S. cerevisiae

Trends Cell Biol, 2001 May, 11(5), 220 - 30
The Ste20 group kinases as regulators of MAP kinase cascades; Dan I et al.; Ste20p (sterile 20 protein) is a putative yeast mitogen-activated protein kinase kinase kinase kinase (MAP4K) involved in the mating pathway . Its homologs in mammals, Drosophila, Caenorhabditis elegans and other organisms make up a large emerging group of protein kinases including 28 members in human . The Ste20 group kinases are further divided into the p21-activated kinase (PAK) and germinal center kinase (GCK) families . They are characterized by the presence of a conserved kinase domain and a noncatalytic region of great structural diversity that enables the kinases to interact with various signaling molecules and regulatory proteins of the cytoskeleton . This review describes the phylogenetic relationships of the Ste20 group kinases based on discussions with many researchers in this field . With the newly established phylogenetic relationships, crucial arguments can be advanced regarding the functions of these kinases as upstream activators of the MAPK pathways and possible activity as MAP4Ks . Their involvement in apoptosis, morphogenesis and cytoskeletal rearrangements is also discussed.

Oncogene, 2001 Mar 1, 20(9), 1128 - 34
Towards a blueprint of the cell cycle; Alberghina L et al.; The understanding of the organisation of cell cycle events is of utmost importance to devise effective therapeutic strategies for cancer . In this article we gather evidences from the literature in support of a system model of the cell cycle, in which a growth-sensitive threshold controls entry into S phase and the sequential activation of cyclin-dependent kinases . The cycle is terminated by an End function, that comprises events from the onset of mitosis to cell division and that may also be modulated by the increase of cell size . This blueprint allows quantitative predictions by computer simulations of steady and transitory states . In fact, we show that the proposed control system applies to budding yeast populations during nutritional shift-up and following hyperactivation of the cAMP signalling pathway . Besides the growth-sensitive control system it is shown to apply to mammalian cells both in the exit from quiescence and in active proliferation . The putative molecular determinants that set the threshold controlling S phase entry are consistently altered in cancer cells . Finally, we discuss an input/output analysis based on the simulated behaviour derived from the blueprint as a new tool to investigate the road to cancer.

Oncogene, 2001 Feb 8, 20(6), 739 - 47
Tumor specific modulation of KU70/80 DNA binding activity in breast and bladder human tumor biopsies; Pucci S et al.; The Ku70/80 heterodimer is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) and its DNA-binding activity mediates DNA double-strand breaks repair . Although Ku80 was recently proposed as a caretaker gene involved in the control of genome integrity, no data are available on Ku70/80 DNA-binding activity in human tumors . Heterodimer DNA-binding activity and protein expression were assayed by electrophoretic-mobility-shift-assay (EMSA) and Western blot analysis, in nuclear and cytoplasmic extracts from eight breast, seven bladder primary tumors and three metastatic nodes from breast cancers . Corresponding normal tissues of the same patients were used as controls . Ten out of 15 tumors showed nuclear Ku-binding activity 3-10 times higher than in the normal tissues, irrespective of bladder or breast origin . Conversely, in 5/15 primary tumors and in all the metastatic nodes analysed, nuclear Ku-activity was 1.5-4.5-fold lower than in the corresponding normal tissues . Cytoplasmic heterodimer activity significantly differed between tumor and normal tissues, displaying a 2-10-fold increase in neoplastic tissues . Three different patterns combining both Ku expression and activity with tumor characteristics were identified . In low aggressive breast tumors p70/p80 proteins were expressed in tumor but not in normal tissues . The heterodimer binding-activity matched the protein levels . In non-invasive bladder carcinomas no significant differences in protein expression between tumor and the corresponding normal tissues were found, however heterodimer binding-activity was increased in tumor samples . In breast and bladder tumors, at the advanced stage and in node metastases, the binding activity was strongly reduced in tumor biopsies, however no differences were demonstrated between normal and tumor protein levels . Our results suggest a different modulation of Ku70/80 DNA-binding activity in human neoplastic tissues, possibly related to tumor progression . Findings provide further data on tissue-specific protein expression and post-translational regulation of heterodimer activity.

Oncogene, 2001 Jan 18, 20(3), 404 - 9
The monocytic leukemia zinc finger protein MOZ is a histone acetyltransferase; Champagne N et al.; The monocytic leukemia zinc finger protein (MOZ) gene is rearranged in t(8;16)(p11;p13), t(8;22)(p11;q13) and inv(8)(p11q13) associated with acute myeloid leukemia . The other fusion partners involved are CBP, p300 and TIF2, transcriptional coactivators with known or potential histone acetyltransferase (HAT) activity . MOZ itself is a 2004-residue protein containing a putative acetyl CoA-binding motif, so it was hypothesized that MOZ is a HAT . Here we present direct evidence that MOZ has intrinsic HAT activity . Moreover, MOZ possesses a transcriptional repression domain at its N-terminal part and an activation domain at its C-terminal part . The activation domain does not show sequence similarity to any yeast proteins, but when tethered, it is able to activate transcription in yeast . Therefore, MOZ is a HAT with characteristics of a transcriptional coregulator, supporting the hypothesis that aberrant acetylation by abnormal MOZ proteins leads to leukemogenesis.

Oncogene, 2001 Jan 18, 20(3), 295 - 302
Runx2: a novel oncogenic effector revealed by in vivo complementation and retroviral tagging; Blyth K et al.; The Runx2 (Cbfa1, Pebp2alphaA, Aml3) gene was previously identified as a frequent target for transcriptional activation by proviral insertion in T-cell lymphomas of CD2-MYC transgenic mice . We have recently shown that over-expression of the full-length, most highly expressed Runx2 isoform in the thymus perturbs T-cell development, leads to development of spontaneous lymphomas at low frequency and is strongly synergistic with Myc . To gain further insight into the relationship of Runx2 to other lymphomagenic pathways, we tested the effect of combining the CD2-Runx2 transgene either with a Pim1 transgene (E(mu)-Pim1) or with the p53 null genotype, as each of these displays independent synergy with Myc . In both cases we observed synergistic tumour development . However, Runx2 appeared to have a dominant effect on the tumour phenotype in each case, with most tumours conforming to the CD3(+), CD8(+), CD4(+/-) phenotype seen in CD2-Runx2 mice . Neonatal infection of CD2-Runx2 mice with Moloney murine leukaemia virus (Moloney MLV) also led to a dramatic acceleration of tumour onset . Analysis of known Moloney MLV target genes in these lymphomas showed a high frequency of rearrangement at c-Myc or N-Myc (82%), and a significant number at Pim1 or Pim2 (23%), and at Pal1/Gfi1 (18%) . These results indicate that Runx2 makes a distinct contribution to T-cell lymphoma development which does not coincide with any of the oncogene complementation groups previously identified by retroviral tagging.

Oncogene, 2001 Jan 11, 20(2), 178 - 87
PRCC, the commonest TFE3 fusion partner in papillary renal carcinoma is associated with pre-mRNA splicing factors; Skalsky YM et al.; In papillary renal cell carcinomas the TFE3 transcription factor becomes fused to the PSF and NonO pre-mRNA splicing factors and most commonly to a protein of unknown function designated PRCC . In this study we have examined the ability of the resulting PRCC-TFE3 and NonO-TFE3 fusions to activate transcription from the plasminogen activator inhibitor-1 (PAI-1) promoter . The results show that only fusion to PRCC enhanced transcriptional activation, indicating that the ability to enhance the level of transcription from endogenous TFE3 promoters is not a consistent feature of TFE3 fusions . In investigations of the normal function of PRCC we observed that PRCC expressed as a green fluorescent fusion protein colocalizes within the nucleus with Sm pre-mRNA splicing factors . It was also found that endogenous PRCC is coimmunoprecipitated by antibodies that recognize a variety of pre-mRNA splicing factors including SC35, PRL1 and CDC5 . Association with the cellular splicing machinery is therefore, a common feature of the proteins that become fused to TFE3 in papillary renal cell carcinomas.

Gene Ther, 2001 Jan, 8(2), 166 - 71
Enhancement of MSH receptor- and GAL4-mediated gene transfer by switching the nuclear import pathway; Chan CK et al.; Efficient nuclear delivery of plasmid DNA represents a major barrier in nonviral gene transfer . One approach has been to use DNA-binding proteins such as GAL4 from yeast as DNA carriers with nuclear targeting properties . We recently showed, however, that GAL4 is inefficient in targeting DNA to the nucleus because its DNA-binding and nuclear targeting activities are mutually exclusive, which relates to the fact that GAL4 nuclear import occurs via a novel pathway . Here, we 'switch' this pathway to a more conventional one by adding a modified poly-lysine to which an optimized nuclear targeting signal, based on that of the SV40 large T-antigen, is linked . We also use a chimeric GAL4-alpha-melanocyte stimulating hormone (MSH) fusion protein to enable gene transfer to cells expressing the MSH receptor . Switching the nuclear import pathway of the transfecting complex significantly enhances receptor-mediated gene transfer through enabling interaction with desired components of the cellular nuclear import machinery . The present study represents the first demonstration that nuclear targeting signals can enhance receptor-mediated gene delivery, the approaches having important relevance to research and clinical applications, such as in generating transgenic or knock-out animals, or in gene therapy.

Cell Death Differ, 2001 Jan, 8(1), 63 - 9
Resting membrane potential as a marker of apoptosis: studies on Xenopus oocytes microinjected with cytochrome c; Bhuyan AK et al.; Observation of the electrical potential difference across the cell membrane is described as a new method for monitoring apoptosis of a single cell . The resting membrane potential (DeltaPsi) of Xenopus oocytes has been recorded in real time following microinjection of cytochrome c . Soon after microinjection, DeltaPsi becomes less negative and attains a new constant value with a half time, t(m), of about 35 (+ /- 5) min at all cytochrome c concentrations greater than 1 microM . The cytosol extract of cytochrome c-injected oocytes shows DEVD proteolytic activity characteristic of aspartate specific proteases, implicating an apoptotic death pathway . In response to the delivery of cytochrome c into the cytosol, caspases are activated within 7 min while the changes in DeltaPsi begin to occur after about 30 min . The method described here will be potentially useful to assess the effectiveness of cell death regulators and modulators of synthetic and biological origin, and the results presented shed light on the currently debated issue of the importance of the redox state of cytochrome c in the initiation of apoptosis.

Science, 2001 May 18, 292(5520), 1376 - 8 Epub 2001 Apr 19.
A GDP/GTP exchange factor involved in linking a spatial landmark to cell polarity; Kang PJ et al.; In both animal and yeast cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate polarized organization of the actin cytoskeleton . In the budding yeast Saccharomyces cerevisiae, the Ras-like GTPase Bud1/Rsr1 and its guanosine 5'-diphosphate (GDP)/guanosine 5'-triphosphate (GTP) exchange factor Bud5 are involved in the selection of a specific site for growth, thus determining cell polarity . We found that Bud5 is localized at the cell division site and the presumptive bud site . Its localization is dependent on potential cellular landmarks, such as Bud3 and Axl2/Bud10 in haploid cells and Bud8 and Bud9 in diploid cells . Bud5 also physically interacts with Axl2/Bud10, a transmembrane glycoprotein, suggesting that a receptor-like transmembrane protein recruits a GDP/GTP exchange factor to connect an intrinsic spatial signal to oriented cell growth.

Science, 2001 Jun 8, 292(5523), 1876 - 82 Epub 2001 Apr 19.
Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution; Gnatt AL et al.; The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution . Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site . Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site . The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking . The 5'-most residue of the RNA is close to the point of entry to an exit groove . Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid . Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.

Science, 2001 Jun 8, 292(5523), 1863 - 76 Epub 2001 Apr 19.
Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution; Cramer P et al.; Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution . Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center . In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription . Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription . A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis . The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

Mol Cell Biol, 2001 May, 21(10), 3564 - 75
Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway; Wang G et al.; The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase . Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain . A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins . We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated . Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites . The latter appeared to correspond to endocytic intermediates, as these structures were not seen in a sla2/end4-1 mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p . The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization . Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein . Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway.

Mol Cell Biol, 2001 May, 21(10), 3558 - 63
Role of DNA polymerase zeta in the bypass of a (6-4) TT photoproduct; Johnson RE et al.; UV light-induced DNA lesions block the normal replication machinery . Eukaryotic cells possess DNA polymerase eta (Poleta), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Poleta result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum . Here, we test Poleta for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than a cis-syn TT dimer . Opposite the 3' T of a (6-4) TT photoproduct, both yeast and human Poleta preferentially insert a G residue, but they are unable to extend from the inserted nucleotide . DNA Polzeta, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3' T of the (6-4) TT lesion by Poleta, and Polzeta inserts the correct nucleotide A opposite the 5' T of the lesion . Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Poleta and Polzeta, wherein Poleta inserts a nucleotide opposite the 3' T of the lesion and Polzeta extends from it . These biochemical observations are in concert with genetic studies in yeast indicating that mutations occur predominantly at the 3' T of the (6-4) TT photoproduct and that these mutations frequently exhibit a 3' T-->C change that would result from the insertion of a G opposite the 3' T of the (6-4) TT lesion.

Mol Cell Biol, 2001 May, 21(10), 3514 - 22
A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex; Sutton A et al.; In the yeast Saccharomyces cerevisiae, a and alpha mating-type information is stored in transcriptionally silenced cassettes called HML and HMR . Silencing of these loci, maintained by the formation of a specialized type of heterochromatin, requires trans-acting proteins and cis-acting elements . Proteins required for silencing include the Sir2 NAD(+)-dependent deacetylase, Sir3, and Sir4 . Factors that bind to the cis elements at HMR and HML and that are important for silencing include the origin recognition complex (ORC) . Mutations of any of these Sir proteins or combinations of cis elements result in loss of silencing . SUM1-1 was previously identified as a dominant mutation that restores silencing to HMR in the absence of either the Sir proteins or some of the cis elements . We have investigated the novel mechanism whereby Sum1-1 causes Sir-independent silencing at HMR and present the following findings: Sum1-1 requires the Sir2 homolog, Hst1, for silencing and most probably requires the NAD(+)-dependent deacetylase activity of this protein . Sum1-1 interacts strongly with ORC, and this strong interaction is dependent on HMR DNA . Furthermore, ORC is required for Sum1-1-mediated silencing at HMR . These observations lead to a model for Sum1-1 silencing of HMR in which Sum1-1 is recruited to HMR by binding to ORC . Sum1-1, in turn, recruits Hst1 . Hst1 then deacetylates histones or other chromatin-associated proteins to cause chromatin condensation and transcriptional silencing.

Mol Cell Biol, 2001 May, 21(10), 3425 - 35
Evidence for biased holliday junction cleavage and mismatch repair directed by junction cuts during double-strand-break repair in mammalian cells; Baker MD et al.; In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination . In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution . According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes . The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency . However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule . The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting . The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.

Mol Cell Biol, 2001 May, 21(10), 3405 - 15
Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p; Gadal O et al.; Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified . Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants . One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits . We find that thermosensitive nmd3 mutants are impaired in large subunit export . Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p . Moreover, we show that export of 60S subunits is Xpo1p dependent . We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.

J Biol Chem, 2001 Jun 15, 276(24), 20817 - 20 Epub 2001 Apr 19.
A new pathway for heavy metal detoxification in animals . Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans; Vatamaniuk OK et al.; Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms . Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, gamma-Glu-Cys-Gly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins . Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH . Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism.

FEMS Microbiol Lett, 2001 Apr 13, 197(2), 209 - 14
A novel actin-related protein gene of Colletotrichum gloeosporioides f . sp . malvae shows altered expression corresponding with spore production; Li J et al.; A novel actin-related protein (arp) was found in the plant pathogenic fungus, Colletotrichum gloeosporioides f . sp . malvae (Cgm), which causes anthracnose disease of round-leaved mallow (Malva pusilla) . Sequence comparisons showed that this gene, arpA, belongs to the highly divergent 'other arps' category in the current arp classification system . ArpA is most similar to the arp11 gene of Mus musculus but has a unique structure with deletions at the C-terminus similar to that of the arp10 gene of Saccharomyces cerevisiae . A portion of another putative arp gene, arpB, was found immediately downstream of arpA . Expression of arpA was compared to the constitutively expressed Cgm actin gene, actA . In culture, the relative expression of arpA increased when growth conditions favored sporulation . During infection, arpA expression was greatest at the late necrotrophic phase, when sporulation occurred . Arps have been shown to be important in nuclear migration in fungal hyphae, and the expression pattern of arpA indicates that it may have a particular role during sporulation.

J Agric Food Chem, 2001 Mar, 49(3), 1607 - 11
Antifungal mechanism of polygodial; Kubo I et al.; The primary antifungal action of polygodial comes in part from its ability to function as a nonionic surfactant, disrupting the lipid-protein interface of integral proteins and denaturing their conformation . As a result, the antifungal mechanism of this sesquiterpene dialdehyde is associated with the membrane functions or derangement of the membrane . For example, the glucose-induced medium acidification process of Saccharomyces cerevisiae was inhibited by polygodial, presumably caused by inhibition of the plasma membrane H(+)-ATPase . However, the potent antifungal activity of polygodial results from its multiple functions.

J Virol, 2001 May, 75(10), 4902 - 6
Identification of critical elements in the tRNA acceptor stem and T(Psi)C loop necessary for human immunodeficiency virus type 1 infectivity; Yu Q et al.; A mutant human immunodeficiency virus type 1 (HIV-1) with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe), which relies on exogenous yeast tRNA(Phe) as reverse transcription primer, was used to investigate elements in the tRNA acceptor stem and T(Psi)C stem-loop required for the tRNA primer selection and use in HIV-1 replication . tRNA(Phe) mutants with two- or four-nucleotide deletions in the 3' end retained the capacity to complement replication of psHIV-Phe . tRNA(Phe) mutants with an extended 5' end had reduced capacity for complementation, which could be restored by extension of the 3' end of these tRNA(Phe) mutants with sequences complementary to the HIV-1 U5 region . Further analysis of mutations in the acceptor stem of tRNA(Phe) suggested that an intact acceptor stem RNA structure is important for complementation . Analysis of single-nucleotide changes in the T(Psi)C stem-loop of tRNA(Phe) revealed an unexpected, essential role of this region for rescue of psHIV-Phe.

J Endocrinol, 2001 May, 169(2), 389 - 96
GH gene expression in the submaxillary gland in normal and Ames dwarf mice; Perez-Romero A et al.; High local GH-releasing hormone (GHRH) levels are capable of inducing transdifferentiation in salivary cells to synthesize GH . However, the factors implicated in this process remain unknown . To study this subject, normal and Ames dwarf mice were implanted in the submaxillary gland with a slow release pellet releasing 21 microgram GHRH (1-29)-NH(2)/day for 2 months . Control animals received placebo pellets at the same site . After 60 days, heart blood was collected and submaxillary glands were removed . Circulating levels of GH and IGF-I were significantly decreased (P<0.05) in dwarf mice in comparison with controls, and GHRH treatment did not modify either of these two parameters . Controls carrying GHRH pellets showed a significantly higher GH content (P<0.05) in the submaxillary gland than the placebo-treated normal mice . There were no differences between the IGF-I concentrations of placebo- and GHRH-treated salivary tissue from normal mice . Analysis of GH mRNA by RT-PCR followed by Southern blot revealed that GH transcripts were present in the salivary gland samples carrying the placebo pellets in both normal and dwarf mice . The expression of GH was significantly (P<0.05) increased by the GHRH pellets in salivary tissue from normal mice, but not in submaxillary glands from dwarf mice . Pit-1 mRNA was not detected in the GHRH-treated glands of normal and dwarf mice by RT-PCR or by Southern blot . Using these highly sensitive methods, we have been able to detect the transcription of both GH and Pit-1 in pituitaries from Pit-1-deficient Ames dwarf mice . The present experiment demonstrates that salivary tissue synthesizes GH when it is exposed to the influence of GHRH . Both basal and GHRH-induced salivary GH expression appear to be independent of Pit-1.

Gene, 2001 Apr 4, 267(1), 101 - 10
Interaction of MCC2, a novel homologue of MCC tumor suppressor, with PDZ-domain Protein AIE-75; Ishikawa S et al.; AIE-75 is a protein identified as an autoantigen in patients with autoimmune enteropathy and as a colon cancer-related antigen . It has recently been assigned to be a causative gene for Usher type 1C congenital syndromic hearing loss . The novel protein has three PSD-95/Dlg/ZO-1 (PDZ) protein-protein interaction domains and is therefore implicated to function as a molecular anchor or sorter . We have identified a novel protein that binds to AIE-75 by yeast two-hybrid screening . The protein has a high homology to the tumor suppressor MCC (mutated in colon cancer; or MCC1 hereafter) and was named MCC2 . MCC2 protein binds the first PDZ domain of AIE-75 with its C-terminal amino acids -DTFL . Since the MCC1 does not bind to AIE-75 and the MCC2 displays different expression patterns in various organs compared to MCC1, they appear to play distinct roles in cells . The MCC2 gene is located on chromosome 19p13 in the vicinity of APCL gene, while MCC1 maps near to APC tumor suppressor gene . Because of negative expression of MCC2 in a panel of cancer cell-lines compared to the corresponding normal tissues, we suggest that further study is necessary to investigate a possible role of MCC2 as a tumor suppressor.

FEBS Lett, 2001 Apr 13, 494(3), 208 - 12
The transcriptional regulator, Arg82, is a hybrid kinase with both monophosphoinositol and diphosphoinositol polyphosphate synthase activity; Zhang T et al.; The Arg82 gene of Saccharomyces cerevisiae encodes a transcriptional regulator that phosphorylates inositol 1,4,5-trisphosphate {Saiardi et al . (1999) Curr . Biol . 9, 1323-1326} . However, some controversy has surrounded the nature of the reaction products . We now show that Arg82 phosphorylates inositol 1,3,4,5-tetrakisphosphate to inositol pentakisphosphate, which is itself converted to two isomers of diphosphoinositol tetrakisphosphate, one of which has never previously been identified . One of the diphosphoinositol phosphates was further phosphorylated by a yeast cell lysate . We propose that Arg82 is an ancestral precursor of two distinct and specific enzyme families: inositol 1,4,5-trisphosphate kinases and diphosphoinositol polyphosphate synthases.

Nature, 2001 Apr 19, 410(6831), 955 - 9
Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability; Rao H et al.; Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin . At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K) . The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule . Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway . Overexpression of a long-lived derivative of the SCC1 fragment is lethal . In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss . The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment . This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue . A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.

Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 5122 - 7 Epub 2001 Apr 17.
The 3'-->5' exonuclease of DNA polymerase delta can substitute for the 5' flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability; Jin YH et al.; Many DNA polymerases (Pol) have an intrinsic 3'-->5' exonuclease (Exo) activity which corrects polymerase errors and prevents mutations . We describe a role of the 3'-->5' Exo of Pol delta as a supplement or backup for the Rad27/Fen1 5' flap endonuclease . A yeast rad27 null allele was lethal in combination with Pol delta mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol delta . The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol delta Exo I and Exo II motifs . However, rad27-p Pol delta Exo III double mutants were viable . They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability . Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5' flaps in the lagging strand . These results suggest that the 3'-->5' Exo activity of Pol delta is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand.

J Cell Biol, 2001 Apr 16, 153(2), 251 - 62
Nuclear import of histone H2A and H2B is mediated by a network of karyopherins; Mosammaparast N et al.; The first step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones . The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers . We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family . An abundant complex of H2A, H2B, and Kap114p was detected in cytosol . In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B . Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B . We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail . Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains . In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo . Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association . These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

J Biol Chem, 2001 Jun 29, 276(26), 24203 - 11 Epub 2001 Apr 17.
A novel snare N-terminal domain revealed by the crystal structure of Sec22b; Gonzalez LC Jr et al.; Intra-cellular membrane fusion is facilitated by the association of SNAREs from opposite membranes into stable alpha-helical bundles . Many SNAREs, in addition to their alpha-helical regions, contain N-terminal domains that likely have essential regulatory functions . To better understand this regulation, we have determined the 2.4-A crystal structure of the 130-amino acid N-terminal domain of mouse Sec22b (mSec22b), a SNARE involved in endoplasmic reticulum/Golgi membrane trafficking . The domain consists of a mixed alpha-helical/beta-sheet fold that resembles a circular permutation of the actin/poly-proline binding protein, profilin, and the GAF/PAS family of regulatory modules . The structure is distinct from the previously characterized N-terminal domain of syntaxin 1A, and, unlike syntaxin 1A, the N-terminal domain of mSec22b has no effect on the rate of SNARE assembly in vitro . An analysis of surface conserved residues reveals a potential protein interaction site . Key residues in this site are distinct in two mammalian Sec22 variants that lack SNARE domains . Finally, sequence analysis indicates that a similar domain is likely present in the endosomal/lysosomal SNARE VAMP7.

