essential for the processive helicase activity . The double heterohexameric complex of Mcm4/6/7, in the presence of a single-strand DNA binding protein, is capable of unwinding duplex DNA region of about 600 bp in length . These results support the hypothesis that the Mcm4/6/7 complex can function as a replication helicase.

Mol Cell Biol, 2001 Jan, 21(2), 476 - 87
Stimulation of CREB binding protein nucleosomal histone acetyltransferase activity by a class of transcriptional activators; Chen CJ et al.; The transcriptional coactivator CREB binding protein (CBP) possesses intrinsic histone acetyltransferase (HAT) activity that is important for gene regulation . CBP binds to and cooperates with numerous nuclear factors to stimulate transcription, but it is unclear if these factors modulate CBP HAT activity . Our previous work showed that CBP interacts with the Epstein-Barr virus-encoded basic region zipper (b-zip) protein, Zta, and augments its transcriptional activity . Here we report that Zta strongly enhances CBP-mediated acetylation of nucleosomal histones . Zta stimulated the HAT activity of CBP that had been partially purified or immunoprecipitated from mammalian cells as well as from affinity-purified, baculovirus expressed CBP . Stimulation of nucleosome acetylation required the CBP HAT domain, the Zta DNA binding and transcription activation domain, and nucleosomal DNA . In addition to Zta, we found that two other b-zip proteins, NF-E2 and C/EBPalpha, strongly stimulated nucleosomal HAT activity . In contrast, several CBP-binding proteins, including phospho-CREB, JUN/FOS, GATA-1, Pit-1, and EKLF, failed to stimulate HAT activity . These results demonstrate that a subset of transcriptional activators enhance the nucleosome-directed HAT activity of CBP and suggest that nuclear factors may regulate transcription by altering substrate recognition and/or the enzymatic activity of chromatin modifying coactivators.

What Is Pcr?, What Is Cell Biology?, What Is Bioremediation?, What Is Genetic Engineering?, What Is Bioengineering?, o, Bacteriology, c, Microorganism, o, Bacterium, s, Microbes, r, Bacteria, a, Cell cultures, a, Microbiological, n, Escherichia coli, s, Multidrug resistant, a, Yeasts, n, Antibiotic resistance, c, Streptococci, e, Botulin, c, Brevibacteria, n, Escherichia coli, n, Staphylococcus, r, Culture medium, i, Vibriosis, o, Botulism, a, Escherichia coli, a, Vibriosis, n, Microbial, r, Gram negative, e, Clostridia, e, Antimicrobial, c, Wastewater treatment

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005