J Biol Chem, 2001 Jun 29, 276(26), 24323 - 30 Epub 2001 Apr 17.
Accurate in vitro end joining of a DNA double strand break with partially cohesive 3'-overhangs and 3'-phosphoglycolate termini: effect of Ku on repair fidelity; Chen S et al.; To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3'-overhangs and a one-base gap in each strand, was constructed . In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence . In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3' --> 5' resection before ligation, was formed . Similar results were seen for a substrate with 5'-overhangs and recessed 3'-phosphoglycolate ends . Supplementation of the xrs6 extracts with purified Ku restored accurate end joining . In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity . The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation.

Mol Microbiol, 2001 Apr, 40(2), 361 - 75
The Aspergillus nidulans GATA transcription factor gene areB encodes at least three proteins and features three classes of mutation; Conlon H et al.; In Aspergillus nidulans, the principal transcription factor regulating nitrogen metabolism, AREA, belongs to the GATA family of DNA-binding proteins . In seeking additional GATA factors, we have cloned areB, which was originally identified via a genetic screen for suppressors of areA loss-of-function mutations . Based on our analysis, areB is predicted to encode at least three distinct protein products . These arise from the use of two promoters, differential splicing and translation initiating at AUG and non-AUG start codons . All the putative products include a GATA domain and a putative Leu zipper . These regions show strong sequence similarity to regulatory proteins from Saccharomyces cerevisiae (Dal80p and Gzf3p), Penicillium chrysogenum (NREB) and Neurospora crassa (ASD4) . We have characterized three classes of mutation in areB; the first are loss-of-function mutations that terminate the polypeptides within or before the GATA domain . The second class truncates the GATA factor either within or upstream of the putative Leu zipper but retains the GATA domain . The third class fuses novel gene sequences to areB with the potential to produce putative chimeric polypeptides . These novel gene fusions transform the putative negative-acting transcription factor into an activator that can partially replace areA.

J Agric Food Chem, 2001 Apr, 49(4), 2030 - 6
Effect of chemical and genetic attachment of polysaccharides to proteins on the production of IgG and IgE; Arita K et al.; To investigate the effect of polysaccharide attachment to proteins on the production of IgG and IgE, the genetic attachment of polysaccharide to lysozymes (G49N and R21T) using the yeast expression system (Saccharomyces cerevisiae AH 22) and the Maillard-type polysaccharide attachment to native lysozyme and soybean P34 protein were attempted . The production of IgG and IgE was investigated by using mice immunized with the protein-polysaccharide conjugates or native proteins . The attachment of polysaccharide to lysozyme using the yeast expression system greatly suppressed the production level of IgG and IgE . The attachment of polysaccharide to native lysozyme and soybean P34 protein using the Maillard-type reaction was also found to be effective in reducing the production level of IgE compared to IgG.

Biol Chem, 2001 Feb, 382(2), 161 - 77
Congenital disorders of glycosylation: glycosylation defects in man and biological models for their study; Marquardt T et al.; Several inherited disorders affecting the biosynthetic pathways of N-glycans have been discovered during the past years . This review summarizes the current knowledge in this rapidly expanding field and covers the molecular bases of these disorders as well as their phenotypical consequences.

J Protein Chem, 2000 Nov, 19(8), 649 - 62
Expression and properties of recombinant HbA2 (alpha2delta2) and hybrids containing delta-beta sequences; Inagaki K et al.; Hemoglobin A2 (alpha2delta2), which is present at low concentration (1-2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic . However, reports on its functional properties regarding O2 binding are conflicting . We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A2 and systematically studying its functional properties . The construct expressing HbA2 contains only alpha and delta genes so that the extensive purification required to isolate natural HbA2 is circumvented . Although natural hemoglobin A2 is expressed at low levels in vivo, the amount of recombinant alpha2delta2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the alpha + beta or the alpha + gamma genes, respectively, are present on the construct . Recombinant HbA2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA . However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin . Molecular modeling studies which attempt to understand these properties of HbA2 are described.

J Biol Chem, 2001 Jul 6, 276(27), 24654 - 60 Epub 2001 Apr 16.
Functional analysis of the protein-interacting domains of chloroplast SRP43; Jonas-Straube E et al.; The chloroplast signal recognition particle (cpSRP) consists of an evolutionarily conserved 54-kDa subunit (cpSRP54) and a dimer of a unique 43-kDa subunit (cpSRP43) . cpSRP binds light-harvesting chlorophyll proteins (LHCPs) to form a cpSRP/LHCP transit complex, which targets LHCP to the thylakoid membrane . Previous studies showed that transit complex formation is mediated through the binding of the L18 domain of LHCP to cpSRP43 . cpSRP43 is characterized by a four-ankyrin repeat domain at the N terminus and two chromodomains at the C terminus . In the present study we used the yeast two-hybrid system and in vitro binding assays to analyze the function of different domains of cpSRP43 in protein complex formation . We report here that the first ankyrin repeat binds to the 18-amino acid domain on LHCP that binds to cpSRP43, whereas the third and fourth ankyrin repeats are involved in the dimerization of cpSRP43 . We show further that the interaction of cpSRP43 with cpSRP54 is mediated via binding of the methionine-rich domain of cpSRP54 to the C-terminally located chromodomains of cpSRP43 . Both chromodomains contain essential elements for binding cpSRP54, indicating that the closely spaced chromodomains together create a single binding site for cpSRP54 . In addition, our data demonstrate that the interaction of cpSRP54 with the chromodomains of cpSRP43 is enhanced indirectly by the dimerization motif of cpSRP43.

J Biol Chem, 2001 Jun 22, 276(25), 22595 - 603 Epub 2001 Apr 16.
An essential role for Mad homology domain 1 in the association of Smad3 with histone deacetylase activity*; Liberati NT et al.; The Smads are a family of sequence-specific DNA-binding proteins that modulate transcription in response to transforming growth factor beta (TGFbeta) by recruiting transcriptional activators like the histone acetyltransferase, p300/CBP, or repressors like the histone deacetylase, HDAC1, to TGFbeta target genes . The association of Smads and HDAC1 is mediated in part by direct binding of Smads to the HDAC1-associated proteins, TG-interacting factor, c-ski, and SnoN . Although ectopic expression of these proteins inhibits Smad-activated transcription, the contribution of histone deacetylase enzymatic activity to transcriptional repression by TGFbeta is unknown . Here, the biological requirements for the interaction between Smads and endogenous histone deacetylase activity are investigated . We identify residues in Mad homology domain 1 of Smad3 that are required for association with histone deacetylase activity . An amino acid change at one of these critical residues does not disrupt the association of Smad3 with c-ski, SnoN, and transforming growth-interacting factor but does abrogate the ability of Smad3 to repress transcription . These findings indicate that the association of Smad3 and histone deacetylase activity relies on additional protein mediators that make contact with Smad3 at its amino terminus . Moreover, these data suggest that the suppressive effect of Smad3 on transcription is dependent upon its association with histone deacetylase enzymatic activity.

J Biol Chem, 2001 Jun 22, 276(25), 22439 - 45 Epub 2001 Apr 16.
Cloning and characterization of MST4, a novel Ste20-like kinase; Qian Z et al.; MST4, a novel member of the germinal center kinase subfamily of human Ste20-like kinases, was cloned and characterized . Composed of a C-terminal regulatory domain and an N-terminal kinase domain, MST4 is most closely related to mammalian Ste20 kinase family member MST3 . Both the kinase and C-terminal regulatory domains of MST4 are required for full activation of the kinase . Northern blot analysis indicates that MST4 is ubiquitously distributed, and the MST4 gene is localized to chromosome Xq26, a disease-rich region, by fluorescence in situ hybridization . Although some members of the MST4 family function as upstream regulators of mitogen-activated protein kinase cascades, expression of MST4 in 293 cells was not sufficient to activate or potentiate extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase . An alternatively spliced isoform of MST4 (MST4a) was isolated by yeast two-hybrid interaction with the catalytic domain of Raf from a human fetal brain cDNA library and also found in a variety of human fetal and adult tissues . MST4a lacks an exon encoding kinase subdomains IX-XI that stabilizes substrate binding . The existence of both MST4 isoforms suggests that the MST4 kinase activity is highly regulated, and MST4a may function as a dominant-negative regulator of the MST4 kinase.

EMBO Rep, 2001 Apr, 2(4), 287 - 91
Homologous recombination in planta is stimulated in the absence of Rad50; Gherbi H et al.; Chromosomal double-strand DNA breaks must be repaired; in the absence of repair the resulting acentromeric (and telomereless) fragments may be lost and/or the broken DNA ends may recombine causing general chromosomal instability . The Rad50/Mre11/Xrs2 protein complex acts at DNA ends and is implicated in both homologous and non-homologous recombination . We have isolated a rad50 mutant of the plant Arabidopsis thaliana and show here that it has a somatic hyper-recombination phenotype in planta . This finding supports the hypothesis of a competition between homologous and illegitimate recombination in higher eukaryotes . To our knowledge, this is the first direct in vivo support for the role of this complex in chromosomal recombination in a multicellular organism and the first description of a mutation of a known gene leading to hyper-recombination in plants.

Cell Growth Differ, 2001 Mar, 12(3), 157 - 67
Signaling mediated by the closely related mammalian Rho family GTPases TC10 and Cdc42 suggests distinct functional pathways; Murphy GA et al.; The mammalian Rho family GTPases TC10 and Cdc42 share many properties . Activated forms of both proteins stimulate transcription mediated by nuclear factor kappaB, serum response factor, and the cyclin D1 promoter; activate c-Jun NH2-terminal kinase; cooperate with activated Raf to transform NIH-3T3 cells; and, by a mechanism independent of all of these effects, induce filopodia formation . In contrast, previously reported differences between TC10 and Cdc42 are not striking . We now present studies of TC10 and Cdc42 in cell culture that reveal clear functional differences: (a) wild-type TC10 localizes predominantly to the plasma membrane and less extensively to a perinuclear membranous compartment, whereas wild-type Cdc42 localizes predominantly to this compartment and less extensively to the plasma membrane; (b) expression of Rho guanine nucleotide dissociation inhibitor alpha results in a redistribution of wild-type Cdc42 to the cytosol but has no effect on the plasma membrane localization of wild-type TC10; (c) TC10 fails to rescue a Saccharomyces cerevisiae cdc42 mutation, unlike mammalian Cdc42; (d) dominant negative Cdc42, but not dominant negative TC10, inhibits neurite outgrowth in PC12 cells stimulated by nerve growth factor; and (e) activation of nuclear factor kappaB-dependent transcription by Cdc42, but not by TC10, is inhibited by sodium salicylate . These findings point to distinct pathways in which TC10 and Cdc42 may act and distinct modes of regulation of these proteins.

Chem Biol, 2001 Mar, 8(3), 231 - 41
Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities; Kasanov J et al.; Introduction: WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins . Five classes of WW domains have been annotated to date, where each class is largely defined by the type of peptide ligand selected, rather than by similarities within WW domains . Class I WW domains bind Pro-Pro-Xxx-Tyr containing ligands, and it would be of interest to determine residues within the domains that determine this specificity.Results: Fourteen WW domains selected Leu/Pro-Pro-Xxx-Tyr containing peptides ligands via phage display and were thus designated as Class 1 WW domains . These domains include those present in human YAP (hYAP) and WWP3, as well as those found in ubiquitin protein ligases of the Nedd4 family, including mouse Nedd4 (mNedd4), WWP1, WWP2 and Rsp5 . Comparing the primary structures of these WW domains highlighted a set of highly conserved residues, in addition to those originally noted to occur within WW domains . Substitutions at two of these conserved positions completely inhibited ligand binding, whereas substitution at a non-conserved position did not . Moreover, mutant WW domains containing substitutions at conserved positions bound novel peptide ligands.Conclusions: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro-Xxx-Tyr containing peptide ligands . The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro-Xxx-Tyr containing peptide ligands.

Trends Endocrinol Metab, 2001 Apr, 12(3), 127 - 34
The TRAP/SMCC/Mediator complex and thyroid hormone receptor function; Ito M et al.; The TRAP/SMCC/Mediator complex is a mammalian transcriptional regulatory complex that contains over 25 polypeptides and is, in part, phylogenetically conserved . It was originally isolated as a thyroid hormone receptor (TR)-associated protein (TRAP) complex that mediates TR-activated transcription from DNA templates in conjunction with the general transcription machinery, and probably acts in vivo after the action of other receptor-interacting coactivators involved in chromatin remodeling . Subsequently, the TRAP complex was identified as a more broadly used coactivator complex for a wide variety of activators . The TRAP220 subunit mediates ligand-dependent interactions of the complex with TR and other nuclear receptors; and genetic ablation of murine TRAP220 has revealed that it is essential both for optimal TR function and for a variety of early developmental and adult homeostasis events in mice, but not for cell viability per se.

Trends Cell Biol, 2001 Mar, 11(3), 136 - 41
Congenital disorders of glycosylation: genetic model systems lead the way; Aebi M et al.; N-linked glycosylation is the most frequent modification of secretory proteins in eukaryotic cells . The highly conserved glycosylation process is initiated in the endoplasmic reticulum (ER), where the Glc(3)Man(9)GlcNAc(2) oligosaccharide is assembled on the lipid carrier dolichylpyrophosphate and then transferred to selected asparagine residues of polypeptide chains . In recent years, several inherited human diseases, congenital disorders of glycosylation (CDG), have been associated with deficiencies in this pathway . The ER-associated glycosylation pathway has been studied in the budding yeast Saccharomyces cerevisiae, and this model system has been invaluable in elucidating the molecular basis of novel types of CDG.

Trends Cell Biol, 2001 Mar, 11(3), 102 - 6
Towards an understanding of complex protein networks; Tucker CL et al.; Large-scale two-hybrid screens have generated a wealth of information describing potential protein--protein interactions . When compiled with data from systematic localizations of proteins, mutant screens and other functional tests, a network of interactions among proteins and between proteins and other components of eukaryotic cells can be deduced . These networks can be viewed as maps of the cell, depicting potential signaling pathways and interactive complexes . Most importantly, they provide potential clues to the function of previously uncharacterized proteins . Focusing on recent experiments, we explore these protein-interaction studies and the maps derived from such efforts.

Genome Biol . 2001;2(4):REVIEWS1013 . Epub 2001 Apr 04.
Genome-wide analysis of protein-DNA interactions in living cells; Pugh BF et al.; Understanding the regulation of gene expression requires an analysis of gene-specific transcription factors . This review highlights recent work that uses protein-DNA crosslinking, immunoprecipitation and DNA microarrays to determine the binding sites for specific transcription factors throughout the yeast genome.

Genome Biol . 2001;2(4):REVIEWS0003 . Epub 2001 Apr 05.
A tale of histone modifications; Grant PA; The modification of chromatin structure is important for a number of nuclear functions, exemplified by the regulation of transcription . This review discusses recent studies of covalent histone modifications and the enzymatic machines that generate them.

Biochemistry, 2001 Apr 24, 40(16), 4893 - 903
Enzymes of sphingolipid metabolism: from modular to integrative signaling; Hannun YA et al.; Many enzymes of sphingolipid metabolism are regulated in response to extra- and intracellular stimuli and in turn serve as regulators of levels of bioactive lipids (such as sphingosine, ceramide, sphingosine 1-phosphate, and diacylglycerol), and as such, they serve a prototypical modular function in cell regulation . However, lipid metabolism is also closely interconnected in that a product of one enzyme serves as a substrate for another . Moreover, many cell stimuli regulate more than one of these enzymes, thus adding to the complexity of regulation of lipid metabolism . In this paper, we review the status of enzymes of sphingolipid metabolism in cell regulation and propose a role for these enzymes in integration of cell responses, a role that builds on the modular organization while also taking advantage of the complexity and interconnectedness of lipid metabolism, thus providing for a combinatorial mechanism of generating diversity in cell responses . This may be a general prototype for the involvement of metabolic pathways in cell regulation.

Anal Chem, 2001 Mar 15, 73(6), 1307 - 15
Nanoflow gradient generator coupled with mu-LC-ESI-MS/MS for protein identification; Le Bihan T et al.; The large-scale identification of proteins from proteomes of complex organisms, and the availability of various types of protein and DNA databases, increasingly require the additional information provided by tandem mass spectrometry . HPLC and microLC coupled to ESI-MS/MS presently dominate the field of protein identification by tandem mass spectrometry and database searching . The analysis of protein digests is typically performed using HPLC or LC columns with 50-100-microm diameters, requiring the delivery of solvent gradients at low to mid nanoliter per minute flow rates . This has been typically achieved using expensive generic HPLC pumping systems for the delivery of microliter per minute gradients that were either flow-split or sampled . Here we present an alternative system for the delivery of nanoliter per minute gradients . The inexpensive nanoflow gradient generator (etagrad) described here can be modulated to reproducibly deliver selected gradients . The performance of the etagrad on-line with a microLC-ESI-MS/MS system has been demonstrated for the identification of standard protein digests . Moreover, the performance of the etagrad-microLC-ESI-MS/MS system, with protein prefractionation by IPG isoelectric focusing, was also evaluated for rapid study of yeast and human proteomes.

J Biol Chem, 2001 Jun 15, 276(24), 20973 - 80 Epub 2001 Apr 13.
Coactivator p300 acetylates the interferon regulatory factor-2 in U937 cells following phorbol ester treatment; Masumi A et al.; Interferon regulatory factor-2 (IRF-2) is a transcription factor of the IRF family that represses interferon-mediated gene expression . In the present study, we show that human monocytic U937 cells express truncated forms of IRF-2 containing the DNA binding domain but lacking much of the C-terminal regulatory domain . U937 cells are shown to respond to phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce expression of histone acetylases p300 and p300/CBP-associated factor (PCAF) . In addition, TPA treatment led to the appearance of full-length IRF-2, along with a reduction of the truncated protein . Interestingly, full-length IRF-2 in TPA-treated U937 cells occurred as a complex with p300 as well as PCAF and was itself acetylated . Consistent with these results, recombinant IRF-2 was acetylated by p300 and to a lesser degree by PCAF in vitro . Another IRF member, IRF-1, an activator of interferon-mediated transcription, was also acetylated in vitro by these acetylases . Finally, we demonstrate that the addition of IRF-2 but not IRF-1 inhibits core histone acetylation by p300 in vitro . The addition of IRF-2 also inhibited acetylation of nucleosomal histones in TPA-treated U937 cells . Acetylated IRF-2 may affect local chromatin structure in vivo by inhibiting core histone acetylation and may serve as a mechanism by which IRF-2 negatively regulates interferon-inducible transcription.

Biochem Biophys Res Commun, 2001 Apr 20, 282(5), 1244 - 50
Rpg1p/Tif32p, a subunit of translation initiation factor 3, interacts with actin-associated protein Sla2p; Palecek J et al.; The yeast two-hybrid system was used to screen for proteins that interact in vivo with Saccharomyces cerevisiae Rpg1p/Tif32p, the large subunit of the translation initiation factor 3 core complex (eIF3) . Eight positive clones encoding portions of the SLA2/END4/MOP2 gene were isolated . They overlapped in the region of amino acids 318-550 . Subsequent deletion analysis of Sla2p showed that amino acids 318-373 were essential for the two-hybrid protein-protein interaction . The N-terminal part of Rpg1p (aa 1-615) was essential and sufficient for the Rpg1p-Sla2p interaction . A coimmunoprecipitation assay provided additional evidence for the physical interaction of Rpg1p/Tif32p with Sla2p in vivo . Using immunofluorescence microscopy, Rpg1p and Sla2p proteins were colocalized at the patch associated with the tip of emerging bud . Considering the essential role of Rpg1p as the large subunit of the eIF3 core complex and the association of Sla2p with the actin cytoskeleton, a putative role of the Rpg1p-Sla2p interaction in localized translation is discussed .

Biochem Biophys Res Commun, 2001 Apr 20, 282(5), 1211 - 9
The role of heat shock protein 70 in vitamin D receptor function; Lutz W et al.; We previously demonstrated that the 1alpha,25-dihydroxyvitamin D(3) receptor (VDR) interacts with the constitutive heat shock protein, hsc70 in vitro, and with DnaK (Biochem . Biophys . Res . Commun . 260, 446-452, 1999) . The biological significance of VDR-heat shock protein interactions, however, is unknown . To examine the role of such interactions in eukaryotic cells, we heterologously expressed VDR and RXRalpha together with a vitamin D-responsive reporter system in Saccharomyces cerevisiae and examined the consequences of heat shock protein 70 gene (SSA) deletion in these cells . We show that heterologously expressed VDR associates with the yeast cytosolic hsp70 protein, Ssa1p . Deletion of the SSA2, SSA3, and SSA4 genes and reduction of Ssa1p activity, reduces the intracellular concentrations of the VDR and its heterodimeric partner, RXRalpha and reduces the activity of a vitamin D-dependent gene . Hsp70-like chaperone proteins play a role in controlling concentrations of the VDR within the cell .

Exp Cell Res, 2001 May 1, 265(2), 195 - 202
Histone acetylation and chromatin remodeling; Gregory PD et al.; Chromatin represents a repressive barrier to the process of transcription . This molecular obstacle is a highly dynamic structure, able to compact the DNA of the entire genome into the confines of a nucleus, and yet it allows access to the genetic information held within . The acetylation of histones has emerged as a regulatory mechanism capable of modulating the properties of chromatin and thus the competence of the DNA template for transcriptional activation . The role of acetylation in chromatin remodeling is therefore of paramount importance to our understanding of gene regulation in vivo .

Eur J Cell Biol, 2001 Feb, 80(2), 126 - 38
Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p; Yang X et al.; Saccharomyces cerevisiae delta3,delta2-enoyl-CoA isomerase (Eci1p), encoded by ECI1, is an essential enzyme for the betaoxidation of unsaturated fatty acids . It has been reported, as well as confirmed in this study, to be a peroxisomal protein . Unlike many other peroxisomal proteins, Ecilp possesses both a peroxisome targeting signal type 1 (PTS1)-like signal at its carboxy-terminus (-HRL) and a PTS2-like signal at its amino-terminus (RIEGPFFIIHL) . We have found that peroxisomal targeting of a fusion protein consisting of Eci1p in front of green fluorescent protein (GFP) is not dependent on Pex7p (the PTS2 receptor), ruling out a PTS2 mechanism, but is dependent on Pex5p (the PTS1 receptor) . This Pex5p-dependence was unexpected, since the putative PTS1 of Ecilp is not at the C-terminus of the fusion protein; indeed, deletion of this signal (-HRL-) from the fusion did not affect the Pex5p-dependent targeting . Consistent with this, Pex5p interacted in two-hybrid assays with both Eci1p and Eci1PdeltaHRL . Ecilp-GFP targeting and Eci1pdeltaHRL interaction were abolished by replacement of Pex5p with Pex5p(N495K), a point-mutated Pex5p that specifically abolishes the PTS1 protein import pathway . Thus, Eci1p peroxisomal targeting does require the Pex5p-dependent PTS1 pathway, but does not require a PTS1 of its own . By disruption of ECI1 and DCI1, we found that Dci1p, a peroxisomal PTS1 protein that shares 50% identity with Eci1p, is necessary for Eci1p-GFP targeting . This suggests that the Pex5p-dependent import of Eci1p-GFP is due to interaction and co-import with Dci1p . Despite the dispensability of the C-terminal HRL for import in wild-type cells, we have also shown that this tripeptide can function as a PTS1, albeit rather weakly, and is essential for targeting in the absence of Dci1p . Thus, Eci1p can be targeted to peroxisomes by its own PTS1 or as a hetero-oligomer with Dcilp . These data demonstrate a novel, redundant targeting pathway for Eci1p.

J Biol Chem, 2001 Jun 29, 276(26), 24261 - 7 Epub 2001 Apr 11.
Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals; Benaroudj N et al.; The disaccharide trehalose, which accumulates dramatically during heat shock and stationary phase in many organisms, enhances thermotolerance and reduces aggregation of denatured proteins . Here we report a new role for trehalose in protecting cells against oxygen radicals . Exposure of Saccharomyces cerevisiae to a mild heat shock (38 degrees C) or to a proteasome inhibitor (MG132) induced trehalose accumulation and markedly increased the viability of the cells upon exposure to a free radical-generating system (H(2)O(2)/iron) . When cells were returned to normal growth temperature (28 degrees C) or MG132 was removed from the medium, the trehalose content and resistance to oxygen radicals decreased rapidly . Furthermore, a mutant unable to synthesize trehalose was much more sensitive to killing by oxygen radicals than wild-type cells . Providing trehalose exogenously enhanced the resistance of mutant cells to H(2)O(2) . Exposure of cells to H(2)O(2) caused oxidative damage to amino acids in cellular proteins, and trehalose accumulation was found to reduce such damage . After even brief exposure to H(2)O(2), the trehalose-deficient mutant exhibited a much higher content of oxidatively damaged proteins than wild-type cells . Trehalose accumulation decreased the initial appearance of damaged proteins, presumably by acting as a free radical scavenger . Therefore, trehalose accumulation in stressed cells plays a major role in protecting cellular constituents from oxidative damage.

Bioinformatics, 2001 Apr, 17(4), 309 - 18
Validating clustering for gene expression data; Yeung KY et al.; MOTIVATION: Many clustering algorithms have been proposed for the analysis of gene expression data, but little guidance is available to help choose among them . We provide a systematic framework for assessing the results of clustering algorithms . Clustering algorithms attempt to partition the genes into groups exhibiting similar patterns of variation in expression level . Our methodology is to apply a clustering algorithm to the data from all but one experimental condition . The remaining condition is used to assess the predictive power of the resulting clusters-meaningful clusters should exhibit less variation in the remaining condition than clusters formed by chance . RESULTS: We successfully applied our methodology to compare six clustering algorithms on four gene expression data sets . We found our quantitative measures of cluster quality to be positively correlated with external standards of cluster quality.

Exp Hematol, 2001 Apr, 29(4), 490 - 8
Novel expression of cyclin-dependent kinase inhibitors in human B-cell precursors; Fink JR et al.; Eukaryotic cell division is regulated by cyclins, cyclin-dependent kinases (CDK), and cyclin-dependent kinase inhibitors (CKI) . Genes encoding these proteins are mutated or deleted in many types of cancer . For example, 20%-30% of B-lineage acute lymphoblastic leukemias (ALL) have deletions in the CKI known as INK4a . The contribution of INK4a deletions to the progression of B-lineage ALL is uncertain, partially due to a paucity of data on expression in normal B-cell precursors . We therefore conducted a comparative analysis of normal and leukemic human B-cell development for the expression of cyclins, CDK, and CKI.Specific stages of human B-cell development from normal bone marrow were purified by fluorescence-activated cell sorting . The sorted populations and B-lineage ALL cell lines (BLIN-1, 2, 3, 4) were examined for expression of cyclins, CDK, and CKI by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting.RT-PCR analysis showed that cyclin D2, cyclin D3, CDK4, and CDK6 were ubiquitously expressed in normal B-cell development and in the BLIN ALL cell lines . The p19(INK4d) CKI was the most commonly expressed member of the INK4 family, whereas p16(INK4a) was more weakly and variably expressed . Expression of the p57(KIP2) CKI varied as a function of the stage of B-cell development . Analysis of normal B-cell precursors by Western blotting indicated that CDK4, CDK6, p19(INK4d), and p57(KIP2) were expressed, whereas p16(INK4a) was not detected.Cyclin D/CDK expression in normal and leukemic human B-cell precursors is similar to expression of these proteins in human and murine mature B cells . In contrast, the ubiquitous expression of p19(INK4d) has not been previously described in human or murine B-lineage cells . Our results suggest that loss of INK4a may only minimally contribute to tumor cell progression in B-lineage ALL, since expression of INK4d could provide a compensatory function as a cyclin-dependent kinase inhibitor.

Plant Physiol, 2001 Apr, 125(4), 1813 - 20
ANT1, an aromatic and neutral amino acid transporter in Arabidopsis; Chen L et al.; A new amino acid transporter was identified from the Arabidopsis expressed sequence tag cDNAs by expressing the cDNA in a yeast amino acid transport mutant . Transport analysis of the expressed protein in yeast showed that it transports aromatic and neutral amino acids, as well as arginine . This transporter (ANT1, aromatic and neutral transporter) also transports indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid . The cDNA is 1.6 kb in length with an open reading frame that codes for a protein with 432 amino acids and a calculated molecular mass of 50 kD . Hydropathy analysis showed ANT1 is an integral membrane protein with 11 putative membrane-spanning domains . Southern analysis and a BLAST search of the Arabidopsis genome database suggests that ANT1 is part of a small gene family containing at least five members . Phylogenetic comparisons with other known amino acid transporters in plants suggests that ANT1 represents a new class of amino acid transporter . RNA gel-blot analysis showed that this transporter is expressed in all organs with highest abundance in flowers and cauline leaves.

Eur J Neurosci, 2001 Apr, 13(7), 1339 - 48
Corticotropin-releasing factor triggers neurite outgrowth of a catecholaminergic immortalized neuron via cAMP and MAP kinase signalling pathways; Cibelli G et al.; Corticotropin-releasing factor (CRF), a neuropeptide of 41 amino acids, acts as the major physiological regulator of the basal and stress-induced release of corticotropin (ACTH), beta-endorphin and other proopiomelanocortin-derived peptides from the anterior pituitary gland . In addition to its endocrine activity, CRF displays extrahypophysiotropic effects, mainly as a regulator of stress responses . We show here that CRF may additionally function as a differentiating factor in immortalized noradrenergic neuronal CATH.a cells that express CRF receptor type I and resemble locus coeruleus-derived neurons . CRF triggers morphological changes in CATH.a cells including the appearance of extended long, slender neurites with prominent growth cones . CRF-treated CATH.a cells exhibit a morphology similar to locus coeruleus neurons in primary culture . CRF-induced neurite outgrowth of CATH.a cells was blocked by addition of inhibitors for cAMP-dependent protein kinase or extracellular signal-regulated protein kinase (ERK), a subtype of the mitogen-activated protein kinases . The participation of ERK within the CRF signalling cascade was further confirmed by Western blot experiments, with antibodies directed against the phosphorylated form of ERK, and also with transcription-based assays . We conclude that CRF functions as a differentiating factor of CATH.a cells via the cAMP and the MAP kinase signalling pathways.

Eur J Biochem, 2001 Apr, 268(8), 2512 - 9
Multisite control of the Crabtree effect in ascites hepatoma cells; Rodriguez-Enriquez S et al.; AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively . To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined . The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8) . Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively) . The cytosolic concentrations of Ca2+ and Mg2+ were not modified . The addition of galactose or glycerol did not modify the concentrations of the metabolites . Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation . The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.

Eur J Immunol, 2001 Apr, 31(4), 1239 - 46
Tumor-associated antigens identified by mRNA expression profiling induce protective anti-tumor immunity; Mathiassen S et al.; Defined tumor-associated antigens (TAA) are attractive targets for anti-tumor immunotherapy . Here, we describe a novel genome-wide approach to identify multiple TAA from any given tumor . A panel of transplantable thymomas was established from an inbred p53-/- mouse strain . The resulting tumors were examined for gene expression by mRNA microarray scanning . This analysis revealed heterogeneity of the tumors in agreement with the assumption that they represent different tumorigenic events . Several genes were overexpressed in one or more of the tumors . To examine whether overexpressed genes might be used to identify TAA, mice were immunized with mixtures of peptides representing putative cytotoxic T cell epitopes derived from one of the gene products . Indeed, such immunized mice were partially protected against subsequent tumor challenge . Despite being immunized with bona fide self antigens, no clinical signs of autoimmune reactions were observed . Thus, it appears possible to evaluate the entire metabolism of any given tumor and use this information rationally to identify multiple epitopes of value in the generation of tumor-specific immunotherapy . We expect that human tumors express similar tumor-specific metabolic imprints, which may be used to identify patient-specific arrays of TAA . This may enable a multi-epitope based immunotherapy with improved prospects of clinical tumor rejection.

Curr Opin Struct Biol, 2001 Apr, 11(2), 163 - 73
Structural insights into the molecular mechanism of calcium-dependent vesicle-membrane fusion; Brunger AT; The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions . Recently determined structures of the SNARE complex, synaptotagmin III, nSec1, domains of the NSF chaperone and its adaptor (SNAP), and Rab3 and some of its effectors provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models . Ultimately, knowledge of the structures of higher-order complexes and their dynamic behavior will be required to obtain a full understanding of the vesicle fusion protein machinery.

FEBS Lett, 2001 Apr 6, 494(1-2), 90 - 4
Functional characterization of sphingolipid C4-hydroxylase genes from Arabidopsis thaliana; Sperling P et al.; In the genome of Arabidopsis thaliana, two genes were identified encoding isoenzymes for C4-hydroxylation of long chain bases (LCB) in plant sphingolipids . Both predicted proteins consist of 258 amino acid residues (77% identity) which show sequence similarity to di-iron-binding enzymes, such as Sur2p and Erg3p from yeast, involved in oxygen-dependent lipid modifications . Heterologous expression of these genes in a yeast sur2Delta-null mutant lacking C4-LCB hydroxylation resulted in the formation of D-ribo-C(18)- and -C(20)-phytosphinganine . The identity and stereochemical configuration of the isolated trihydroxybases was confirmed by electrospray ionization-mass spectroscopy, gas-liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance spectroscopy . These results represent the first functional identification of SUR2 genes from plants as well as from any organism other than yeast.

FEBS Lett, 2001 Apr 6, 494(1-2), 64 - 8
Why Ppr1p is a weak activator of transcription; Patzold AJ et al.; Upon uracil depletion, the transcriptional activator Ppr1p stimulates expression of the Saccharomyces cerevisiae URA3 gene only four-fold . We performed a split-ubiquitin screen with Tup1p as bait, and we found that the global repressor Tup1p interacts with the transcriptional activator Ppr1p both in vivo and in vitro . The interaction is biologically significant, since the deletion of the TUP1 gene as well as the removal of the Tup1p-binding domain from Ppr1p results in an increased expression of the URA3 gene . Our results suggest that Tup1p blocks Ppr1p directly, and that Ppr1p is a weak activator of transcription because of its interaction with Tup1p . Thus we were able to demonstrate that the global repressor Tup1p can modulate transcription by interacting with an activator.

FEBS Lett, 2001 Apr 6, 494(1-2), 38 - 43
The second cysteine-rich domain of Mac1p is a potent transactivator that modulates DNA binding efficiency and functionality of the protein; Voutsina A et al.; Mac1p is a Saccharomyces cerevisiae DNA binding transcription factor that activates genes involved in copper uptake . A copper-induced N-C-terminal intramolecular interaction and copper-independent homodimerization affect its function . Here, we present a functional analysis of Mac1p deletion derivatives that attributes new roles to the second cysteine-rich (REPII) domain of the protein . This domain exhibits the copper-responsive potent transactivation function when assayed independently and, in the context of the entire protein, modulates the efficiency of Mac1p binding to DNA . The efficiency of binding to both copper-response promoter elements can determine the in vivo functionality of Mac1p independent of homodimerization.

FEBS Lett, 2001 Apr 6, 494(1-2), 6 - 10
A putative proton binding site of plasma membrane H(+)-ATPase identified through homology modelling; Bukrinsky JT et al.; We have used the 2.6 A structure of the rabbit sarcoplasmic reticulum Ca(2+)-ATPase isoform 1a, SERCA1a {Toyoshima, C., Nakasako, M., Nomura, H . and Ogawa, H . (2000) Nature 405, 647-655}, to build models by homology modelling of two plasma membrane (PM) H(+)-ATPases, Arabidopsis thaliana AHA2 and Saccharomyces cerevisiae PMA1 . We propose that in both yeast and plant PM H(+)-ATPases a strictly conserved aspartate in transmembrane segment (M)6 (D684(AHA2)/D730(PMA1)), and three backbone carbonyls in M4 (I282(AHA2)/I331(PMA1), G283(AHA2)/I332(PMA1) and I285(AHA2)/V334(PMA1)) comprise a binding site for H3O(+), suggesting a previously unknown mechanism for transport of protons . Comparison with the structure of the SERCA1a made it feasible to suggest a possible receptor region for the C-terminal auto-inhibitory domain extending from the phosphorylation and anchor domains into the transmembrane region.

FEBS Lett, 2001 Apr 6, 494(1-2), 1 - 5
The rotor in the membrane of the ATP synthase and relatives; Arechaga I et al.; In recent years, structural information on the F(1) sector of the ATP synthase has provided an insight into the molecular mechanism of ATP catalysis . The structure strongly supports the proposal that the ATP synthase works as a rotary molecular motor . Insights into the membrane domain have just started to emerge but more detailed structural information is needed if the molecular mechanism of proton translocation coupled to ATP synthesis is to be understood . This review will focus mainly on the ion translocating rotor in the membrane domain of the F-type ATPase, and the related vacuolar and archaeal relatives.

J Clin Endocrinol Metab, 2001 Apr, 86(4), 1539 - 44
Novel assay for determination of androgen bioactivity in human serum; Raivio T et al.; We have developed a mammalian cell (COS-1) bioassay, which can measure androgen bioactivity directly from a small amount (10 microL) of human serum . The recombinant assay is based on androgen-dependent interaction between the ligand-binding domain and the N-terminal region of the androgen receptor (AR), which were fused to Gal4 DNA-binding domain of Saccharomyces cerevisiae and transcriptional activation domain of herpes simplex VP16 protein, respectively . The interaction is amplified by coexpression of AR-interacting protein 3 in the cells . The reporter plasmid contains 5 Gal4-binding sites upstream of the luciferase gene; luciferase activity in cell lysates is derived from androgen bioactivity in human serum . Saturating concentration of testosterone in FCS induced more than 700-fold induction in relative luciferase activity . The sensitivity was less than 1.0 nmol/L testosterone in FCS . The intra- and interassay coefficients of variation were 8.3% and 21%, respectively . Interaction between the AR termini was blocked by nonsteroidal antiandrogens, and the assay exhibited minimal cross-reactivity with 17 beta-estradiol . Serum androgen bioactivity was studied in 23 boys (13.9--16.8 yr old) with constitutional delay of puberty and in 9 prepubertal boys with cryptorchidism (1.0--6.4 yr old) . Androgen bioactivity was detectable in 15 boys with constitutional delay of puberty and in all boys with cryptorchidism during treatment with human CG (range, 1.0-14.5 nmol/L testosterone equivalents) . Serum androgen bioactivity measured by the bioassay correlated strongly with serum testosterone concentration (r = 0.93, P < 0.0001, n = 22) but not to 5 alpha-dihydrotestosterone, dehydroepiandrosterone, or androstenedione levels . We conclude that our novel bioassay enables quantitation of mammalian cell response to bioactive androgens in human serum, even in pediatric patients with relatively low androgen levels.

J Biol Chem, 2001 Apr 27, 276(17), 14257 - 63 Epub 2001 Jan 31.
N-methyl-D-aspartate receptors regulate a group of transiently expressed genes in the developing brain; Sugiura N et al.; Mammalian brain development requires the transmission of electrical signals between neurons via the N-methyl-d-aspartate (NMDA) class of glutamate receptors . However, little is known about how NMDA receptors carry out this role . Here we report the first genes shown to be regulated by physiological levels of NMDA receptor function in developing neurons in vivo: NMDA receptor-regulated gene 1 (NARG1), NARG2, and NARG3 . These genes share several striking regulatory features . All three are expressed at high levels in the neonatal brain in regions of neuronal proliferation and migration, are dramatically down-regulated during early postnatal development, and are down-regulated by NMDA receptor function . NARG2 and NARG3 appear to be novel, while NARG1 is the mammalian homologue of a yeast N-terminal acetyltransferase that regulates entry into the G(o) phase of the cell cycle . The results suggest that highly specific NMDA receptor-dependent regulation of gene expression plays an important role in the transition from proliferation of neuronal precursors to differentiation of neurons.

J Biol Chem, 2001 May 11, 276(19), 16520 - 7 Epub 2001 Jan 31.
5-Lipoxygenase interacts with coactosin-like protein; Provost P et al.; We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait . In this report, we demonstrate a direct interaction between 5LO and CLP . 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner . Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates . In reciprocal experiments, 5LO was detected in CLP immunoprecipitates . Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca(2+)-independent manner . Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding . In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments . However, no F-actin-CLP.5LO ternary complex was observed . In contrast, 5LO appeared to compete with F-actin for the binding of CLP . Moreover, 5LO was found to interfere with actin polymerization . Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics.

Biochemistry, 2001 Mar 27, 40(12), 3657 - 65
Direct measurement of acylenzyme hydrolysis demonstrates rate-limiting deacylation in cleavage of physiological sequences by the processing protease Kex2; Rockwell NC et al.; Saccharomyces cerevisiae Kex2 protease is the prototype for the family of eukaryotic proprotein convertases that includes furin, PC1/3, and PC2 . These enzymes belong to the subtilase superfamily of serine proteases and are distinguished from degradative subtilisins by structural features and by their much more stringent substrate specificity . Pre-steady-state studies have shown that both Kex2 and furin exhibit an initial burst of 7-amino-4-methylcoumarin release in cleavage of peptidyl methylcoumarinamide substrates that are based on physiological cleavage sites . Thus, in cleavage of such substrates, formation of the acylenzyme intermediate is fast relative to some later step (deacylation or N-terminal product release) . This behavior is significant, because Kex2 also exhibits burst kinetics in cleavage of peptide bonds . k(cat) for cleavage of a tetrapeptidyl methylcoumarinamide substrate based on the physiological yeast substrate pro-alpha-factor exhibits a weak solvent isotope effect, but neither this isotope effect nor temperature dependence studies with this substrate conclusively identify the rate-limiting step for Kex2 cleavage of this substrate . We therefore developed an assay to measure deacylation directly by pulse-chase incorporation of H(2)(18)O in a rapid-quenched-flow mixer followed by mass spectrometric quantitation . The results given by this assay rule out rate-limiting product release for cleavage of this substrate by Kex2 . These experiments demonstrate that cleavage of the acylenzyme ester bond, as opposed to either the initial attack on the amide bond or product release, is rate-limiting for the action of Kex2 at physiological sequences . This work demonstrates a fundamental difference in the catalytic strategy of proprotein processing enzymes and degradative subtilisins.

Biochemistry, 2001 Mar 27, 40(12), 3544 - 52
Energetics of coiled coil folding: the nature of the transition states; Bosshard HR et al.; Coiled coils are simple models for studying the association of two polypeptide chains to form a folded protein . Previous work has shown that the folding of a coiled coil can be described by a two-state transition between two unfolded monomeric peptide chains and a folded coiled coil dimer . Here we report the thermodynamic activation parameters for the folding and unfolding of two unrelated coiled coils: C62GCN4 and A(2) . C62GCN4 corresponds to the 62 C-terminal residues of yeast transcription factor GCN4 . The peptide forms a dimeric coiled coil through its 33 C-terminal residues . A(2) is a designed 30-residue dimeric coiled coil whose folding is induced by low pH {Durr, E., Jelesarov, I., and Bosshard, H . R . (1999) Biochemistry 38, 870-880} . Folding and unfolding were assessed under identical native buffer conditions so that the microscopic reversibility applied and the transition state was the same for folding and unfolding . The time course of folding was followed from the self-quenching of a C-terminal fluorescent label (Texas Red) . The overall folding of both peptides is enthalpy-driven and opposed by a loss of entropy . The main energetic changes occur after the system has passed the transition state . In the folding of C62GCN4, only 10-20% of the heat capacity change is attained between the monomeric state and the dimeric transition state . For coiled coil A(2), the fractional heat capacity change preceding the transition state is 30-40% . The results indicate that the activated states of folding of coiled coils are not well structured and differ considerably from the folded coiled coil conformation . These findings are in agreement with a rate-limiting transition state in which the coiled coil helices and the hydrophobic coiled coil interface are poorly developed.

EMBO J, 2001 Apr 17, 20(8), 2028 - 40
MOT1-catalyzed TBP-DNA disruption: uncoupling DNA conformational change and role of upstream DNA; Darst RP et al.; SNF2/SWI2-related ATPases employ ATP hydrolysis to disrupt protein-DNA interactions, but how ATP hydrolysis is coupled to disruption is not understood . Here we examine the mechanism of action of MOT1, a yeast SNF2/SWI2-related ATPase that uses ATP hydrolysis to remove TATA binding protein (TBP) from DNA . MOT1 function requires a 17 bp DNA 'handle' upstream of the TATA box, which must be double stranded . Remarkably, MOT1-catalyzed disruption of TBP-DNA does not appear to require DNA strand separation, DNA bending or twisting of the DNA helix . Thus, TBP-DNA disruption is accomplished in a reaction apparently not driven by a change in DNA structure . MOT1 action is supported by DNA templates in which the handle is connected to the TATA box via single-stranded DNA, indicating that the upstream duplex DNA can be conformationally uncoupled from the TATA box . Combining these results with proposed similarities between SNF2/SWI2 ATPases and helicases, we suggest that MOT1 uses ATP hydrolysis to translocate along the handle and thereby disrupt interactions between TBP and DNA.

EMBO J, 2001 Apr 17, 20(8), 1984 - 92
Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity; Soutoglou E et al.; CREB-binding protein (CBP) and CBP-associated factor (P/CAF) are coactivators possessing an intrinsic histone acetyltransferase (HAT) activity . They are positioned at promoter regions via association with sequence-specific DNA-binding factors and stimulate transcription in a gene-specific manner . The current view suggests that coactivator function depends mainly on the strength and specificity of transcription factor-coactivator interactions . Here we show that two dominant-negative mutants of hepatocyte nuclear factor-1alpha (HNF-1alpha), P447L and P519L, occurring in maturity onset diabetes of the young (MODY3) patients, exhibit paradoxically stronger interactions than the wild-type protein with either CBP or P/CAF . However, CBP and P/CAF recruited by these mutants lack HAT activity . In contrast, wild-type HNF-1alpha and other transcription factors, such as Sp1 or HNF-4, stimulated the HAT activity of CBP . The results suggest a more dynamic role for DNA-binding proteins in the transcription process than was considered previously . They are not only required for the recruitment of coactivators to the promoter but they may also modulate their enzymatic activity.

Cancer Lett, 2001 May 10, 166(1), 55 - 64
Paradoxical effects of trichostatin A: inhibition of NF-Y-associated histone acetyltransferase activity, phosphorylation of hGCN5 and downregulation of cyclin A and B1 mRNA; Nair AR et al.; Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), is widely used to study the role of histone acetylation in gene expression, since genes that use histone acetylation as a means of regulating expression may be up regulated when TSA is added . In this study, however, we show that TSA has an unexpected paradoxical effect leading to inhibition of NF-Y-associated histone acetyl transferase (HAT) activity and phosphorylation of the HAT, hGCN5 . TSA treatment of cells resulted in diminished levels of NF-Y-associated HAT activity without changes in NF-Y(A) amount . hGCN5 is one of the HATs known to associate with NF-Y . The association of hGCN5 with NF-Y was not altered by TSA treatment . The enzymatic activity of hGCN5 is known to be inhibited by phosphorylation . TSA treatment of Hela cells resulted in phosphorylation of hGCN5 . Exposure of the NF-Y immunoprecipitates from TSA-treated cells to a phosphatase resulted in enhanced HAT activity . We have also shown that the mRNA levels of several genes, cyclin B1 and cyclin A, are downregulated by TSA; these effects do not require protein synthesis and the downregulation of cyclin B1 by TSA occurs through transcription . These results suggest that TSA can have contradictory effects, on one hand stimulating HAT activity in general by inhibition of HDACs, but also resulting in inhibition of NF-Y-associated HAT activity and phosphorylation of hGCN5.

Brain Res Mol Brain Res, 2001 Mar 31, 88(1-2), 124 - 34
Localization of mRNAs for six ARFs (ADP-ribosylation factors) in the brain of developing and adult rats and changes in the expression in the hypoglossal nucleus after its axotomy; Suzuki I et al.; ADP-ribosylation factors (ARFs) play crucial roles in the intracellular vesicular transport and in regulation of phospholipid-modifying enzyme activities and cytoskeletons . Using in situ hybridization histochemistry, the gene expression for six isoforms of ARF was examined during normal development of rats and in the hypoglossal nucleus following its axotomy . In the embryonic brain, the expression for ARF-1, -4, -5, -6 mRNAs was distinct in the ventricular germinal zone while that for ARF-3, -4, -5 in the mantle zone . In early postnatal brain, the expression for six ARFs was seen widely in various loci of the gray matter with different intensity, and the expression of ARF-4, -5, -6 mRNAs was evident in the cerebellar external granule cell layer . In the adult brain, the gene expression for all ARF isoforms decreased more or less in most gray matters and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex . The expression levels of ARF-2 and -4 mRNAs in affected hypoglossal nucleus increased after 24 h up to 7 days following axotomy of the hypoglossal nerve, while no such changes were seen in the expression levels for the other ARFs . The present findings suggest that ARFs are differentially involved in some processes essential to nerve regeneration as well as neuronal differentiation and maturation.

Mol Biol Cell, 2001 Apr, 12(4), 1093 - 101
O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation; Harty C et al.; Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes . It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins . We asked whether misfolded secretory proteins are covalently modified in the ER before export . We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by protein O-mannosyl transferase 2 (Pmt2p) . O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane . Deletion of PMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells . In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen . Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage . We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.

Mol Biol Cell, 2001 Apr, 12(4), 1035 - 45
The ADP ribosylation factor-nucleotide exchange factors Gea1p and Gea2p have overlapping, but not redundant functions in retrograde transport from the Golgi to the endoplasmic reticulum; Spang A et al.; The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors . Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p . We identified GEA2 as a multicopy suppressor of a sec21-3 temperature-sensitive mutant . SEC21 encodes the gamma-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat . GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable . Conditional mutants defective in both GEA1 and GEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions . The two genes do not serve completely overlapping functions because a Deltagea1 Deltaarf1 mutant is not more sickly than a Deltaarf1 strain, whereas Deltagea2 Deltaarf1 is inviable . Biochemical experiments revealed similar distributions and activities for the two proteins . Gea1p and Gea2p exist both in membrane-bound and in soluble forms . The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum . In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

J Biol Chem, 2001 Jun 22, 276(25), 22788 - 96 Epub 2001 Apr 09.
Activation and functional characterization of the mosaic receptor SorLA/LR11; Jacobsen L et al.; We previously isolated and sequenced the approximately 250-kDa type 1 receptor sorLA/LR11, a mosaic protein with elements characterizing the Vps10p domain receptor family as well as the low density lipoprotein receptor family . The N terminus of the Vps10p domain comprises a consensus sequence for cleavage by furin ((50)RRKR(53)) that precedes a truncation found in sorLA isolated from human brain . Here we show that sorLA, like sortilin-1/neurotensin receptor-3, whose lumenal domain consists of a Vps10p domain only, is synthesized as a proreceptor that is cleaved by furin in late Golgi compartments . We show that the truncation conditions the Vps10p domain for propeptide inhibitable binding of neuropeptides and the receptor-associated protein . We further demonstrate that avid binding of the receptor-associated protein, apolipoprotein E, and lipoprotein lipase not inhibited by propeptide occurs to sites located in other lumenal domains . In transfected cells, about 10% of full-length sorLA were expressed on the cell surface capable of mediating endocytosis . However, the major pool of receptors was found in late Golgi compartments, suggesting possible interaction with newly synthesized ligands . The results show that sorLA, following activation by truncation, binds multiple ligands and may mediate both endocytosis and sorting.

J Biol Chem, 2001 Jun 22, 276(25), 22810 - 8 Epub 2001 Apr 05.
Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery; Suvorova ES et al.; The roles of the components of the Sec34p protein complex in intracellular membrane trafficking, first identified in the yeast Saccharomyces cerevisiae, have yet to be characterized in higher eukaryotes . We cloned a human cDNA whose predicted amino acid sequence showed 41% similarity to yeast Sec34p with homology throughout the entire coding region . Affinity-purified antibodies raised against the human SEC34 protein (hSec34p) recognized a cellular protein of 94 kDa in both soluble and membrane fractions . Like yeast Sec34p, cytosolic hSec34p migrated with an apparent molecular mass of 300 kDa on a glycerol velocity gradient, suggesting that it is part of a protein complex . Immunofluorescence microscopy localized hSec34p to the Golgi compartment in cells of all species examined, where it co-localized well with the cis/medial Golgi marker membrin and partially co-localized with cis-Golgi network marker p115 and trans-Golgi marker TGN38 . The co-localization with membrin was maintained at 15 degrees C and after microtubule depolymerization with nocodazole . During transport of the tsO45 vesicular stomatitis virus G protein through the Golgi, there was significant overlap with the hSec34p compartment . Green fluorescent protein-hSec34 expressed in HeLa cells was restricted to Golgi cisternae, and its membrane association was sensitive to brefeldin A treatment . Taken together, our findings indicate that hSec34p is part of a peripheral membrane protein complex localized on cis/medial Golgi cisternae where it may participate in tethering intra-Golgi transport vesicles.

J Immunol Methods, 2001 May 1, 251(1-2), 53 - 61
A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera; Jafri F et al.; Sera from patients with systemic lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease are frequently characterized by the presence of high levels of autoantibodies directed against linked sets of nuclear proteins . One of these autoantigen systems is made up of Ku and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), proteins that are essential for double-strand DNA break repair and for the related process of V(D)J recombination . Ku and DNA-PKcs bind avidly to DNA ends in vivo and in vitro and form an active protein kinase complex . One hypothesis is that this assembled nucleoprotein particle, rather than its component proteins, is a primary trigger for the autoimmune response and thus a major target for the resulting autoantibodies . To screen for particle-specific antibodies, we developed an assay in which the fully native nucleoprotein particle is reconstituted in vitro and is tethered to the surface of an ELISA plate via a streptavidin-biotin linkage . These particles are recognized efficiently by monoclonal antibodies and by autoantibodies present in patient sera . The assay may detect a broader spectrum of epitopes than a conventional ELISA in which Ku and DNA-PKcs are adsorbed directly to a plastic surface . The method will be advantageous for high-throughput screening for antibodies and other ligands that bind the assembled DNA-dependent protein kinase complex.

Semin Cell Dev Biol, 2001 Apr, 12(2), 183 - 91
Phosphatidylinositol transfer proteins couple lipid transport to phosphoinositide synthesis; Cockcroft S; Phosphatidylinositol transfer proteins (PITPs) are lipid binding proteins that can catalyse the transfer of phosphatidylinositol (PI) from membranes enriched in PI to PI-deficient membranes . Three soluble forms of PITP of 35--38 kDa (PITP alpha, PITP beta and rdgB beta) and two larger integral proteins of 160 kDa (rdgB alpha I and II), which contain a PITP domain, are found in mammalian cells . PITPs are intimately associated with the compartmentalised synthesis of different phosphorylated inositol lipids . PI is the primary inositol lipid that is synthesised at the endoplasmic reticulum and is further phosphorylated in distinct membrane compartments by many specific lipid kinases to generate seven phosphorylated inositol lipids which are required for both signalling and for membrane traffic . PITPs play essential roles in both signalling via phospholipase C and phosphoinositide 3-kinases and in multiple aspects of membrane traffic including regulated exocytosis and vesicle biogenesis .

J Mol Biol, 2001 Apr 13, 307(5), 1427 - 50
Socket: a program for identifying and analysing coiled-coil motifs within protein structures; Walshaw J et al.; The coiled coil is arguably the simplest protein-structure motif and probably the most ubiquitous facilitator of protein-protein interactions . Coiled coils comprise two or more alpha-helices that wind around each other to form "supercoils" . The hallmark of most coiled coils is a regular sequence pattern known as the heptad repeat . Despite this apparent simplicity and relatedness at the sequence level, coiled coils display a considerable degree of structural diversity: the helices may be arranged parallel or anti-parallel and may form a variety of oligomer states . To aid studies of coiled coils, we developed SOCKET, a computer program to identify these motifs automatically in protein structures . We used SOCKET to gather a set of unambiguous coiled-coil structures from the RCSB Protein Data Bank . Rather than searching for sequence features, the algorithm recognises the characteristic knobs-into-holes side-chain packing of coiled coils; this proved to be straightforward to implement and was able to distinguish coiled coils from the great majority of helix-helix packing arrangements observed in globular domains . SOCKET unambiguously defines coiled-coil helix boundaries, oligomerisation states and helix orientations, and also assigns heptad registers . Structures retrieved from the Protein Data Bank included parallel and anti-parallel variants of two, three and four-stranded coiled coils, one example of a parallel pentamer and a small number of structures that extend the classical description of a coiled coil . We anticipate that our structural database and the associated sequence data that we have gathered will be of use in identifying principles for coiled-coil assembly, prediction and design . To illustrate this we give examples of sequence and structural analyses of the structures that are possible using the new data bases, and we present amino acid profiles for the heptad repeats of different motifs .

J Mol Biol, 2001 Apr 13, 307(5), 1247 - 60
Supporting the structural basis of prion strains: induction and identification of {PSI} variants; King CY; The {PSI} genetic element, which enhances the nonsense suppression efficiency in the yeast Saccharomyces cerevisiae, is thought to be amyloid-like aggregates of the Sup35 protein, and to self-propagate by a prion-like mechanism . Analogous to strains of the mammalian prion, variants of {PSI}, with different nonsense suppression efficiencies and mitotic stabilities, can be isolated from the same yeast genetic background . In the framework of the "protein-only" hypothesis, variants of prion are assumed to be distinct conformers of the same prion polypeptide . This study aims to provide further support for the structural basis of {PSI} variation . Three variants of {PSI} were induced and distinguished by a panel of 11 single point mutations of the Sup35 protein . The variant phenotypes are intrinsically associated with {PSI} elements, presumably structurally different amyloids, rather than produced from variations in the genetic background . Differential incorporation to {PSI} variants of a Sup35 point mutation as well as N and C-terminally truncated Sup35 fragments is further demonstrated in vivo, suggesting that distinct patches of amino acid residues are involved in the assembly of {PSI} variants . These results establish a method for {PSI} variant-typing and indicate that heritable variations of amyloid structures can be derived from the same polypeptide .

Immunol Rev, 2001 Feb, 179, 156 - 62
Regulation of eosinophil and neutrophil apoptosis--similarities and differences; Simon HU; Apoptosis is the most common form of physiologic cell death and a necessary process to maintain cell numbers in multicellular organisms . In many chronic inflammatory diseases, reduced cell death of different types of granulocytes is one important mechanism for cell accumulation . Granulocytes are constantly produced in large amounts in the bone marrow and the same numbers die, under normal circumstances, within a defined time period . Changing the rate of apoptosis rapidly changes cell numbers in such systems . Overexpression of IL-5 appears to be crucial for delaying eosinophil apoptosis in many allergic disorders, whereas overexpression of GM-CSF and G-CSF is associated with suppression of neutrophil apoptosis in bacterial and non-bacterial inflammations . Cytokine withdrawal leads to the induction of apoptosis both in vitro and in vivo . In contrast to the role of survival cytokines, little is known about the role of death factors and their receptors in the regulation of granulocyte apoptosis . Recent observations suggest a role for mitochondria in both eosinophil and neutrophil apoptosis, although the mechanisms that trigger mitochondria to release pro-apoptotic factors remain to be determined . Besides similarities, there are differences in the regulation of apoptosis between these granulocyte subtypes that include both expression and function of Bcl-2 and caspase family members . The identification of differences in the apoptosis regulation may help to define new molecular targets that allow specific induction of either eosinophil or neutrophil apoptosis by pharmacological means.

J Neurobiol, 2001 May, 47(2), 81 - 92
Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons; Kitamoto T; Behavior is a manifestation of temporally and spatially defined neuronal activities . To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit . I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila . In this method, a temperature-sensitive allele of shibire (shi(ts1)) is overexpressed in neuronal subsets using the GAL4/UAS system . Because the shi gene product is essential for synaptic vesicle recycling, and shi(ts1) is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi(ts1) expressing neurons . When shi(ts1) expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30 degrees C . This temperature-induced paralysis was reversible . After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min . When shi(ts1) was expressed in photoreceptor cells, adults and larvae exhibited temperature-dependent blindness . These observations show that the GAL4/UAS system can be used to express shi(ts1) in a specific subset of neurons to cause temperature-dependent changes in behavior . Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals .

Biotechnol Bioeng, 2000-2001, 71(3), 217 - 22
Detection of toxic chemicals with high sensitivity by measuring the quantity of induced P450 mRNAs based on surface plasmon resonance; Oyama M et al.; In this study we describe a novel sensor system to detect toxic chemicals based on measurement of the quantity of Saccharomyces cerevisiae P450 mRNAs induced by them . Detection was conducted using a flow-injection-type sensor system based on surface plasmon resonance (SPR) . The DNA and peptide nucleic acid (PNA) probes containing a complementary sequence to a part of P450 mRNA were immobilized on the sensor chip and the P450 mRNAs hybridized to the probes were quantified . We succeeded in detecting 10 ng/L (10 ppt) of atrazine using both DNA and PNA probes . Using this sensor system, we were able to detect bisphenol A in addition to atrazine . Furthermore, we achieved higher sensitivity by amplifying the target P450 mRNA based on nucleic acid sequence-based amplification (NASBA) . This method allows for sensitive, rapid, and easy detection of some toxic chemicals .

Genetics, 2001 Apr, 157(4), 1639 - 48
Transgenic analysis of the Smad family of TGF-beta signal transducers in Drosophila melanogaster suggests new roles and new interactions between family members; Marquez RM et al.; Smad signal transducers are required for transforming growth factor-beta-mediated developmental events in many organisms including humans . However, the roles of individual human Smad genes (hSmads) in development are largely unknown . Our hypothesis is that an hSmad performs developmental roles analogous to those of the most similar Drosophila Smad gene (dSmad) . We expressed six hSmad and four dSmad transgenes in Drosophila melanogaster using the Gal4/UAS system and compared their phenotypes . Phylogenetically related human and Drosophila Smads induced similar phenotypes supporting the hypothesis . In contrast, two nearly identical hSmads generated distinct phenotypes . When expressed in wing imaginal disks, hSmad2 induced oversize wings while hSmad3 induced cell death . This observation suggests that a very small number of amino acid differences, between Smads in the same species, confer distinct developmental roles . Our observations also suggest new roles for the dSmads, Med and Dad, in dActivin signaling and potential interactions between these family members . Overall, the study demonstrates that transgenic methods in Drosophila can provide new information about non-Drosophila members of developmentally important multigene families.

Gene, 2001 Mar 21, 266(1-2), 45 - 56
Identification and characterization of the human MOG1 gene; Marfatia KA et al.; Many of the proteins that mediate transport into and out of the nucleus have been structurally and functionally conserved throughout evolution . Here we describe the sequence and characterization of the human MOG1 gene . The MOG1 gene was originally identified in Saccharomyces cerevisiae as a multi-copy suppressor of conditional alleles of the yeast nuclear transport factor, GSP1 (scRan) (Oki and Nishimoto (1998) Proc . Natl . Acad . Sci . USA 95, 15388-15393) . A search of the expressed sequence tag database identified a putative human protein that is 29% identical and 47% similar to the yeast protein . Our experiments demonstrate that the human MOG1 message is expressed in a variety of tissue samples . Several experiments indicate that the human MOG1 protein binds to both yeast and human Ran suggesting functional conservation between the yeast and human MOG1 proteins . Furthermore, hMOG1a, like scMOG1, is localized throughout the cell but is concentrated within the nucleus . Consistent with these findings, hMOG1a can partially complement the growth defect present in yeast MOG1 deletion cells . Taken together, our findings suggest that MOG1 is an evolutionarily conserved Ran binding protein that could play a role in regulating nuclear protein trafficking.

Cell, 2001 Mar 23, 104(6), 839 - 47
Modulation of a transcription factor counteracts heterochromatic gene silencing in Drosophila; Ahmad K et al.; Variegation is a common feature of gene silencing phenomena, yet the basis for stochastic on/off expression is unknown . We used a conditional system that allows probing of heterochromatin at a reporter GFP gene by altering GAL4 transcription factor levels during Drosophila eye development . Surprisingly, the frequency of gene silencing is exquisitely sensitive to GAL4 levels, as though binding site occupancy affects the silenced state . The silent state is plastic, as spontaneous derepression occasionally occurs in both mitotically active and differentiating cells . By simultaneously assaying expression of a nearby gene, we further show that the size of an activated region within heterochromatin is small . We propose that variegation occurs because heterochromatin inhibits the transient exposure of factor binding sites.

Cell, 2001 Mar 23, 104(6), 817 - 27
Histone acetyltransferase complexes stabilize swi/snf binding to promoter nucleosomes; Hassan AH et al.; To investigate the function of SWI/SNF in site-specific chromatin remodeling at promoters, we have used a purified system to analyze its distribution, function, and retention following recruitment by a sequence-specific transcription activator . Activator recruitment of SWI/SNF bound the complex to promoter proximal nucleosomes and led to localized nucleosome disruption . However, retention of SWI/SNF on the promoter required either the continued binding of the transcription activator or acetylated histones . Histone acetylation by either the SAGA or NuA4 HAT complexes increased the retention of SWI/SNF on the promoter . These data illustrate a functional link between HAT complexes and the SWI/SNF chromatin remodeling complex and provide a mechanistic basis for the ordered recruitment of these complexes.

Development, 2001 May, 128(9), 1697 - 707
Orc mutants arrest in metaphase with abnormally condensed chromosomes; Pflumm MF et al.; The origin recognition complex (ORC) is a six subunit complex required for eukaryotic DNA replication initiation and for silencing of the heterochromatic mating type loci in Saccharomyces cerevisiae . Our discovery of the Drosophila ORC complex concentrated in the centric heterochromatin of mitotic cells in the early embryo and its interactions with heterochromatin protein 1 (HP-1) lead us to speculate that ORC may play some general role in chromosomal folding . To explore the role of ORC in chromosomal condensation, we have identified a mutant of subunit 5 of the Drosophila melanogaster origin recognition complex (Orc5) and have characterized the phenotypes of both the Orc5 and the previously identified Orc2 mutant, k43 . Both Orc mutants died at late larval stages and surprisingly, despite a reduced number of S-phase cells, an increased fraction of cells were also detected in mitosis . For this latter population of cells, Orc mutants arrest in a defective metaphase with shorter and thicker chromosomes that fail to align at the metaphase plate within a poorly assembled mitotic spindle . In addition, sister chromatid cohesion was frequently lost . PCNA and MCM4 mutants had similar phenotypes to Orc mutants . We propose that DNA replication defects trigger the mitotic arrest, due to the fact that frequent fragmentation was observed . Thus, cells have a mitotic checkpoint that senses chromosome integrity . These studies also suggest that the density of functional replication origins and completion of S phase are requirements for proper chromosomal condensation.

Development, 2001 May, 128(9), 1559 - 72
Mesodermal patterning defect in mice lacking the Ste20 NCK interacting kinase (NIK); Xue Y et al.; We have previously shown that the Drosophila Ste20 kinase encoded by misshapen (msn) is an essential gene in Drosophila development . msn function is required to activate the Drosophila c-Jun N-terminal kinase (JNK), basket (Bsk), to promote dorsal closure of the Drosophila embryo . Later in development, msn expression is required in photoreceptors in order for their axons to project normally . A mammalian homolog of msn, the NCK-interacting kinase (NIK) (recently renamed to mitogen-activated protein kinase kinase kinase kinase 4; Map4k4), has been shown to activate JNK and to bind the SH3 domains of the SH2/SH3 adapter NCK . To determine whether NIK also plays an essential role in mammalian development, we created mice deficient in NIK by homologous recombination at the Nik gene . Nik(-/-) mice die postgastrulation between embryonic day (E) 9.5 and E10.5 . The most striking phenotype in Nik(-/-) embryos is the failure of mesodermal and endodermal cells that arise from the anterior end of the primitive streak (PS) to migrate to their correct location . As a result Nik(-/- )embryos fail to develop somites or a hindgut and are truncated posteriorly . Interestingly, chimeric analysis demonstrated that NIK has a cell nonautonomous function in stimulating migration of presomitic mesodermal cells away from the PS and a second cell autonomous function in stimulating the differentiation of presomitic mesoderm into dermomyotome . These findings indicate that despite the large number of Ste20 kinases in mammalian cells, members of this family play essential nonredundant function in regulating specific signaling pathways . In addition, these studies provide evidence that the signaling pathways regulated by these kinases are diverse and not limited to the activation of JNK because mesodermal and somite development are not perturbed in JNK1-, and JNK2-deficient mice.

Planta, 2001 Feb, 212(3), 466 - 9
Cortical actin filaments in guard cells respond differently to abscisic acid in wild-type and abi1-1 mutant Arabidopsis; Eun SO et al.; Cortical actin filaments in guard cells of Commelina communis L . show signal-specific organization during stomatal movements {S.-O . Eun and Y . Lee (1997) Plant Physiol 115: 1491-1498; S.-O . Eun and Y . Lee (2000) Planta 210: 1014-1017} . To study the roles of actin in signal transduction, it is advantageous to use Arabidopsis thaliana (L.) Heynh., an excellent model plant with numerous well-characterized mutants . Using an immunolocalization technique, we found that actin deployments in guard cells of A . thaliana were basically identical to those in C . communis: actin proteins were assembled into radial filaments under illumination, and were disassembled by ABA . In addition, we examined actin organization in an ABA-insensitive mutant (abi1-1) to test the involvement of protein phosphatase 2C (PP2C) in the control of actin structure . A clear difference was observed after ABA treatment, namely, neither stomatal closing nor depolymerization of actin filaments was observed in guard cells of the mutant . Our results indicate that PP2C participates in ABA-induced actin changes in guard cells.

Plant Mol Biol, 2001 Jan, 45(2), 201 - 14
Characterization of a novel class of plant homeodomain proteins that bind to the C4 phosphoenolpyruvate carboxylase gene of Flaveria trinervia; Windhovel A et al.; We are interested in the regulatory mechanisms responsible for the mesophyll-specific expression of C4 phosphoenolpyruvate carboxylase (PEPCase) . A one-hybrid screen resulted in the cloning of four different members of a novel class of plant homeodomain proteins, which are most likely involved in the mesophyll-specific expression of the C4 PEPCase gene in C4 species of the genus Flaveria . Inspection of the homeodomains of the four proteins reveals that they share many common features with homeodomains described so far, but there are also significant differences . Interestingly, this class of homeodomain proteins occurs also in Arabidopsis thaliana and other C3 plants . One-hybrid experiments as well as in vitro DNA binding studies confirmed that these novel homeodomain proteins specifically interact with the proximal region of the C4 PEPCase gene . The N-terminal domains of the homeodomain proteins contain highly conserved sequence motifs . Two-hybrid experiments show that these motifs are sufficient to confer homo- or heterodimer formation between the proteins . Mutagenesis of conserved cysteine residues within the dimerization domain indicates that these residues are essential for dimer formation . Therefore, we designate this novel class of homeobox proteins ZF-HD, for zinc finger homeodomain protein . Our data suggest that the ZF-HD class of homeodomain proteins may be involved in the establishment of the characteristic expression pattern of the C4 PEPCase gene.

Anal Chem, 2001 Mar 1, 73(5), 978 - 86
Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer; Griffin TJ et al.; We describe an approach to the quantitative analysis of complex protein mixtures using a MALDI quadrupole time-of-flight (MALDI QqTOF) mass spectrometer and isotope coded affinity tag reagents (Gygi, S . P.; et al . Nat . Biotechnol . 1999, 17, 994-9.) . Proteins in mixtures are first labeled on cysteinyl residues using an isotope coded affinity tag reagent, the proteins are enzymatically digested, and the labeled peptides are purified using a multidimensional separation procedure, with the last step being the elution of the labeled peptides from a microcapillary reversed-phase liquid chromatography column directly onto a MALDI sample target . After addition of matrix, the sample spots are analyzed using a MALDI QqTOF mass spectrometer, by first obtaining a mass spectrum of the peptides in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptides, followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptides for protein identification . The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources.

Electrophoresis, 2001 Jan, 22(2), 289 - 93
Indirect micromanipulation of single molecules in water-in-oil emulsion; Katsura S et al.; Based on real-time observation and micromanipulation, analytical methods for single DNA molecules have been under development for some time . Precise manipulation, however, is still difficult because single molecules are too small for conventional techniques . We have developed a chemical reaction system that uses water droplets in oil as containers of materials . The water droplets can be manipulated by optical force . The manipulation of the water droplets permits the fusion of two selected droplets . This process corresponds to mixing of different samples . We designate this system as "w/o (water-in-oil emulsion) microreactor system", and each droplet can be thought of as a "microreactor" . In this system, single molecules can be manipulated readily, as a molecule can be contained in a microm-sized microreactor . The microreactor utilizes extremely small quantities of samples, therefore, reactions are rapid, as diffusion times in the microreactor are very short . The manipulation technique of the microreactors based on optical force has been applied to induce fusion between microreactors loaded with DNA and YOYO, a fluorescent dye that binds to DNA . This fusion induced a rapid binding of YOYO.

Mol Cell Biol, 2001 May, 21(9), 3096 - 104
High-mobility-group proteins NHP6A and NHP6B participate in activation of the RNA polymerase III SNR6 gene; Lopez S et al.; Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC . Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes . Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro . The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6ADelta nhp6BDelta double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6ADelta nhp6BDelta cells at 37 degrees C . In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6ADelta nhp6BDelta cells or reconstituted transcription systems . Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay . These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxillary factors are required for the optimal transcription of at least some specific Pol III genes.

J Biol Chem, 2001 Jun 15, 276(24), 21317 - 24 Epub 2001 Apr 03.
Ikappa b-alpha, the NF-kappa B inhibitory subunit, interacts with ANT, the mitochondrial ATP/ADP translocator; Bottero V et al.; The transcription factor NF-kappaB regulates a wide set of genes involved in the establishment of many cellular processes that control cell activation, proliferation, and apoptosis . IkappaB inhibitory subunits integrate NF-kappaB activation signals through phosphorylation and ubiquitination of its N-terminal domain . Using the two-hybrid system in yeast, we searched for IkappaB-alpha N-terminal domain interactors and therefore potential NF-kappaB regulators . An interaction of IkappaB-alpha with the mitochondrial ATP/ADP translocator ANT was detected in yeast and confirmed in glutathione S-transferase pull-down assays and co-precipitation experiments in transfected cells . Subcellular cell fractionation, resistance to proteinase K treatment, and electron microscopy experiments demonstrated the presence of IkappaB-alpha and associated p65 NF-kappaB in the mitochondrial intermembrane space . IkappaB-alpha.NF-kappaB appeared to be released from mitochondria upon the induction of apoptosis by engagement of the Fas receptor . These data suggest that the mitochondrial IkappaB-alpha.NF-kappaB pool participates in the regulation of apoptosis.

Am J Physiol Cell Physiol, 2001 May, 280(5), C1284 - 92
A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol; Su Z et al.; Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx . While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated . We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels . Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF) . In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations . Furthermore, CRANC channels are also directly activated by diacylglycerol . These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.

Mech Dev, 2001 Apr, 102(1-2), 17 - 32
Homeotic Complex (Hox) gene regulation and homeosis in the mesoderm of the Drosophila melanogaster embryo: the roles of signal transduction and cell autonomous regulation; Miller DF et al.; In this paper we evaluate homeosis and Homeotic Complex (Hox) regulatory hierarchies in the somatic and visceral mesoderm . We demonstrate that both Hox control of signal transduction and cell autonomous regulation are critical for establishing normal Hox expression patterns and the specification of segmental identity and morphology . We present data identifying novel regulatory interactions associated with the segmental register shift in Hox expression domains between the epidermis/somatic mesoderm and visceral mesoderm . A proposed mechanism for the gap between the expression domains of Sex combs reduced (Scr) and Antennapedia (Antp) in the visceral mesoderm is provided . Previously, Hox gene interactions have been shown to occur on multiple levels: direct cross-regulation, competition for binding sites at downstream targets and through indirect feedback involving signal transduction . We find that extrinsic specification of cell fate by signaling can be overridden by Hox protein expression in mesodermal cells and propose the term autonomic dominance for this phenomenon.

Mech Dev, 2001 Apr, 102(1-2), 3 - 16
Cross-regulation of Hox genes in the Drosophila melanogaster embryo; Miller DF et al.; Cross-regulation of Homeotic Complex (Hox) genes by ectopic Hox proteins during the embryonic development of Drosophila melanogaster was examined using Gal4 directed transcriptional regulation . The expression patterns of the endogenous Hox genes were analyzed to identify cross-regulation while ectopic expression patterns and timing were altered using different Gal4 drivers . We provide evidence for tissue specific interactions between various Hox genes and demonstrate the induction of endodermal labial (lab) by ectopically expressed Ultrabithorax outside the visceral mesoderm (VMS) . Similarly, activation and repression of Hox genes in the VMS from outside tissues seems to be mediated by decapentaplegic (dpp) gene activation . Additionally, we find that proboscipedia (pb) is activated in the epidermis by ectopically driven Sex combs reduced (Scr) and Deformed (Dfd); however, mesodermal pb expression is repressed by ectopic Scr in this tissue . Mutant analyses demonstrate that Scr and Dfd regulate pb in their normal domains of expression during embryogenesis . Ectopic Ultrabithorax and Abdominal-A repress only lab and Scr in the central nervous system (CNS) in a timing dependent manner; otherwise, overlapping expression in the CNS in tolerated . A summary of Hox gene cross-regulation by ectopically driven Hox proteins is tabulated for embryogenesis.

Toxicol In Vitro, 2001 Apr, 15(2), 131 - 42
Fluoroquinolones as chemical tools to define a strategy for photogenotoxicity in vitro assessment; Marrot L et al.; Today's lifestyle is often associated with frequent exposure to sunlight, but some xenobiotics used in drugs, cosmetics or food chemicals can produce adverse biological effects when irradiated . In particular, they can increase the risk of photogenotoxicity already due to UV radiation itself . There is thus a need to design appropriate approaches in order to obtain relevant data at the molecular and cellular level in this field . For ethical and practical reasons, in vitro models can be very convenient at least for first evaluation tests . Here, we propose a strategy based on complementary experiments to study the photogenotoxic potential of a compound . The fluoroquinolones BAYy3118 and lomefloxacin were used as standards to demonstrate the performance of each test: photoinduced interaction with supercoiled circular DNA, photomutagenicity in the yeast Saccharomyces cerevisae, induction of DNA photodamage in cultured human skin cells as revealed by comet assay, and finally induction of specific phototoxic stress responses such as p53 activation or melanogenesis stimulation . Such a strategy should help to ensure the safety of products likely to undergo environmental sunlight exposure.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 73 - 7
DDSE: downstream targets of the SNF3 signal transduction pathway; Theodoris G et al.; Mutations in the yeast SNF3 gene affect glucose sensing and snf3 mutants show defective growth on glucose . DNA sequence dependent suppressing elements (DDSEs) are regions located in the promoters of yeast glucose transporter (HXT) genes that when present in high copy suppress the snf3 growth defect . Here we provide evidence that the multicopy DDSE suppression is due to the titration of the Rgt1p transcriptional repressor . The DDSE region from HXT4 was found to function as a UAS sequence rendering a UAS(gal)-less LacZ gene fused to the GAL1 promoter responsive to glucose induction . Expression mediated by the UAS(DDSE) was dependent upon the presence of Snf3p . Expression was elevated to a high level in an rgt1 mutant in the absence of Snf3p suggesting that this DDSE region contains binding sites for the Rgt1p transcriptional repressor/activator . The UAS(DDSE) led to expression in a grr1 mutant background, which confers a defect in inactivation of Rgt1p, as predicted from the model . The presence of tandem repeats of the putative Rgt1p binding site gave results similar to those of the DDSE, suggesting that loss of repression is due to the presence of Rgt1p footprint in the multicopy DDSE.

Trends Mol Med, 2001 Mar, 7(3), 94 - 6
A new chapter in Rh research: Rh proteins are ammonium transporters; Avent ND; Occasionally, an original research paper has an unusually significant impact on a particular research field . Such a paper, published recently in Nature Genetics, describes the uncovering of the functional role of the Rh protein family--the proteins that express the Rh blood group antigens . Marini et al . (1) demonstrate how two human Rh glycoproteins can correct ammonium transport deficiency in mutant yeast cells . Rh proteins are therefore ammonium transporters--a role that, in vertebrates, has remained previously uncharacterized . These data herald a new era in Rh protein research, beyond their role as blood group antigens, and into the characterization of ammonium transport mechanisms, notably in the kidney.

J Mol Biol, 2001 Apr 6, 307(4), 977 - 85
Effects of histone acetylation on the equilibrium accessibility of nucleosomal DNA target sites; Anderson JD et al.; Posttranslational acetylation of the conserved core histone N-terminal tail domains is linked to gene activation, but the molecular mechanisms involved are not known . In an earlier study we showed that removing the tail domains altogether by trypsin proteolysis (which leaves nucleosomes nevertheless intact) leads to 1.5 to 14-fold increases in the dynamic equilibrium accessibility of nucleosomal DNA target sites . These observations suggested that, by modestly increasing the equilibrium accessibility of buried DNA target sites, histone acetylation could result in an increased occupancy by regulatory proteins, ultimately increasing the probability of transcription initiation . Here, we extend these observations to a more natural system involving intact but hyperacetylated nucleosomes . We find that histone hyperacetylation leads to 1.1 to 1.8-fold increases in position-dependent equilibrium constants for exposure of nucleosomal DNA target sites, with an average increase of 1.4(+/-0.1)-fold . The mechanistic and biological implications of these results are discussed .

Physiol Genomics, 2001 Apr 02, 5(3), 137 - 45
Cloning and functional characterization of an uncoupling protein homolog in hummingbirds; Vianna CR et al.; The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura . The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs . The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5' and 3' ends of the open reading frame . The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being approximately 72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively . The uncoupling activity of this novel protein was characterized in yeast . In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction . Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3',3-dihexyloxacarbocyanine iodide . The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis . Lowering the room's temperature to 12-14 degrees C triggered the cycle torpor/rewarming, typical of hummingbirds . Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor . In conclusion, this is the first report of an UCP homolog in birds . The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

J Clin Invest, 2001 Apr, 107(7), 813 - 22
A novel mouse model of lipotoxic cardiomyopathy; Chiu HC et al.; Inherited and acquired cardiomyopathies are associated with marked intracellular lipid accumulation in the heart . To test the hypothesis that mismatch between myocardial fatty acid uptake and utilization leads to the accumulation of cardiotoxic lipid species, and to establish a mouse model of metabolic cardiomyopathy, we generated transgenic mouse lines that overexpress long-chain acyl-CoA synthetase in the heart (MHC-ACS) . This protein plays an important role in vectorial fatty acid transport across the plasma membrane . MHC-ACS mice demonstrate cardiac-restricted expression of the transgene and marked cardiac myocyte triglyceride accumulation . Lipid accumulation is associated with initial cardiac hypertrophy, followed by the development of left-ventricular dysfunction and premature death . Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and cytochrome c release in transgenic hearts suggest that cardiac myocyte death occurs, in part, by lipid-induced programmed cell death . Taken together, our data demonstrate that fatty acid uptake/utilization mismatch in the heart leads to accumulation of lipid species toxic to cardiac myocytes . This novel mouse model will provide insight into the role of perturbations in myocardial lipid metabolism in the pathogenesis of inherited and acquired forms of heart failure.

J Cell Biol, 2001 Apr 2, 153(1), 191 - 206
Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP; Reczek D et al.; The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1-like) domains followed by a COOH-terminal sequence that binds to active ERM family members . Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50 . Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50-PDZ interactor of 64 kD) . The gene for human EPI64 lies on chromosome 22 where nine exons specify a protein of 508 residues that contains a Tre/Bub2/Cdc16 (TBC)/rab GTPase-activating protein (GAP) domain . EPI64 terminates in DTYL, which is necessary for binding to the PDZ domains of EBP50, as a mutant ending in DTYLA no longer interacts . EPI64 colocalizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell microvilli, and this localization in cultured cells is abolished by introduction of the DTYLA mutation . In addition to EPI64, immobilized EBP50 PDZ domains retain several polypeptides from placental microvilli, including an isoform of nadrin, a rhoGAP domain-containing protein implicated in regulating vesicular transport . Nadrin binds EBP50 directly, probably through its COOH-terminal STAL sequence . Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.

J Biol Chem, 2001 Jun 15, 276(24), 21870 - 7 Epub 2001 Apr 02.
Abrin triggers cell death by inactivating a thiol-specific antioxidant protein; Shih SF et al.; Abrin A-chain (ABRA) inhibits protein synthesis by its N-glycosidase activity as well as induces apoptosis, but the molecular mechanism of ABRA-induced cell death has been obscure . Using an ABRA mutant that lacks N-glycosidase activity as bait in a yeast two-hybrid system, a 30-kDa antioxidant protein-1 (AOP-1) was found to be an ABRA(E164Q)-interacting protein . The interaction was further confirmed in vitro by a glutathione S-transferase pull-down assay . The colocalization of endogenous AOP-1 and exogenous ABR proteins in the cell was demonstrated by confocal immunofluorescence . We also demonstrated that ABRA attenuates AOP-1 antioxidant activity in a dose-dependent manner and the intracellular level of reactive oxygen species (ROS) increases in ABR-treated cells . Moreover, ROS scavengers N-acetylcysteine and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl delayed programmed cell death . This indicates that ROS are important mediators of ABR-induced apoptosis . When ectopically expressed, AOP-1 blocked the release of cytochrome c and prevented apoptosis in ABR-treated cells . These findings suggest that the binding of ABRA to AOP-1 promotes apoptosis by inhibiting the mitochondrial antioxidant protein AOP-1, resulting in the increase of intracellular ROS and the release of cytochrome c from the mitochondria to the cytosol, which activates caspase-9 and caspase-3.

EMBO J, 2001 Apr 2, 20(7), 1807 - 17
Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA; MacAlpine DM et al.; Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA . Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage . Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds . We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites . Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates . We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA.

Traffic, 2001 Apr, 2(4), 268 - 76
Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering; Moyer BD et al.; Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways . We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis-SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE . Science 2000; 289: 444-448) . However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown . Here, we demonstrate that the cis-Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis-Golgi . We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.

Biochem J, 2001 Apr 15, 355(Pt 2), 339 - 46
Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase; Oda K et al.; Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61) . In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I . We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis . The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion . The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs) . Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase . The reactive site of the inhibitor was determined to be -Thr(69)-Lys(70) downward arrowGlu(71)-, where downward arrow indicates the reactive site . Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P(1) and P(2) sites, conversion of the Thr(69)-Lys(70) segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities . Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) sequence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Arg(70) sequences using PCR-based mutagenesis, and analysed them kinetically . Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibitor . The K(i) for Kex2 proteinase was 3.2x10(-10) M, which was 140-fold lower than that of the inhibitor with the Thr(69)-Lys(70) sequence . Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition.

Yeast, 2001 Apr, 18(6), 523 - 31
Assessment of prediction accuracy of protein function from protein--protein interaction data; Hishigaki H et al.; Functional prediction of open reading frames coded in the genome is one of the most important tasks in yeast genomics . Among a number of large-scale experiments for assigning certain functional classes to proteins, experiments determining protein-protein interaction are especially important because interacting proteins usually have the same function . Thus, it seems possible to predict the function of a protein when the function of its interacting partner is known . However, in vitro experiments often suffer from artifacts and a protein can often have multiple binding partners with different functions . We developed an objective prediction method that can systematically include the information of indirect interaction . Our method can predict the subcellular localization, the cellular role and the biochemical function of yeast proteins with accuracies of 72.7%, 63.6% and 52.7%, respectively . The prediction accuracy rises for proteins with more than three binding partners and thus we present the open prediction results for 16 such proteins .

Nat Cell Biol, 2001 Apr, 3(4), 417 - 20
A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint; Harrison JC et al.; In many cells the timing of entry into mitosis is controlled by the balance between the activity of inhibitory Wee1-related kinases (Swe1p in budding yeast) and the opposing effect of Cdc25-related phosphatases (Mih1p in budding yeast) that act on the cyclin-dependent kinase Cdc2 (Cdc28p in budding yeast) . Wee1 and Cdc25 are key elements in the G2 arrest mediated by diverse checkpoint controls . In budding yeast, a 'morphogenesis checkpoint' that involves Swe1p and Mih1p delays mitotic activation of Cdc28p . Many environmental stresses (such as shifts in temperature or osmolarity) provoke transient depolarization of the actin cytoskeleton, during which bud construction is delayed while cells adapt to environmental conditions . During this delay, the morphogenesis checkpoint halts the cell cycle in G2 phase until actin can repolarize and complete bud construction, thus preventing the generation of binucleate cells . A similar G2 delay can be triggered by mutations or drugs that specifically impair actin organization, indicating that it is probably actin disorganization itself, rather than specific environmental stresses, that triggers the delay . The G2 delay involves stabilization of Swe1p in response to various actin perturbations, although this alone is insufficient to produce a long G2 delay.

Nat Cell Biol, 2001 Apr, 3(4), 353 - 60
Spatial regulation of the exocyst complex by Rho1 GTPase; Guo W et al.; Spatial regulation of membrane traffic is fundamental to many biological processes, including epithelial cell polarization and neuronal synaptogenesis . The multiprotein exocyst complex is localized to sites of polarized exocytosis, and is required for vesicle targeting and docking at specific domains of the plasma membrane . One component of the complex, Sec3, is thought to be a spatial landmark for polarized exocytosis . We have searched for proteins that regulate the polarized localization of the exocyst in the budding yeast Saccharomyces cerevisiae . Here we report that certain rho1 mutant alleles specifically affect the localization of the exocyst proteins . Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3 . Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho1 and Sec3 . These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity.

Nat Biotechnol, 2001 Apr, 19(4), 375 - 8
A systematic approach to the analysis of protein phosphorylation; Zhou H et al.; Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities . Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems . However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis . Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures . Here we describe such an approach to protein phosphorylation analysis . It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography-tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases . By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture.

Nat Biotechnol, 2001 Apr, 19(4), 342 - 7
Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer; Hughes TR et al.; We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry . We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method . We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques . Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays . Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides . Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Science, 2001 Mar 30, 291(5513), 2600 - 2
Sterility of Drosophila with mutations in the Bloom syndrome gene--complementation by Ku70; Kusano K et al.; The Drosophila Dmblm locus is a homolog of the human Bloom syndrome gene, which encodes a helicase of the RECQ family . We show that Dmblm is identical to mus309, a locus originally identified in a mutagen-sensitivity screen . One mus309 allele, which carries a stop codon between two of the helicase motifs, causes partial male sterility and complete female sterility . Mutant males produce an excess of XY sperm and nullo sperm, consistent with a high frequency of nondisjunction and/or chromosome loss . These phenotypes of mus309 suggest that Dmblm functions in DNA double-strand break repair . The mutant Dmblm phenotypes were partially rescued by an extra copy of the DNA repair gene Ku70, indicating that the two genes functionally interact in vivo.

Science, 2001 Apr 6, 292(5514), 110 - 3 Epub 2001 Mar 15.
Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly; Nakayama J et al.; The assembly of higher order chromatin structures has been linked to the covalent modifications of histone tails . We provide in vivo evidence that lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast . Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo . Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation . Moreover, an H3-specific deacetylase Clr3 and a beta-propeller domain protein Rik1 are required for H3 Lys9 methylation by Clr4 and Swi6 localization . These data define a conserved pathway wherein sequential histone modifications establish a "histone code" essential for the epigenetic inheritance of heterochromatin assembly.

Mol Cell Biol, 2001 Apr, 21(8), 2726 - 35
Histone acetylation at promoters is differentially affected by specific activators and repressors; Deckert J et al.; We analyzed the relationship between histone acetylation and transcriptional regulation at 40 Saccharomyces cerevisiae promoters that respond to specific activators and repressors . In accord with the general correlation between histone acetylation and transcriptional activity, Gcn4 and the general stress activators (Msn2 and Msn4) cause increased acetylation of histones H3 and H4 . Surprisingly, Gal4-dependent activation is associated with a dramatic decrease in histone H4 acetylation, whereas acetylation of histone H3 is unaffected . A specific decrease in H4 acetylation is also observed, to a lesser extent, at promoters activated by Hap4, Adr1, Met4, and Ace1 . Activation by heat shock factor has multiple effects; H4 acetylation increases at some promoters, whereas other promoters show an apparent decrease in H3 and H4 acetylation that probably reflects nucleosome loss or gross alteration of chromatin structure . Repression by targeted recruitment of the Sin3-Rpd3 histone deacetylase is associated with decreased H3 and H4 acetylation, whereas repression by Cyc8-Tup1 is associated with decreased H3 acetylation but variable effects on H4 acetylation; this suggests that Cyc8-Tup1 uses multiple mechanisms to reduce histone acetylation at promoters . Thus, individual activators confer distinct patterns of histone acetylation on target promoters, and transcriptional activation is not necessarily associated with increased acetylation . We speculate that the activator-specific decrease in histone H4 acetylation is due to blocking the access or function of an H4-specific histone acetylase such as Esa1.

Genome Res, 2001 Apr, 11(4), 540 - 6
Genome-scale compositional comparisons in eukaryotes; Gentles AJ et al.; We examined dinucleotide relative abundances and their biases in recent sequences of eukaryotic genomes and chromosomes, including human chromosomes 21 and 22, Saccharomyces cerevisiae, Arabidopsis thaliana, and Drosophila melanogaster . We found that dinucleotide relative abundances are remarkably constant across human chromosomes and within the DNA of a particular species . The dinucleotide biases differ between species, providing a genome signature that is characteristic of the bulk properties of an organism's DNA . We detail the relations between species genome signatures and suggest possible mechanisms for their origin and maintenance.

Appl Environ Microbiol, 2001 Apr, 67(4), 1607 - 12
Regulation of fumonisin B(1) biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like (C-type) gene, FCC1; Shim WB et al.; Fumonisins are a group of mycotoxins produced in corn kernels by the plant-pathogenic fungus Fusarium verticillioides . A mutant of the fungus, FT536, carrying a disrupted gene named FCC1 (for Fusarium cyclin C1) resulting in altered fumonisin B(1) biosynthesis was generated . FCC1 contains an open reading frame of 1,018 bp, with one intron, and encodes a putative 319-amino-acid polypeptide . This protein is similar to UME3 (also called SRB11 or SSN8), a cyclin C of Saccharomyces cerevisiae, and contains three conserved motifs: a cyclin box, a PEST-rich region, and a destruction box . Also similar to the case for C-type cyclins, FCC1 was constitutively expressed during growth . When strain FT536 was grown on corn kernels or on defined minimal medium at pH 6, conidiation was reduced and FUM5, the polyketide synthase gene involved in fumonisin B(1) biosynthesis, was not expressed . However, when the mutant was grown on a defined minimal medium at pH 3, conidiation was restored, and the blocks in expression of FUM5 and fumonisin B(1) production were suppressed . Our data suggest that FCC1 plays an important role in signal transduction regulating secondary metabolism (fumonisin biosynthesis) and fungal development (conidiation) in F . verticillioides.

Front Biosci, 2001 Apr 01, 6, D610 - 29
Histone acetylation and the cell-cycle in cancer; Wang C et al.; A number of distinct surveillance systems are found in mammalian cells that have the capacity to interrupt normal cell-cycle progression . These are referred to as cell cycle check points . Surveillance systems activated by DNA damage act at three stages, one at the G1/S phase boundary, one that monitors progression through S phase and one at the G2/M boundary . The initiation of DNA synthesis and irrevocable progression through G1 phase represents an additional checkpoint when the cell commits to DNA synthesis . Transition through the cell cycle is regulated by a family of protein kinase holoenzymes, the cyclin-dependent kinases (Cdks), and their heterodimeric cyclin partner . Orderly progression through the cell-cycle checkpoints involves coordinated activation of the Cdks that, in the presence of an associated Cdk-activating kinase (CAK), phosphorylate target substrates including members of the "pocket protein" family . One of these, the product of the retinoblastoma susceptibility gene (the pRB protein), is phosphorylated sequentially by both cyclin D/Cdk4 complexes and cyclin E/Cdk2 kinases . Recent studies have identified important cross talk between the cell-cycle regulatory apparatus and proteins regulating histone acetylation . pRB binds both E2F proteins and histone deacetylase (HDAC) complexes . HDAC plays an important role in pRB tumor suppression function and transcriptional repression . Histones are required for accurate assembly of chromatin and the induction of histone gene expression is tightly coordinated . Recent studies have identified an important alternate substrate of cyclin E/Cdk2, NPAT (nuclear protein mapped to the ATM locus) which plays a critical role in promoting cell-cycle progression in the absence of pRB, and contributes to cell-cycle regulated histone gene expression . The acetylation of histones by a number of histone acetyl transferases (HATs) also plays an important role in coordinating gene expression and cell-cycle progression . Components of the cell-cycle regulatory apparatus are both regulated by HATs and bind directly to HATs . Finally transcription factors have been identified as substrate for HATs . Mutations of these transcription factors at their sites of acetylation has been associated with constitutive activity and enhanced cellular proliferation, suggesting an important role for acetylation in transcriptional repression as well as activation . Together these studies provide a working model in which the cell-cycle regulatory kinases phosphorylate and inactivate HDACs, coordinate histone gene expression and bind to histone acetylases themselves . The recent evidence for cross-talk between the cyclin-dependent kinases and histone gene expression on the one hand and cyclin-dependent regulation of histone acetylases on the other, suggests chemotherapeutics targeting histone acetylation may have complex and possibly complementary effects with agents targeting Cdks.

Curr Opin Chem Biol, 2001 Apr, 5(2), 120 - 9
Dehydrogenases and transaminases in asymmetric synthesis; Stewart JD; Improved stereoselectivity in dehydrogenase-mediated reductions has been achieved by rationally designed gene overexpression and knockouts in Saccharomyces cerevisiae cells and by isolating and characterizing novel dehydrogenases from other organisms . Transaminases have been used to prepare unnatural amines and amino acids in good yields, particularly when the equilibria are shifted by selective product removal.

J Cell Sci, 2001 Apr, 114(Pt 8), 1447 - 54
Initiating DNA synthesis: from recruiting to activating the MCM complex; Lei M et al.; The exact duplication of a genome once per cell division is required of every proliferating cell . To achieve this goal, eukaryotes adopt a strategy that limits every replication origin to a single initiation event within a narrow window of the cell cycle by temporally separating the assembly of the pre-replication complex (pre-RC) from the initiation of DNA synthesis . A key component of the pre-RC is the hexameric MCM complex, which is also the presumed helicase of the growing forks . An elaborate mechanism recruits the MCM complex to replication origins, and a regulatory chain reaction converts the poised, but inactive, MCM complex into an enzymatically active helicase . A growing list of proteins, including Mcm10 and Cdt1, are involved in the recruitment process . Two protein kinases, the Cdc7-Dbf4 kinase (DDK) and the cyclin-dependent kinase (CDK), trigger a chain reaction that results in the phosphorylation of the MCM complex and finally in the initiation of DNA synthesis . A composite picture from recent studies suggests that DDK is recruited to the pre-RC during G1 phase but must wait until S phase to phosphorylate the MCM complex . CDK is required for the recruitment of Cdc45 and other downstream components of the elongation machinery.

Exp Cell Res, 2001 Apr 15, 265(1), 90 - 103
dSIR2 and dHDAC6: two novel, inhibitor-resistant deacetylases in Drosophila melanogaster; Barlow AL et al.; We have identified new members of the histone deacetylase enzyme family in Drosophila melanogaster . dHDAC6 is a class II deacetylase with two active sites, and dSIR2 is an NAD-dependent histone deacetylase . These proteins, together with two class I histone deacetylases, dHDAC1 and dHDAC3, have been expressed and characterized as epitope-tagged recombinant proteins in Schneider SL2 cells . All these proteins have in vitro deacetylase activity and are able to deacetylate core histone H4 at all four acetylatable lysine residues (5, 8, 12, and 16) . Recombinant dHDAC6 and dSIR2 are both insensitive to TSA and HC toxin and resistant, relative to dHDAC1 and dHDAC3, to inhibition by sodium butyrate . Indirect immunofluorescence microscopy of stably transfected SL2 lines reveals that dHDAC1 and dSIR2 are nuclear, dHDAC6 is cytosolic, and dHDAC3 is detectable in both cytosol and nucleus . dHDAC6 and dSIR2 elute from Superose 6 columns with apparent molecular weights of 90 and 200 kDa, respectively . In contrast, dHDAC1 and dHDAC3elute at 800 and 700 kDa, respectively, suggesting that they are components of multiprotein complexes . Consistent with this, recombinant dHDAC1 coimmunoprecipitates with components of the Drosophila NuRD complex and dHDAC3 with an as yet unknown 45-kDa protein .

Am J Gastroenterol, 2001 Mar, 96(3), 758 - 65
Clinical utility of serodiagnostic testing in suspected pediatric inflammatory bowel disease; Dubinsky MC et al.; OBJECTIVES: Confronted with nonspecific symptoms, accurate screening tests would be useful to clinicians to distinguish between functional childhood disorders and inflammatory bowel disease (IBD), thus avoiding invasive diagnostic testing . Traditional ulcerative colitis-specific perinuclear antineutrophil cytoplasmic antibody (pANCA) and Crohn's disease-specific anti-Saccharomyces cerevisiae antibody (ASCA) serodiagnostic assays have recently been modified, with ELISA cut-off values recalculated to maximize sensitivity . The aim of this study was to determine whether the combination of these serodiagnostic tests could maximize diagnostic accuracy and minimize invasive investigations in pediatric patients presenting with nonspecific symptoms suggestive of IBD . METHODS: With investigators blinded to clinical diagnoses, ASCA, ANCA, and pANCA profiles were obtained prospectively from 128 patients undergoing complete diagnostic evaluation for IBD . In phase I, diagnostic accuracy and predictive values of the modified and traditional assays were compared for the IBD (n = 54) and non-IBD groups (n = 74) . In phase II, the overall accuracy of a novel sequential diagnostic testing strategy was determined . Additionally, the potential number of invasive investigations avoided with the hypothetical application of this strategy to the cohort was determined . RESULTS: For phase I, the modified serodiagnostic assay was more sensitive (81 vs 69%), whereas the traditional assay had a higher specificity (96 vs 72%) for IBD (p < 0.05) For phase II, false-positive diagnoses would have been reduced by 81%, yielding an overall sequential testing strategy accuracy of 84% . CONCLUSIONS: The incorporation of sequential noninvasive testing into a diagnostic strategy may avoid unnecessary and costly evaluations and facilitate clinical decision making when the diagnosis of IBD in children is initially uncertain.

Genes Genet Syst, 2000 Dec, 75(6), 319 - 26
Different domains of Sgs1 are required for mitotic and meiotic functions; Miyajima A et al.; The SGS1 of Saccharomyces cerevisiae is a homologue for human Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome causative genes . Disruptants of SGS1 show high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea, and hyper recombination phenotypes including interchromosomal homologous recombination in mitotic growth . In addition, sgs1 disruptants show poor sporulation and a reduced level of meiotic recombination as assayed by return-to-growth . We examined domains of Sgs1 required for mitotic and meiotic functions of Sgs1 by transfecting variously mutated SGS1 into sgs1 disruptants . The N-terminal 1-401 amino acid region was required for complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations of sgs1 disruptants in mitotic growth and for complementation of poor sporulation and of reduced meiotic recombination . Although the N-terminal 1-125 amino acid region was absolutely required for the complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations in mitotic growth, it was dispensable for the meiotic functions . In contrast, the highly acidic region (400-596 amino acid) was dispensable for the mitotic functions but a deletion of this region affected the meiotic functions . The C-terminal 1271-1350 amino acid region containing a HRDC (helicase and RNaseD C-terminal) domain was dispensable for the mitotic and meiotic functions . Although DNA helicase activity of Sgs1 was not required for Sgs1 to complement the meiotic functions, a deletion of helicase motifs III-IV (842-1046 amino acid) abolished the complementing activity of Sgs1, indicating that a structurally intact helicase domain is necessary for Sgs1 to fulfill its meiotic functions.

J Biol Chem, 2001 Jun 15, 276(24), 21555 - 61 Epub 2001 Mar 09.
Substrate recognition by UDP-galactose and CMP-sialic acid transporters . Different sets of transmembrane helices are utilized for the specific recognition of UDP-galactose and CMP-sialic acid; Aoki K et al.; Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi membrane proteins with 10 transmembrane helices . We have constructed chimeras between these proteins in order to identify submolecular regions responsible for the determination of substrate specificity . To assess the UGT and CST activities, chimeric cDNAs were transiently expressed in either UGT-deficient mutant Lec8 cells or CST-deficient mutant Lec2 cells, and the binding of plant lectins, GS-II or PNA, respectively, to these cells was examined . During the course of analysis of various chimeric transporters, we found that chimeras whose submolecular regions contained helices 1, 8, 9, and 10, and helices 2, 3, and 7 derived from hUGT1 and hCST sequences, respectively, exhibited both UGT and CST activities . The dual substrate specificity for UDP-galactose and CMP-sialic acid of one such representative chimera was directly confirmed by in vitro measurement of the nucleotide sugar transport activity using a heterologous expression system in the yeast Saccharomyces cerevisiae . These findings indicated that the regions which are critical for determining the substrate specificity of UGT and CST resided in different submolecular sites in the two transporters, and that these different determinants could be present within one protein without interfering with each other's function.

J Biol Chem, 2001 May 25, 276(21), 18407 - 14 Epub 2001 Feb 26.
Novel members of the human oxysterol-binding protein family bind phospholipids and regulate vesicle transport; Xu Y et al.; Oxysterol-binding proteins (OSBPs) are a family of eukaryotic intracellular lipid receptors . Mammalian OSBP1 binds oxygenated derivatives of cholesterol and mediates sterol and phospholipid synthesis through as yet poorly undefined mechanisms . The precise cellular roles for the remaining members of the oxysterol-binding protein family remain to be elucidated . In yeast, a family of OSBPs has been identified based on primary sequence similarity to the ligand binding domain of mammalian OSBP1 . Yeast Kes1p, an oxysterol-binding protein family member that consists of only the ligand binding domain, has been demonstrated to regulate the Sec14p pathway for Golgi-derived vesicle transport . Specifically, inactivation of the KES1 gene resulted in the ability of yeast to survive in the absence of Sec14p, a phosphatidylinositol/phosphatidylcholine transfer protein that is normally required for cell viability due to its essential requirement in transporting vesicles from the Golgi . We cloned the two human members of the OSBP family, ORP1 and ORP2, with the highest degree of similarity to yeast Kes1p . We expressed ORP1 and ORP2 in yeast lacking Sec14p and Kes1p function and found that ORP1 complemented Kes1p function with respect to cell growth and Golgi vesicle transport, whereas ORP2 was unable to do so . Phenotypes associated with overexpression of ORP2 in yeast were a dramatic decrease in cell growth and a block in Golgi-derived vesicle transport distinct from that of ORP1 . Purification of ORP1 and ORP2 for ligand binding studies demonstrated ORP1 and ORP2 did not bind 25-hydroxycholesterol but instead bound phospholipids with both proteins exhibiting strong binding to phosphatidic acid and weak binding to phosphatidylinositol 3-phosphate . In Chinese hamster ovary cells, ORP1 localized to a cytosolic location, whereas ORP2 was associated with the Golgi apparatus, consistent with our vesicle transport studies that indicated ORP1 and ORP2 function at different steps in the regulation of vesicle transport.

J Biol Chem, 2001 May 18, 276(20), 17448 - 54 Epub 2001 Feb 22.
A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix and associates with p68 RNA helicase; Akileswaran L et al.; The cell nucleus is structurally and functionally organized by the nuclear matrix . We have examined whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for targeting to the subnuclear compartment and for interaction with other proteins . AKAP95 was expressed in mammalian cells and found to localize exclusively to the nuclear matrix . Mutational analysis was used to identify determinants for nuclear localization and nuclear matrix targeting of AKAP95 . These sites were found to be distinct from previously identified DNA and protein kinase A binding domains . The nuclear matrix-targeting site is unique but conserved among members of the AKAP95 family . Direct binding of AKAP95 to isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting site . Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix isolated from rat brain . Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase . The helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were precipitated as a complex from solubilized cell extracts . The results define novel protein-protein interactions among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly of hormonally responsive transcription complexes.

J Biol Chem, 2001 May 18, 276(20), 16635 - 40 Epub 2001 Feb 13.
Cloning of a human type II phosphatidylinositol 4-kinase reveals a novel lipid kinase family; Minogue S et al.; Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes . The versatility of this phospholipid is provided by combinations of phosphorylation on the 3', 4', and 5' positions of the inositol head group . Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity . Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization . We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data . The results allowed the cDNA containing the full open reading frame to be cloned . The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases . We have named the purified protein type IIalpha and a second human isoform, type IIbeta . The type IIalpha mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes.

J Biol Chem, 2001 May 18, 276(20), 16804 - 9 Epub 2001 Feb 14.
p300-mediated acetylation of human transcriptional coactivator PC4 is inhibited by phosphorylation; Kumar BR et al.; The human positive coactivator 4 (PC4) acts as a general coactivator for activator-dependent transcription, the activity of which is regulated negatively by phosphorylation . We report here that PC4 can be acetylated specifically by another coactivator, p300 . Interestingly, phosphorylation of PC4 by casein kinase II inhibits the p300-mediated acetylation . Mass spectral analysis revealed that there are at least two lysine residues acetylated in PC4, as a result of which its DNA binding activity is stimulated.

J Biol Chem, 2001 May 18, 276(20), 16840 - 7 Epub 2001 Feb 20.
Cyclin D1 represses STAT3 activation through a Cdk4-independent mechanism; Bienvenu F et al.; STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation . Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate into the nucleus, and activate specific target genes . Activation is transient, and down-regulation of STAT3 signaling occurs within a few hours . In this study, we show that cyclin D1 inhibits STAT3 activation . In co-immunoprecipitation and pull-down assays, cyclin D1 was found to associate with the activation domain of STAT3 upon interleukin-6 stimulation . Overexpression of cyclin D1 inhibited transcriptional activation by STAT3 proteins . This effect was not shared by cyclin E, was independent of association with Cdk4, and was unaffected by inhibitors of Cdk4 . Whereas cyclin D1 had no effect on the steady-state level of STAT3 proteins in the cytoplasm, it was found to reduce the STAT3 nuclear level in HepG2 cells . These results suggest a model by which cyclin D1 is part of a feedback network controlling the down-regulation of STAT3 activity and highlight a new activity for this cell cycle regulatory protein.

J Biol Chem, 2001 May 18, 276(20), 16848 - 56 Epub 2001 Feb 20.
The binding of Ku antigen to homeodomain proteins promotes their phosphorylation by DNA-dependent protein kinase; Schild-Poulter C et al.; The Ku antigen (70- and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PKcs) to DNA ends and to specific DNA sequences from which the kinase is activated . Ku and DNA-PKcs plays essential roles in double-stranded DNA break repair and V(D)J recombination and have been implicated in the regulation of specific gene transcription . In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a specific interaction between the 70-kDa subunit of Ku heterodimer and the homeodomain of HOXC4, a homeodomain protein expressed in the hematopoietic system . Unexpectedly, a similar interaction with Ku was observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins . Ku-homeodomain binding was mediated through the extreme C terminus of Ku70 and was abrogated by amino acid substitutions at Lys595/Lys596 . Ku binding allowed the recruitment of the homeodomain to DNA ends and dramatically enhanced the phosphorylation of homeodomain-containing proteins by DNA-PK . These results suggest that Ku functions as a substrate docking protein for signaling by DNA-PK to homeodomain proteins from DNA ends.

J Biol Chem, 2001 Jun 8, 276(23), 20413 - 8 Epub 2001 Mar 05.
Identification of a novel chloride channel expressed in the endoplasmic reticulum, golgi apparatus, and nucleus; Nagasawa M et al.; MID-1 is a Saccharomyces cerevisiae gene encoding a stretch-activated channel . Using MID-1 as a molecular probe, we isolated rat cDNA encoding a protein with four putative transmembrane domains . This gene encoded a protein of 541 amino acids . We also cloned the human homologue, which encoded 551 amino acids . Messenger RNA for this gene was expressed abundantly in the testis and moderately in the spleen, liver, kidney, heart, brain, and lung . In the testis, immunoreactivity of the gene product was detected both in the cytoplasm and the nucleus . When expressed in Chinese hamster ovary cells, the gene product was located in intracellular compartments including endoplasmic reticulum and the Golgi apparatus . When microsome fraction obtained from the transfected cells, but not from mock-transfected cells, was incorporated into the lipid bilayer, an anion channel activity was detected . Unitary conductance was 70 picosiemens in symmetric 150 mm KCl solution . We designated this gene Mid-1-related chloride channel (MCLC) . MCLC encodes a new class of chloride channel expressed in intracellular compartments.

J Biol Chem, 2001 May 18, 276(20), 16767 - 71 Epub 2001 Feb 07.
Thiazolidinediones but not metformin directly inhibit the steroidogenic enzymes P450c17 and 3beta -hydroxysteroid dehydrogenase; Arlt W et al.; Androgen biosynthesis requires 3beta-hydroxysteroid dehydrogenase type II (3betaHSDII) and the 17alpha-hydroxylase and 17,20-lyase activities of cytochrome P450c17 . Thiazolidinedione and biguanide drugs, which are used to increase insulin sensitivity in type 2 diabetes, lower serum androgen concentrations in women with polycystic ovary syndrome . However, it is unclear whether this is secondary to increased insulin sensitivity or to direct effects on steroidogenesis . To investigate potential actions of these drugs on P450c17 and 3betaHSDII, we used "humanized yeast" that express these steroidogenic enzymes in microsomal environments . The biguanide metformin had no effect on either enzyme, whereas the thiazolidinedione troglitazone inhibited 3betaHSDII (K(I) = 25.4 +/- 5.1 microm) and both activities of P450c17 (K(I) for 17alpha-hydroxylase, 8.4 +/- 0.6 microm; K(I) for 17,20-lyase, 5.3 +/- 0.7 microm) . The action of troglitazone on P450c17 was competitive, but it was mainly a noncompetitive inhibitor of 3betaHSDII . The thiazolidinediones rosiglitazone and pioglitazone exerted direct but weaker inhibitory effects on both P450c17 and 3betaHSDII . These differential effects of the thiazolidinediones do not correlate with their effects on insulin sensitivity, suggesting that distinct regions of the thiazolidinedione molecule mediate these two actions . Thus, thiazolidinediones inhibit two key enzymes in human androgen synthesis contributing to their androgen-lowering effects, whereas metformin affects androgen synthesis indirectly, probably by lowering circulating insulin concentrations.

J Biol Chem, 2001 May 4, 276(18), 15397 - 408 Epub 2001 Jan 24.
Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1; Burke TW et al.; The minichromosome maintenance (MCM) proteins, together with the origin recognition complex (ORC) proteins and Cdc6, play an essential role in eukaryotic DNA replication through the formation of a pre-replication complex at origins of replication . We used a yeast two-hybrid screen to identify MCM2-interacting proteins . One of the proteins we identified is identical to the ORC1-interacting protein termed HBO1 . HBO1 belongs to the MYST family, characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain . Biochemical studies confirmed the interaction between MCM2 and HBO1 in vitro and in vivo . An N-terminal domain of MCM2 is necessary for binding to HBO1, and a C2HC zinc finger of HBO1 is essential for binding to MCM2 . A reverse yeast two-hybrid selection was performed to isolate an allele of MCM2 that is defective for interaction with HBO1; this allele was then used to isolate a suppressor mutant of HBO1 that restores the interaction with the mutant MCM2 . This suppressor mutation was located in the HBO1 zinc finger . Taken together, these findings strongly suggest that the interaction between MCM2 and HBO1 is direct and mediated by the C2HC zinc finger of HBO1 . The biochemical and genetic interactions of MYST family protein HBO1 with two components of the replication apparatus, MCM2 and ORC1, suggest that HBO1-associated HAT activity may play a direct role in the process of DNA replication.

J Biol Chem, 2001 Apr 20, 276(16), 12712 - 7 Epub 2001 Jan 17.
Interaction sites of the G protein beta subunit with brain G protein-coupled inward rectifier K+ channel; Albsoul-Younes AM et al.; G protein-coupled inward rectifier K(+) channels (GIRK channels) are activated directly by the G protein betagamma subunit . The crystal structure of the G protein betagamma subunits reveals that the beta subunit consists of an N-terminal alpha helix followed by a symmetrical seven-bladed propeller structure . Each blade is made up of four antiparallel beta strands . The top surface of the propeller structure interacts with the Galpha subunit . The outer surface of the betagamma torus is largely made from outer beta strands of the propeller . We analyzed the interaction between the beta subunit and brain GIRK channels by mutating the outer surface of the betagamma torus . Mutants of the outer surface of the beta(1) subunit were generated by replacing the sequences at the outer beta strands of each blade with corresponding sequences of the yeast beta subunit, STE4 . The mutant beta(1)gamma(2) subunits were expressed in and purified from Sf9 cells . They were applied to inside-out patches of cultured locus coeruleus neurons . The wild type beta(1)gamma(2) induced robust GIRK channel activity with an EC(50) of about 4 nm . Among the eight outer surface mutants tested, blade 1 and blade 2 mutants (D1 and CD2) were far less active than the wild type in stimulating GIRK channels . However, the ability of D1 and CD2 to regulate type I and type II adenylyl cyclases was not very different from that of the wild type beta(1)gamma(2) . As to the activities to stimulate phospholipase Cbeta(2), D1 was more potent and CD2 was less potent than the wild type beta(1)gamma(2) . Additionally we tested four beta(1) mutants in which mutated residues are located in the top Galpha/beta interacting surface . Among them, mutant W332A showed far less ability than the wild type to activate GIRK channels . These results suggest that the outer surface of blade 1 and blade 2 of the beta subunit might specifically interact with GIRK and that the beta subunit interacts with GIRK both over the outer surface and over the top Galpha interacting surface.

J Biol Chem, 2001 Apr 20, 276(16), 13395 - 401 Epub 2001 Jan 24.
Involvement of the pro-oncoprotein TLS (translocated in liposarcoma) in nuclear factor-kappa B p65-mediated transcription as a coactivator; Uranishi H et al.; In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor kappaB (NF-kappaB) using a yeast two-hybrid screen . We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo . TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas . TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II . However, the role of TLS in transcriptional regulation has not yet been clearly elucidated . We found that TLS enhanced the NF-kappaB-mediated transactivation induced by physiological stimuli such as tumor necrosis factor alpha, interleukin-1beta, and overexpression of NF-kappaB-inducing kinase . TLS augmented NF-kappaB-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-beta gene . These results suggest that TLS acts as a coactivator of NF-kappaB and plays a pivotal role in the NF-kappaB-mediated transactivation.

J Biol Chem, 2001 May 25, 276(21), 18442 - 9 Epub 2001 Feb 21.
Tropomyosin-troponin regulation of actin does not involve subdomain 2 motions; Gerson JH et al.; Dynamic properties of F-actin structure prompted suggestions (Squire, J . M., and Morris, E . P . (1998) FASEB J . 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation . Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes in subdomain 2 . These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C(AEDANS)/C374S and D51C(AEDANS)/C374S filaments did not reveal any calcium-dependent changes . Fluorescence energy transfer in these F-actins mostly occurred from Trp(340) and Trp(356) to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys(41) or Cys(51) of adjacent same strand protomers . Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (-Ca(2+)) and closed (+Ca(2+)) states . Ca(2+) also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys(41) and Cys(374) . ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca(2+) nor S1 binding to regulated alpha-actin affects the phalloidin-probe distance . Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked alpha-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.

J Biol Chem, 2001 May 25, 276(21), 18115 - 21 Epub 2001 Feb 22.
H+-coupled pantothenate transport in the intracellular malaria parasite; Saliba KJ et al.; Pantothenate, the precursor of coenzyme A, is an essential nutrient for the intraerythrocytic stage of the malaria parasite Plasmodium falciparum . Pantothenate enters the malaria-infected erythrocyte via new permeation pathways induced by the parasite in the host cell membrane (Saliba, K . J., Horner, H . A., and Kirk, K . (1998) J . Biol . Chem . 273, 10190-10195) . We show here that pantothenate is taken up by the intracellular parasite via a novel H(+)-coupled transporter, quite different from the Na(+)-coupled transporters that mediate pantothenate uptake into mammalian cells . The plasmodial H(+):pantothenate transporter has a low affinity for pantothenate (K(m) approximately 23 mm) and a stoichiometry of 1 H(+):1 pantothenate . It is inhibited by low concentrations of the bioflavonoid phloretin and the thiol-modifying agent p-chloromercuribenzene sulfonate . On entering the parasite, pantothenate is phosphorylated (and thereby trapped) by an unusually high affinity pantothenate kinase (K(m) approximately 300 nm) . The combination of H(+)-coupled transporter and kinase provides the parasite with an efficient, high affinity pantothenate uptake system, which is distinct from that of the host and is therefore an attractive target for antimalarial chemotherapy.

J Biol Chem, 2001 May 25, 276(21), 17712 - 7 Epub 2001 Mar 09.
Nuclear import of Spo12p, a protein essential for meiosis; Chaves SR et al.; In Saccharomyces cerevisiae, Spo12p is involved in mitosis and is essential for meiosis . We found that Spo12p is imported into the nucleus by the karyopherin Kap121p . A complex containing Spo12p and Kap121p was isolated from cytosol and was also reconstituted with recombinant proteins, indicating that this interaction is direct . Spo12p was mislocalized to the cytosol in pse1-1, a temperature-sensitive strain harboring a mutation of Kap121p, at the permissive temperature, confirming an essential role for Kap121p in Spo12p import . Spo12p was also mislocalized in a pse1-1/pse1-1 homozygous strain, suggesting it is imported via the same pathway in diploid cells . Furthermore, we found that pse1-1/pse1-1 shows a sporulation defect similar to that of spo12Delta/spo12Delta . In addition, we have characterized the Spo12p nuclear localization signal, mapped it to residues 76-130, and identified residues within this region that are important for nuclear localization signal function.

J Biol Chem, 2001 May 4, 276(18), 15423 - 33 Epub 2001 Feb 02.
Distinct roles for Ku protein in transcriptional reinitiation and DNA repair; Woodard RL et al.; Transcriptional reinitiation is a distinct phase of the RNA polymerase II transcription cycle . Prior work has shown that reinitiation is deficient in nuclear extracts from Chinese hamster ovary cells lacking the 80-kDa subunit of Ku, a double-strand break repair protein, and that activity is rescued by expression of the corresponding cDNA . We now show that Ku increases the amount or availability of a soluble factor that is limiting for reinitiation, that the factor increases the number of elongation complexes associated with the template at all times during the reaction, and that the factor itself does not form a tight complex with DNA . The factor may consist of a preformed complex of transcription proteins that is stabilized by Ku . A Ku mutant, lacking residues 687-728 in the 80-kDa subunit, preferentially suppresses transcription in Ku-containing extracts, suggesting that Ku interacts directly with proteins required for reinitiation . The Ku mutant functions normally in a DNA end-joining system, indicating that the functions of Ku in transcription and repair are genetically separable . Based on our results, we present a model in which Ku is capable of undergoing a switch between a transcription factor-associated and a repair-active state.

J Biol Chem, 2001 Apr 27, 276(17), 14195 - 203 Epub 2001 Jan 30.
Mechanisms governing subcellular localization and function of human RGS2; Heximer SP et al.; RGS proteins negatively regulate heterotrimeric G proteins at the plasma membrane . RGS2-GFP localizes to the nucleus, plasma membrane, and cytoplasm of HEK293 cells . Expression of activated G(q) increased RGS2 association with the plasma membrane and decreased accumulation in the nucleus, suggesting that signal-induced redistribution may regulate RGS2 function . Thus, we identified and characterized a conserved N-terminal domain in RGS2 that is necessary and sufficient for plasma membrane localization . Mutational and biophysical analyses indicated that this domain is an amphipathic alpha-helix that binds vesicles containing acidic phospholipids . However, the plasma membrane targeting function of the amphipathic helical domain did not appear to be essential for RGS2 to attenuate signaling by activated G(q) . Nevertheless, truncation mutants indicated that the N terminus is essential, potentially serving as a scaffold that binds receptors, signaling proteins, or nuclear components . Indeed, the RGS2 N terminus directs nuclear accumulation of GFP . Although RGS2 possesses a nuclear targeting motif, it lacks a nuclear import signal and enters the nucleus by passive diffusion . Nuclear accumulation of RGS2 does not limit its ability to attenuate G(q) signaling, because excluding RGS2 from the nucleus was without effect . RGS2 may nonetheless regulate signaling or other processes in the nucleus.

J Biol Chem, 2001 May 18, 276(20), 17172 - 80 Epub 2001 Feb 22.
Identification of a novel human tankyrase through its interaction with the adaptor protein Grb14; Lyons RJ et al.; Tankyrase is an ankyrin repeat-containing poly(ADP-ribose) polymerase originally isolated as a binding partner for the telomeric protein TRF1, but recently identified as a mitogen-activated protein kinase substrate implicated in regulation of Golgi vesicle trafficking . In this study, a novel human tankyrase, designated tankyrase 2, was isolated in a yeast two-hybrid screen as a binding partner for the Src homology 2 domain-containing adaptor protein Grb14 . Tankyrase 2 is a 130-kDa protein, which lacks the N-terminal histidine/proline/serine-rich region of tankyrase, but contains a corresponding ankyrin repeat region, sterile alpha motif module, and poly(ADP-ribose) polymerase homology domain . The TANKYRASE 2 gene localizes to chromosome 10q23.2 and is widely expressed, with mRNA transcripts particularly abundant in skeletal muscle and placenta . Upon subcellular fractionation, both Grb14 and tankyrase 2 associate with the low density microsome fraction, and association of these proteins in vivo can be detected by co-immunoprecipitation analysis . Deletion analyses implicate the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10-19 of tankyrase 2 in mediating this interaction . This study supports a role for the tankyrases in cytoplasmic signal transduction pathways and suggests that vesicle trafficking may be involved in the subcellular localization or signaling function of Grb14.

J Biol Chem, 2001 May 25, 276(21), 17908 - 13 Epub 2001 Mar 02.
Interaction of p130 with, and consequent inhibition of, the catalytic subunit of protein phosphatase 1alpha; Yoshimura K et al.; The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but which lacks phospholipase C activity . Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1alpha (PP1calpha) as a p130-binding protein . The association between p130 and PP1calpha was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis . The interaction of p130 with PP1calpha resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner . Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1calpha also detected the presence of a complex of p130 and PP1calpha . The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1calpha, was higher in COS-1 cells that stably express p130 than in control COS-1 cells . These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca(2+) signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1calpha.

J Biol Chem, 2001 May 25, 276(21), 17679 - 85 Epub 2001 Mar 06.
Assembly of Tom6 and Tom7 into the TOM core complex of Neurospora crassa; Dembowski M et al.; Translocation of preproteins across the mitochondrial outer membrane is mediated by the translocase of the outer mitochondrial membrane (TOM) complex . We report the molecular identification of Tom6 and Tom7, two small subunits of the TOM core complex in the fungus Neurospora crassa . Cross-linking experiments showed that both proteins were found to be in direct contact with the major component of the pore, Tom40 . In addition, Tom6 was observed to interact with Tom22 in a manner that depends on the presence of preproteins in transit . Precursors of both proteins are able to insert into the outer membrane in vitro and are assembled into authentic TOM complexes . The insertion pathway of these proteins shares a common binding site with the general import pathway as the assembly of both Tom6 and Tom7 was competed by a matrix-destined precursor protein . This assembly was dependent on the integrity of receptor components of the TOM machinery and is highly specific as in vitro-synthesized yeast Tom6 was not assembled into N . crassa TOM complex . The targeting and assembly information within the Tom6 sequence was found to be located in the transmembrane segment and a flanking segment toward the N-terminal, cytosolic side . A hybrid protein composed of the C-terminal domain of yeast Tom6 and the cytosolic domain of N . crassa Tom6 was targeted to the mitochondria but was not taken up into TOM complexes . Thus, both segments are required for assembly into the TOM complex . A model for the topogenesis of the small Tom subunits is discussed.

J Biol Chem, 2001 May 11, 276(19), 16561 - 6 Epub 2001 Feb 05.
Cotranscription and intergenic splicing of human P2Y11 and SSF1 genes; Communi D et al.; The P2Y(11) receptor is an ATP receptor positively coupled to the cAMP and phosphoinositide pathways . Ssf1 is a Saccharomyces cerevisiae nuclear protein, which plays an important role in mating . The gene encoding the human orthologue of SSF1 is adjacent to the P2Y(11) gene on chromosome 19 . During the screening of placenta cDNA libraries, we isolated a chimeric clone resulting from the intergenic splicing between the P2Y(11) and SSF1 genes . The fusion protein was stably expressed in CHO-K1 cells where it generated a cAMP response to ATP qualitatively indistinguishable from that of the P2Y(11) receptor . According to both Western blotting and cAMP response, the expression of the fusion protein in the transfected cells was clearly lower than that of the P2Y(11) receptor . Both P2Y(11) and SSF1 probes detected a 5.6-kb messenger RNA with a similar pattern of intensity in each of 11 human tissues . The ubiquitous presence of chimeric transcripts and their up-regulation during granulocytic differentiation indicate that the transgenic splicing between the P2Y(11) and the SSF1 genes is a common and regulated phenomenon . There are very few examples of intergenic splicing in mammalian cells, and this is the first case involving a G-protein-coupled receptor.

J Biol Chem, 2001 Apr 27, 276(17), 14019 - 26 Epub 2001 Jan 18.
Role of the N-terminal forkhead-associated domain in the cell cycle checkpoint function of the Rad53 kinase; Pike BL et al.; Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases . Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks . Here we show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1) . This phenotype was highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro . Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain resulted in an increased DNA damage sensitivity in vivo . Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1).

J Biol Chem, 2001 Jun 29, 276(26), 24212 - 22 Epub 2001 Mar 16.
The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in vivo and in vitro studies; Tarabykina S et al.; The S100 calcium-binding proteins are implicated in signal transduction, motility, and cytoskeletal dynamics . The three-dimensional structure of several S100 proteins revealed that the proteins form non-covalent dimers . However, the mechanism of the S100 dimerization is still obscure . In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo . Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible equilibrium (K(d) = 4 +/- 2 microm) . The binding of calcium promoted dimerization . Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer . Helix IV is known to form the major part of the dimerization interface in homologous S100 proteins . By using the yeast two-hybrid system we showed that only a few residues of helix IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in vivo . A homology model demonstrated that these residues form a hydrophobic cluster on helix IV . Their role is to stabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface . Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent data indicating that S100 proteins can form heterodimers.

J Biol Chem, 2001 Jun 1, 276(22), 19375 - 81 Epub 2001 Feb 08.
Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51; Wu L et al.; Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers . The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases . The precise role played by these enzymes in the cell remains to be determined . However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells . In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein . In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair . Here, we report that purified BLM and human RAD51 interact in vitro and in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction . Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells . Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation . Nevertheless, the formation of RAD51 foci does not require functional BLM . Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci . Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci . The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51 . Furthermore, genetic analysis reveals that the SGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway . Potential roles for BLM in the RAD51 recombinational repair pathway are discussed.

J Biol Chem, 2001 Apr 20, 276(16), 13127 - 35 Epub 2001 Jan 25.
Gaf-1, a gamma -SNAP-binding protein associated with the mitochondria; Chen D et al.; The role of alpha/beta-SNAP (Soluble NSF Attachment Protein) in vesicular trafficking is well established; however, the function of the ubiquitously expressed gamma-SNAP remains unclear . To further characterize the cellular role of this enigmatic protein, a two-hybrid screen was used to identify new, gamma-SNAP-binding proteins and to uncover potentially novel functions for gamma-SNAP . One such SNAP-binding protein, termed Gaf-1 (gamma-SNAP associate factor-1) specifically binds gamma- but not alpha-SNAP . The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically associated with gamma-SNAP in cellular extracts . This binding is distinct from other SNAP interactions since no alpha-SNAP or NSF coprecipitated with Gaf-1 . Subcellular fractionation and immunofluorescence analysis show that Gaf-1 is peripherally associated with the outer mitochondrial membrane . Only a fraction of gamma-SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions . GFP-gamma-SNAP and the C-terminal domain of Gaf-1 both show a reticular distribution in HEK-293 cells . This reticular structure colocalizes with Gaf-1 and mitochondria as well as with microtubules but not with other cytoskeletal elements . These data identify a class of gamma-SNAP interactions that is distinct from other members of the SNAP family and point to a potential role for gamma-SNAP in mitochondrial dynamics.

J Biol Chem, 2001 May 11, 276(19), 15886 - 92 Epub 2001 Feb 20.
Taf(II) 250 phosphorylates human transcription factor IIA on serine residues important for TBP binding and transcription activity; Solow S et al.; Transcription factor IIA (TFIIA) is a positive acting general factor that contacts the TATA-binding protein (TBP) and mediates an activator-induced conformational change in the transcription factor IID (TFIID) complex . Previously, we have found that phosphorylation of yeast TFIIA stimulates TFIIA.TBP.TATA complex formation and transcription activation in vivo . We now show that human TFIIA is phosphorylated in vivo on serine residues that are partially conserved between yeast and human TFIIA large subunits . Alanine substitution mutation of serine residues 316 and 321 in TFIIA alphabeta reduced TFIIA phosphorylation significantly in vivo . Additional alanine substitutions at serines 280 and 281 reduced phosphorylation to undetectable levels . Mutation of all four serine residues reduced the ability of TFIIA to stimulate transcription in transient transfection assays with various activators and promoters, indicating that TFIIA phosphorylation is required globally for optimal function . In vitro, holo-TFIID and TBP-associated factor 250 (TAF(II)250) phosphorylated TFIIA on the beta subunit . Mutation of the four serines required for in vivo phosphorylation eliminated TFIID and TAF(II)250 phosphorylation in vitro . The NH(2)-terminal kinase domain of TAF(II)250 was sufficient for TFIIA phosphorylation, and this activity was inhibited by full-length retinoblastoma protein but not by a retinoblastoma protein mutant defective for TAF(II)250 interaction or tumor suppressor activity . TFIIA phosphorylation had little effect on the TFIIA.TBP.TATA complex in electrophoretic mobility shift assay . However, phosphorylation of TFIIA containing a gamma subunit Y65A mutation strongly stimulated TFIIA.TBP.TATA complex formation . TFIIA-gammaY65A is defective for binding to the beta-sheet domain of TBP identified in the crystal structure . These results suggest that TFIIA phosphorylation is important for strengthening the TFIIA.TBP contact or creating a second contact between TFIIA and TBP that was not visible in the crystal structure.

J Biol Chem, 2001 May 18, 276(20), 16960 - 8 Epub 2001 Feb 26.
Phosphorylation-independent association of CXCR2 with the protein phosphatase 2A core enzyme; Fan GH et al.; Protein phosphatase 2A (PP2A) is postulated to be involved in the dephosphorylation of G protein-coupled receptors . In the present study, we demonstrate that the carboxyl terminus of CXCR2 physically interacts with the PP2A core enzyme, a dimer formed by PP2Ac and PR65, but not with the PP2Ac monomer, suggesting direct interaction of the receptor with PR65 . The integrity of a sequence motif in the C terminus of CXCR2, KFRHGL, which is conserved in all CC and CXC chemokine receptors, is required for the receptor binding to the PP2A core enzyme . CXCR2 co-immunoprecipitates with the PP2A core enzyme in HEK293 cells and in human neutrophils . Overexpression of dominant negative dynamin 1 (dynamin 1 K44A) in CXCR2-expressing cells blocks the receptor association with the PP2A core enzyme, and an internalization-deficient mutant form of CXCR2 (I323A,L324A) also exhibits impaired association with the PP2A core enzyme, suggesting that the receptor internalization is required for the receptor binding to PP2A . A phosphorylation-deficient mutant of CXCR2 (331T), which has previously been shown to undergo internalization in HEK293 cells, binds to an almost equal amount of the PP2A core enzyme in comparison with the wild-type CXCR2, suggesting that the interaction of the receptor with PP2A is phosphorylation-independent . The dephosphorylation of CXCR2 is reversed by treatment of the cells with okadaic acid . Moreover, pretreatment of the cells with okadaic acid increases basal phosphorylation of CXCR2 and attenuates CXCR2-mediated calcium mobilization and chemotaxis . Taken together, these data indicate that PP2A is involved in the dephosphorylation of CXCR2 . We postulate that this interaction results from direct binding of the regulatory subunit A (PR65) of PP2A to the carboxyl terminus of CXCR2 after receptor sequestration and internalization.

J Biol Chem, 2001 May 25, 276(21), 17871 - 7 Epub 2001 Feb 26.
Filamin associates with Smads and regulates transforming growth factor-beta signaling; Sasaki A et al.; Members of the Smad proteins transmit signals triggered by the ligands of transforming growth factor (TGF)-beta superfamily . Ligand-activated receptors induce phosphorylation of so-called receptor-regulated Smads, which then accumulate in the nucleus to participate in target gene transcription, in collaboration with Smad-interacting proteins . We performed yeast two-hybrid screening and identified filamin, a cytoskeletal actin-binding protein 280, as a Smad5-interacting protein . Filamin was found to be associated not only with Smad5 but also with other Smad proteins, including TGF-beta/activin receptor-regulated Smad2 . TGF-beta signaling was defective in filamin-deficient human melanoma cells M2 compared with a filamin-transfected subline A7, as determined by TGF-beta-responsive reporter gene activation and Smad2 nuclear accumulation . M2 cells restored TGF-beta responsiveness following transient transfection of full-length filamin encoding vector . The defective TGF-beta signaling in M2 cells seemed to be due to impaired receptor-induced serine phosphorylation of Smad2 . These results suggest that filamin plays an important role in Smad-mediated signaling.

J Biol Chem, 2001 May 11, 276(19), 16310 - 7 Epub 2001 Feb 22.
Stimulation of p300-mediated transcription by the kinase MEKK1; See RH et al.; p300 and CREB-binding protein (CBP) are related transcriptional coactivators that possess histone acetyltransferase activity . Inactivation of p300/CBP is part of the mechanism by which adenovirus E1A induces oncogenic transformation of cells . Recently, the importance of p300/CBP has been demonstrated directly in several organisms including mouse, Drosophila, and Caenorhabditis elegans where p300/CBP play an indispensable role in differentiation, in patterning, and in cell fate determination and proliferation during development . CBP/p300s are modified by phosphorylation during F9 cell differentiation as well as adenovirus infection, suggesting that phosphorylation may play a role in the regulation of p300/CBP activity . Here we show that the mitogen-activated/extracellular response kinase kinase 1 (MEKK1) enhances p300-mediated transcription . We identify several domains within p300 that can respond to MEKK1-induced transcriptional activation . Interestingly, activation of p300-mediated transcription by MEKK1 does not appear to require the downstream kinase JNK and may involve either a direct phosphorylation of p300 by MEKK1 or by other non-JNK MEKK1-directed downstream kinases . Finally, we present evidence that p300 is important for MEKK1 to induce apoptosis . Taken together, these results identify MEKK1 as a kinase that is likely to be involved in the regulation of the transactivation potential of p300 and support a role of p300 in MEKK1-induced apoptosis.

J Biol Chem, 2001 May 4, 276(18), 15051 - 8 Epub 2001 Jan 22.
Deoxycytidyl transferase activity of the human REV1 protein is closely associated with the conserved polymerase domain; Masuda Y et al.; The REV1 protein is a member of the growing family of translesion DNA polymerases . A cDNA of the human REV1 gene that we had originally isolated encoded 1250 amino acids residues, which was one amino acid shorter than previously reported ones . The shorter form of REV1 was named REV1S . All individuals examined expressed equivalent amounts of REV1S and REV1 mRNA, suggesting that the REV1S mRNA is a splicing variant . We show that the REV1S protein also possesses deoxycytidyl transferase activity that inserts a dCMP opposite a DNA template apurinic/apyrimidinic site . Deletion and point mutation analysis of the REV1S protein revealed that the domain required for deoxycytidyl transferase and DNA binding activities of the REV1S protein are located in a conserved domain of translesion DNA polymerases . This result indicates that the structure of the catalytic site of the deoxycytidyl transferase closely resembles that of the translesion DNA polymerases . Therefore, the molecular mechanism of the dCMP transfer reaction of the REV1S protein and maybe also the REV1 protein might be the same as that of the dNTP transfer reaction of the translesion DNA polymerases.

J Biol Chem, 2001 May 25, 276(21), 18513 - 8 Epub 2001 Feb 15.
Sumo-1 modification regulates the DNA binding activity of heat shock transcription factor 2, a promyelocytic leukemia nuclear body associated transcription factor; Goodson ML et al.; Heat shock transcription factor 2 (HSF2) is a transcription factor that regulates heat shock protein gene expression, but the mechanisms regulating the function of this factor are unclear . Here we report that HSF2 is a substrate for modification by the ubiquitin-related protein SUMO-1 and that HSF2 colocalizes in cells with SUMO-1 in nuclear granules . Staining with anti-promyelocytic leukemia antibodies indicates that these HSF2-containing nuclear granules are PML bodies . Our results identify lysine 82 as the major site of SUMO-1 modification in HSF2, which is located in a "wing" within the DNA-binding domain of this protein . Interestingly, SUMO-1 modification of HSF2 results in conversion of this factor to the active DNA binding form . This is the first demonstration that SUMO-1 modification can directly alter the DNA binding ability of a transcription factor and reveals a new mechanism by which SUMO-1 modification can regulate protein function.

J Biol Chem, 2001 Apr 6, 276(14), 10853 - 60 Epub 2001 Jan 12.
The regulation of uncoupling protein-2 gene expression by omega-6 polyunsaturated fatty acids in human skeletal muscle cells involves multiple pathways, including the nuclear receptor peroxisome proliferator-activated receptor beta; Chevillotte E et al.; Fatty acids have been postulated to regulate uncoupling protein (UCP) gene expression in skeletal muscle in vivo . We have identified, at least in part, the mechanism by which polyunsaturated fatty acids increase UCP-2 expression in primary culture of human muscle cells . omega-6 fatty acids and arachidonic acid induced a 3-fold rise in UCP-2 mRNA levels possibly through transcriptional activation . This effect was prevented by indomethacin and mimicked by prostaglandin (PG) E(2) and carbaprostacyclin PGI(2), consistent with a cyclooxygenase-mediated process . Incubation of myotubes for 6 h with 100 micrometer arachidonic acid resulted in a 150-fold increase in PGE(2) and a 15-fold increase in PGI(2) in the culture medium . Consistent with a role of cAMP and protein kinase A, both prostaglandins induced a marked accumulation of cAMP in human myotubes, and forskolin reproduced the effect of arachidonic acid on UCP-2 mRNA expression . Inhibition of protein kinase A with H-89 suppressed the effect of PGE(2), whereas cPGI(2) and arachidonic acid were still able to increase ucp-2 gene expression, suggesting additional mechanisms . We found, however, that the MAP kinase pathway was not involved . Prostaglandins, particularly PGI(2), are potent activators of the peroxisome proliferator-activated receptors . A specific agonist of peroxisome proliferator-activated receptor (PPAR) beta (L165041) increased UCP-2 mRNA levels in myotubes, whereas activation of PPARalpha or PPARgamma was ineffective . These results suggest thus that ucp-2 gene expression is regulated by omega-6 fatty acids in human muscle cells through mechanisms involving at least protein kinase A and the nuclear receptor PPARbeta.

J Biol Chem, 2001 May 11, 276(19), 16193 - 200 Epub 2001 Feb 02.
Endoplasmic reticulum (ER)-associated degradation of T cell receptor subunits . Involvement of ER-associated ubiquitin-conjugating enzymes (E2s); Tiwari S et al.; Degradation of proteins from the endoplasmic reticulum is fundamental to quality control within the secretory pathway, serves as a way of regulating levels of crucial proteins, and is utilized by viruses to enhance pathogenesis . In yeast two ubiquitin-conjugating enzymes (E2s), UBC6p and UBC7p are implicated in this process . We now report the characterization of murine homologs of these E2s . MmUBC6 is an integral membrane protein that is anchored via its hydrophobic C-terminal tail to the endoplasmic reticulum . MmUBC7, which is not an integral membrane protein, shows significant endoplasmic reticulum colocalization with MmUBC6 . Overexpression of catalytically inactive MmUBC7 significantly delayed degradation from the endoplasmic reticulum of two T cell antigen receptor subunits, alpha and CD3-delta, and suggests a role for the ubiquitin conjugating system at the initiation of retrograde movement from the endoplasmic reticulum . These findings also implicate, for the first time, a specific E2 in degradation from the endoplasmic reticulum in mammalian cells.

J Biol Chem, 2001 Apr 20, 276(16), 12749 - 55 Epub 2001 Jan 18.
Glutathione S-transferase mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1; Cho SG et al.; Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that can activate the c-Jun N-terminal kinase and the p38 signaling pathways . It plays a critical role in cytokine- and stress-induced apoptosis . To further characterize the mechanism of the regulation of the ASK1 signal, we searched for ASK1-interacting proteins employing the yeast two-hybrid method . The yeast two-hybrid assay indicated that mouse glutathione S-transferase Mu 1-1 (mGSTM1-1), an enzyme involved in the metabolism of drugs and xenobiotics, interacted with ASK1 . We subsequently confirmed that mGSTM1-1 physically associated with ASK1 both in vivo and in vitro . The in vitro binding assay indicated that the C-terminal portion of mGSTM1-1 and the N-terminal region of ASK1 were crucial for binding one another . Furthermore, mGSTM1-1 suppressed stress-stimulated ASK1 activity in cultured cells . mGSTM1-1 also blocked ASK1 oligomerization . The ASK1 inhibition by mGSTM1-1 occurred independently of the glutathione-conjugating activity of mGSTM1-1 . Moreover, mGSTM1-1 repressed ASK1-dependent apoptotic cell death . Taken together, our findings suggest that mGSTM1-1 functions as an endogenous inhibitor of ASK1 . This highlights a novel function for mGSTM1-1 insofar as mGSTM1-1 may modulate stress-mediated signals by repressing ASK1, and this activity occurs independently of its well-known catalytic activity in intracellular glutathione metabolism.

J Biol Chem, 2001 May 4, 276(18), 14614 - 22 Epub 2001 Jan 26.
DNA bends in TATA-binding protein-TATA complexes in solution are DNA sequence-dependent; Wu J et al.; The TATA-binding protein (TBP) initiates assembly of transcription preinitiation complexes on eukaryotic class II promoters, binding to and restructuring consensus and variant "TATA box" sequences . The sequence dependence of the DNA structure in TBP-TATA complexes has been investigated in solution using fluorescence resonance energy transfer . The mean 5'dye-3'dye distance varies significantly among oligomers bearing the adenovirus major late promoter sequence (AdMLP) and five single-site variants bound to Saccharomyces cerevisiae TBP, consistent with solution bend angles for AdMLP of 76 degrees and for the variants ranging from 30 degrees to 62 degrees . These solution bends contrast sharply with the corresponding co-crystal structures, which show approximately 80 degrees bends for all sequences . Transcription activities for these TATA sequences are strongly correlated with the solution bend angles but not with TBP-DNA binding affinities . Our results support a model in which transcription efficiency derives primarily from the sequence-dependent structure of the TBP-TATA binary complex . Specifically, the distance distribution for the average solution structure of the TBP-TATA complex may reflect the sequence-dependent probability for the complex to assume a conformation in which the TATA box DNA is severely bent . Upon assumption of this geometry, the binary complex becomes a target for binding and correctly orienting the other components of the preinitiation complex.

J Biol Chem, 2001 Apr 20, 276(16), 12497 - 500 Epub 2001 Mar 07.
Biochemical analysis of transcriptional repression by Drosophila histone deacetylase 1; Huang X et al.; To study the mechanisms by which deacetylases regulate transcription by RNA polymerase II, we investigated the biochemical properties of purified recombinant Drosophila histone deacetylase 1 (dHDAC1, also known as dRPD3) . We found that purified dHDAC1 and Gal4-dHDAC1 polypeptides possess substantial deacetylase activity . Thus, deacetylation by dHDAC1 does not require any additional cofactors . Gal4-dHDAC1, but not dHDAC1, was observed to repress transcription in vitro by about 2-3-fold from chromatin templates, but not from naked DNA templates, in a Gal4 site-dependent manner . This magnitude of repression is similar to that commonly seen by deacetylases in vivo, as assessed by treatment of cells with deacetylase inhibitors . Transcriptional repression by Gal4-dHDAC1 was blocked by the deacetylase inhibitor, FR901228, and thus, deacetylase activity correlates with repression . Single round transcription analyses showed that Gal4-dHDAC1 reduces the absolute number of productive initiation complexes with chromatin templates . Moreover, with chromatin templates that were assembled with completely purified components, Gal4-dHDAC1 was found to deacetylate nucleosomal histones as well as to repress transcription . These experiments provide biochemical evidence for the requirement of chromatin for transcriptional repression by dHDAC1 and further show that dHDAC1 acts to repress the transcription initiation process.

Genome Biol . 2000;1(3):REVIEWS1022 . Epub 2000 Sep 15.
The fork'ed path to mitosis; Jorgensen P et al.; A concurrence of genomic, reverse genetic and biochemical approaches has cracked the decade-long enigma concerning the identity of the transcription factors that control gene expression at the G2/M transition in the budding yeast cell cycle.

Curr Microbiol, 2001 Mar, 42(3), 217 - 24
Molecular characterization of the host-adapted pathogen Verticillium longisporum on the basis of a group-I intron found in the nuclear SSU-rRNA gene; Karapapa VK et al.; Verticillium wilt of oilseed rape is caused by the host-adapted pathogen Verticillium longisporum comb . nov . With one set of nuclear SSU-rRNA gene primers, a PCR amplification product of ca . 2.5 kb was generated from all isolates of V . longisporum tested (36 from Europe, Japan, and USA), with the exception of two recombinant isolates . On the contrary, all the other phytopathogenic and nonphytopathogenic species of Verticillium tested (18 species, 46 isolates), with the exception of one isolate of V . lecanii and two of Cordyceps sp., generated a product of ca . 1.65 kb . Sequence analysis of the SSU-rRNA gene of two typical isolates of V . longisporum (wild radish, Japan, and oilseed rape, Germany) revealed that this dimorphism was due to the presence of an identical 839-bp intron located in a highly conserved insertion position (nt 1165 of Saccharomyces cerevisiae) . The intron sequence was classified as group-I intron on the basis of conserved sequence and secondary structural elements . Primers designed from the 839-bp intron sequence amplified only the V . longisporum . Phylogenetic analysis based on SSU-rDNA sequences showed that V . longisporum was closely related to the genera of other filamentous Ascomycetes with fruiting bodies.

Genome Biol . 2001;2(3):REVIEWS1010 . Epub 2001 Mar 09.
Rhesus factors and ammonium: a function in efflux?
Ludewig U, von Wiren N, Rentsch D, Frommer WB.
Completion of fungal, plant and human genomes paved the way to the identification of erythrocytic rhesus proteins and their kidney homologs as ammonium transporters.

Nat Struct Biol, 2001 Apr, 8(4), 361 - 70
Multistep assembly of the protein import channel of the mitochondrial outer membrane; Model K et al.; Proteins targeted to mitochondria are transported into the organelle through a high molecular weight complex called the translocase of the outer mitochondrial membrane (TOM) . At the core of this machinery is a multisubunit general import pore (GIP) of 400 kDa . Here we report the assembly of the yeast GIP that involves two successive intermediates of 250 kDa and 100 kDa . The precursor of the channel-lining Tom40 is first targeted to the membrane via the receptor proteins Tom20 and Tom22; it then assembles with Tom5 to form the 250 kDa intermediate exposed to the intermembrane space . The 250 kDa intermediate is followed by the formation of the 100 kDa intermediate that associates with Tom6 . Maturation to the 400 kDa complex occurs by association of Tom7 and Tom22 . Tom7 functions by promoting both the dissociation of the 400 kDa complex and the transition from the 100 kDa intermediate to the mature complex . These results indicate that the dynamic conversion between the 400 kDa complex and the 100 kDa late intermediate allows integration of new precursor subunits into pre-existing complexes.

Proteins, 2001 May 1, 43(2), 227 - 32
Transcriptional activity of the TFIIA four-helix bundle in vivo; Stargell LA et al.; TFIIA contributes to transcription initiation by stabilizing the TBP-TATA interaction and by mediating the response to transcriptional activators and inhibitors . TFIIA contains a six-stranded beta-sheet domain and a four-helix bundle . The beta-domain makes functional contacts with DNA and TBP . The role of the four-helix bundle was investigated using a structure-based model of this domain (called 4HB) . 4HB adopts a highly stable, helical fold, consistent with its structure in the context of TFIIA . Like TBP and other intact transcription factors, 4HB is able to activate transcription in vivo when artificially recruited to a promoter via a heterologous DNA-binding domain . Thus, in addition to making important contacts with TBP and DNA via the beta-domain, TFIIA makes other specific, functional contacts with the transcriptional machinery via the four-helix bundle . Proteins 2001;43:227-232 .

J Biochem (Tokyo), 2001 Apr, 129(4), 635 - 41
Tip60 is a cell-type-specific transcriptional regulator; Hlubek F et al.; Tip60 was originally identified as cellular HIV-Tat interacting protein and has been shown to augment Tat-dependent transcription . It has also been shown to interact with various cellular transcription factors and to belong to the nuclear histone acetyltransferase (HAT) family . To further elucidate the function of Tip60 and its HAT domain in transcription regulation, we compared Tip60 activity in HeLa and Jurkat T lymphoma cells . Here we show that Tip60 augments the HIV-1 Tat activity at the HIV-LTR promoter in HeLa but inhibits it in Jurkat cells . Moreover, we isolated two new variants of the Tip60 protein (Tip60Delta1, Tip60Delta2) from Jurkat cells . The Tip60Delta2 variant lacks the entire HAT domain but modulates HIV-1 Tat activity like full-length Tip60 . In addition, Tip60 and the transcriptional repressor ZEB (zinc finger E box binding protein) interact specifically in the yeast two-hybrid system and additively inhibit the CD4 enhancer/promoter activity in Jurkat cells . Thus, Tip60 may function as corepressor of the ZEB protein . In summary, these data show that Tip60 functions as a cell-type-specific transcriptional regulator and that the HAT domain is not required for either transcriptional activation or inhibition . This indicates that Tip60 may function by recruiting additional cell-type-specific cofactors.

J Biochem (Tokyo), 2001 Apr, 129(4), 491 - 9
Participation of histones and histone-modifying enzymes in cell functions through alterations in chromatin structure; Nakayama T et al.; Alterations in the chromatin structure are preferentially involved in the regulation of cell functions, including gene expression, in eukaryotes . Three types of mechanisms, by which the alterations are caused have been reported: (i) variants of histone subtypes, (ii) chromatin remodeling, and (iii) post-translational modification . This review focuses mainly on the first and third mechanisms, especially on the acetylation of core histones, one of the third mechanisms . Using the gene targeting technique for the DT40 chicken B cell line, we systematically generated a number of mutants, respectively, devoid of particular genes encoding histones and histone deacetylase(s) (HDACs) . Most of the H1 and core histone variants should be involved positively or negatively in the transcription regulation of particular genes . Of the chicken HDACs (chHDACs), chHDAC-2 controls the amount of the IgM H-chain at the steps of both transcription and alternative pre-mRNA processing, and chHDAC-3 is essential for cell viability, whereas chHDAC-1 merely affects gene expression in DT40 cells . These results indicate that HDAC family members should participate, in combination with one another, and/or histone acetyltransferase(s) (HATs), in the acetylation of core histones that regulates gene expression through alterations in the chromatin structure.

J Biol Chem, 2001 Jun 15, 276(24), 20858 - 65 Epub 2001 Mar 26.
Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein . Implication for heme oxygenase-1 gene regulation; He CH et al.; Nrf2 regulates expression of genes encoding enzymes with antioxidant (e.g . heme oxygenase-1 (HO-1)) or xenobiotic detoxification (e.g . NAD(P)H:quinone oxidoreductase, glutathione S-transferase) functions via the stress- or antioxidant-response elements (StRE/ARE) . Nrf2 heterodimerizes with small Maf proteins, but the role of such dimers in gene induction is controversial, and other partners may exist . By using the yeast two-hybrid assay, we identified activating transcription factor (ATF) 4 as a potential Nrf2-interacting protein . Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation and mammalian two-hybrid assays . Furthermore, Nrf2.ATF4 dimers bound to an StRE sequence from the ho-1 gene . CdCl(2), a potent inducer of HO-1, increased expression of ATF4 in mouse hepatoma cells, and detectable induction of ATF4 protein preceded that of HO-1 (30 min versus 2 h) . A dominant-negative mutant of ATF4 inhibited basal and CdCl(2)-stimulated expression of a StRE-dependent/luciferase fusion construct (pE1-luc) in hepatoma cells but only basal expression in mammary epithelial MCF-7 cells . A dominant mutant of Nrf2 was equally inhibitory in both cell types in the presence or absence of CdCl(2) . These results indicate that ATF4 regulates basal and CdCl(2)-induced expression of the ho-1 gene in a cell-specific manner and possibly in a complex with Nrf2.

Mol Ther, 2001 Mar, 3(3), 278 - 83
Augmenting transgene expression from carcinoembryonic antigen (CEA) promoter via a GAL4 gene regulatory system; Koch PE et al.; Though extensively studied, the use of tissue- or cell-type-specific promoters to target transgene expression is hampered by their weak activity . We hypothesized that this problem could be addressed by using a GAL4 gene regulatory system, wherein a weak, tissue-specific promoter would drive expression of the GAL4/VP16 fusion protein (GV16), which in turn would transactivate a minimal synthetic promoter, GAL4/TATA (GT), upstream of a transgene . To test this hypothesis, we constructed adenoviral vectors expressing a lacZ or GV16 gene driven by a carcinoembryonic antigen (CEA) promoter (Ad/CEA-LacZ or Ad/CEA-GV16) and evaluated levels of transgene expression they produced in cultured cells and in subcutaneous tumors after intratumoral administration . In CEA-positive cells, treatment with Ad/CEA-GV16 + Ad/GT-LacZ versus Ad/CEA-LacZ increased transgene expression 20- to 100-fold . In CEA-negative cells, treatment with Ad/CEA-GV16 + Ad/GT-LacZ increased transgene expression to a much lower degree (6- to 8-fold) . In addition, analysis of Bax gene-mediated cell death revealed that this system can be used to avoid Bax's toxic effects on CEA-negative cells without compromising its ability to kill CEA-positive cells in vitro and in vivo . Thus, the combination of a tissue-specific promoter with the GAL4 gene regulatory system could be useful for targeting transgene expression.

J Mol Biol, 2001 Mar 30, 307(3), 815 - 25
The mitochondrial proteins Ssq1 and Jac1 are required for the assembly of iron sulfur clusters in mitochondria; Lutz T et al.; Mitochondria of the yeast Saccharomyces cerevisiae contain three different Hsp70 chaperones, Ssc1, Ecm10 and Ssq1 . Ssc1 is an essential protein that mediates the import of nuclear-encoded proteins into the organelle and their subsequent folding . The nucleotide state of Ssc1 is thereby regulated by the nucleotide exchange factor Mge1 . Here, we show that Mge1 interacts with Ssq1 in an ATP-dependent manner, suggesting that Mge1 also regulates Ssq1 function . In contrast to Ssc1, Ssq1 does not associate with the Tim44 subunit of the protein translocating complex, indicating a different function of both chaperones . Mutants in Ssq1 were reported to have low levels of iron sulfur (FeS) cluster-containing enzymes . Employing an assay that allowed us to monitor the conversion of the apoform of mitochondrial ferredoxin into its FeS-containing holoform, Ssq1 was demonstrated to be required for the FeS cluster assembly in mitochondria . The mitochondrial DnaJ homolog Jac1 is crucial for this process, whereas Mdj1 function is dispensable . Furthermore, the presence of frataxin is necessary for FeS cluster assembly into ferredoxin suggesting a role for frataxin at the level of the formation of holo-ferredoxin .

Curr Genet, 2001 Jan, 38(6), 314 - 22
Regulation of the Aspergillus nidulans hisB gene by histidine starvation; Busch S et al.; The hisB gene of the filamentous fungus Aspergillus nidulans encodes imidazole glycerol-phosphate dehydratase (E.C . 4.2.1.19), which catalyses the seventh enzymatic step in histidine biosynthesis . The gene was isolated and its deduced peptide sequence of 247 amino acids showed up to 54% identity with the IGPD enzymes of organisms comprising all three kingdoms . Expression of hisB cDNA in a Saccharomyces cerevisiae his3delta mutant strain functionally complemented the growth phenotype under histidine limitation . Addition of histidine did not affect hisB mRNA levels in A . nidulans wild-type cells . Histidine starvation conditions increased the hisB transcript level four-fold, suggesting regulation by a cross-pathway regulatory network . Deletion of the complete hisB open reading frame in A . nidulans strain A234 resulted in histidine auxotrophy . Additionally, hisB deletion strains were blocked from sexual fruiting body formation on medium containing low concentrations of histidine . This developmental phenotype of the hisB deletion mutant strain correlated with the induction of the cross-pathway control system.

EMBO Rep, 2000 Oct, 1(4), 319 - 22
The Cdc7/Dbf4 protein kinase: target of the S phase checkpoint?
Jares P, Donaldson A, Blow JJ.
Cdc7/Dbf4 is a protein kinase that is required for the initiation of DNA replication in eukaryotes . Recent work has provided new clues to the role that Cdc7/Dbf4 plays in this process . A range of other observations suggest that Cdc7/Dbf4 also plays another, less well characterized, role in checkpoint function and in the maintenance of genomic integrity . In this review we attempt to bring together new information to explain how Cdc7/Dbf4 may perform these two distinct functions.

Bioessays, 2001 Apr, 23(4), 333 - 43
The molecular machinery for lysosome biogenesis; Mullins C et al.; The lysosome serves as a site for delivery of materials targeted for removal from the eukaryotic cell . The mechanisms underlying the biogenesis of this organelle are currently the subject of renewed interest due to advances in our understanding of the protein sorting machinery . Genetic model systems such as yeast and Drosophila have been instrumental in identifying both protein and lipid components of this machinery . Importantly, many of these components, as well as the processes in which they are involved, are proving conserved in mammals . Other recently identified components, however, appear to be unique to higher eukaryotes . BioEssays 23:333-343, 2001 . Published 2001 John Wiley & Sons, Inc.

J Cell Physiol, 2001 May, 187(2), 236 - 43
Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity; VanderWaal RP et al.; The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress-inducing agents is the subject of much research . We have investigated the relationship between heat-induced damage to DNA replication machinery and the lethal effects of heat shock, in S-phase cells, which are more sensitive to heat shock than either G1 or G2 . We found that maintaining cells in aphidicolin, which prevents the passage of cells through S-phase, can rescue S-phase HeLa cells from the lethal effects of heat shock . When S-phase, HeLa cells were held for 5-6 h in 3 microM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells . It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co-isolate with the nuclear matrix . Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S-phase cells following heat shock under four different experimental conditions . Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S-phase correlated with cell killing . Heat-induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation . These results support the hypothesis that heat-induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S-phase progression but are repairable if S-phase progression is delayed .

Curr Biol, 2001 Mar 6, 11(5), R185 - 97
Chromatin remodeling enzymes: who's on first?
Fry CJ, Peterson CL.
A central problem in the regulation of eukaryotic gene expression is understanding how gene-specific transcriptional activators orchestrate the recruitment of the myriad proteins that are required for transcription initiation . An emerging view indicates that activators must first target two types of chromatin remodeling enzyme to the promoter region: an ATP-dependent SWI/SNF-like complex and a histone acetyltransferase . These two enzymes appear to act synergistically to establish a local chromatin structure that is permissive for subsequent events . Furthermore, several recent studies indicate that the recruitment of chromatin remodeling enzymes must follow an obligatory, sequential order of events that is determined by either promoter context or cell-cycle position . Here we review recent developments concerning the role of chromatin remodeling enzymes in gene regulation, and propose several models to explain how different chromatin remodeling activities can be functionally coupled.

Curr Biol, 2001 Mar 6, 11(5), R178 - 81
Cell cycle: Flies teach an old dogma new tricks; Cayirlioglu P et al.; E2F transcription factors are thought to influence the G1-S cell-cycle transition by controlling expression of genes required for growth and DNA synthesis . But emerging evidence suggests E2F complexes can control the cell cycle independently of transcription by directly regulating DNA replication origin usage during S phase.

Curr Biol, 2001 Mar 6, 11(5), 295 - 307
A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis; Steffensen S et al.; BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes . Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase . Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood . RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila . DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family . DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase . During decondensation in telophase, most of the DmSMC4 leaves the chromosomes . An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages . A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted . This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis . CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.

Mutat Res, 2001 Apr 4, 485(3), 237 - 53
Isolation and genetic characterisation of the Drosophila homologue of (SCE)REV3, encoding the catalytic subunit of DNA polymerase zeta; Eeken JC et al.; In Drosophila, about 30 mutants are known that show hypersensitivity to the methylating agent methyl methane sulfonate (MMS) . Addition of this agent to the medium results in an increased larval mortality of the mutants . Using a P-insertion mutagenesis screen, three MMS-sensitive mutants on chromosome II were isolated . One of these is allelic to the known EMS-induced mus205 (mutagen sensitive) mutant . In the newly isolated mutant, a P-element is detected in region 43E by in situ hybridisation . The localisation of mus205 to this region was confirmed by deficiency mapping . The gene was cloned and shows strong homology to the Saccharomyces cerevisiae REV3 gene . The REV3 gene encodes the catalytic subunit of DNA polymerase zeta, involved in translesion synthesis . The P-element is inserted in the first exon of the mus205 gene resulting in an aberrant mRNA, encoding a putative truncated protein containing only the first 13 of the 2130 aa native Drosophila protein . The mus205 mutant is hypersensitive to alkylating agents and UV, but not to ionising radiation . In contrast to reported data, in germ cells, the mutant has no effect on mutability by X-rays, NQO and alkylating agents . In somatic cells, the mutant shows no effect on MMS-induced mutations and recombinations . This phenotype of the Drosophila mus205 mutant is strikingly different from the phenotype of the yeast rev3 mutant, which is hypomutable after UV, X-rays, NQO and alkylating agents.

Biochim Biophys Acta, 2001 Mar 19, 1518(1-2), 7 - 18
Cloning and functional characterization of chicken p160 coactivator family members; Arai S et al.; The factors SRC-1, TIF2 and ACTR were identified as interacting with nuclear receptors in a highly ligand-dependent manner . Because the molecular mass of each of these factors is approximately 160 kDa, they are collectively termed p160 coactivators . So far, p160 coactivators have been cloned from human, mouse and Xenopus . We report here the cloning of the chicken homologues of p160 coactivator members . As in human and mouse, chicken has three p160 coactivators . Each gene encodes an approximately 160 kDa protein which exhibits 70-80% amino acid sequence identity to human and mouse p160 coactivators . Chicken p160 coactivators also have the property of interacting with several liganded nuclear receptors . Moreover, we describe an imperfect LXXLL sequence, termed NR box 4, which is located downstream of NR box 3 and conserved between evolutionarily diverse species . The loss of NR box 4 results in a decrease of interaction with the nuclear receptor, which indicates that NR box 4 is required for efficient interaction.

Protein Sci, 2001 Jan, 10(1), 24 - 33
An engineered leucine zipper a position mutant with an unusual three-state unfolding pathway; Zhu H et al.; The leucine zipper is a dimeric coiled-coil structural motif consisting of four to six heptad repeats, designated (abcdefg)(n) . In the GCN4 leucine zipper, a position 16 in the third heptad is occupied by an Asn residue whereas the other a positions are Val residues . Recently, we have constructed variants of the GCN4 leucine zipper in which the a position Val residues were replaced by Ile . The folding and unfolding of the wild-type GCN4 leucine zipper and the Val to Ile variant both adhere to a simple two-state mechanism . In this study, another variant of the GCN4 leucine zipper was constructed by moving the single Asn residue from a position 16 to a position 9 . This switch causes the thermal unfolding of the GCN4 leucine zipper to become three state . The unfolding pathway of this variant was determined by thermal denaturation, limited proteinase K digestion, and sedimentation equilibrium analysis . Our data are consistent with a model in which the variant first unfolds from its N terminus and changes the oligomerization specificity from a native dimer to a partially unfolded intermediate containing a mixture of dimers and trimers and then completely unfolds to unstructured monomers.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1638 - 46
Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase; Purugganan MM et al.; V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA . Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity . The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors . We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system . One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed . Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT . Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice . In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active . Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1524 - 33
Transcriptional adaptor and histone acetyltransferase proteins in Arabidopsis and their interactions with CBF1, a transcriptional activator involved in cold-regulated gene expression; Stockinger EJ et al.; The ARABIDOPSIS CBF transcriptional activators bind to the CRT/DRE regulatory element present in the promoters of many cold-regulated genes and stimulate their transcription . Expression of the CBF1 proteins in yeast activates reporter genes carrying a minimal promoter with the CRT/DRE as an upstream regulatory element . Here we report that this ability of CBF1 is dependent upon the activities of three key components of the yeast Ada and SAGA complexes, namely the histone acetyltransferase (HAT) Gcn5 and the transcriptional adaptor proteins Ada2 and Ada3 . This result suggested that CBF1 might function through the action of similar complexes in ARABIDOPSIS In support of this hypothesis we found that ARABIDOPSIS has a homolog of the GCN5 gene and two homologs of ADA2, the first report of multiple ADA2 genes in an organism . The ARABIDOPSIS GCN5 protein has intrinsic HAT activity and can physically interact in vitro with both the ARABIDOPSIS ADA2a and ADA2b proteins . In addition, the CBF1 transcriptional activator can interact with the ARABIDOPSIS GCN5 and ADA2 proteins . We conclude that ARABIDOPSIS encodes HAT-containing adaptor complexes that are related to the Ada and SAGA complexes of yeast and propose that the CBF1 transcriptional activator functions through the action of one or more of these complexes.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1410 - 9
All Tcf HMG box transcription factors interact with Groucho-related co-repressors; Brantjes H et al.; Tcf/Lef family transcription factors are the downstream effectors of the Wingless/Wnt signal transduction pathway . Upon Wingless/Wnt signalling, beta-catenin translocates to the nucleus, interacts with Tcf (1-3) and thus activates transcription of target genes (4,5) . Tcf factors also interact with members of the Groucho (Grg/TLE) family of transcriptional co-repressors (6) . We have now tested all known mammalian Groucho family members for their ability to interact specifically with individual Tcf/Lef family members . Transcriptional activation by any Tcf could be repressed by Grg-1, Grg-2/TLE-2, Grg-3 and Grg-4 in a reporter assay . Specific interactions between Tcf and Grg proteins may be achieved in vivo by tissue- or cell type-limited expression . To address this, we determined the expression of all Tcf and Grg/TLE family members in a panel of cell lines . Within any cell line, several Tcfs and TLEs are co-expressed . Thus, redundancy in Tcf/Grg interactions appears to be the rule . The 'long' Groucho family members containing five domains are repressors of Tcf-mediated transactivation, whereas Grg-5, which only contains the first two domains, acts as a de-repressor . As previously shown for DROSOPHILA: Groucho, we show that long Grg proteins interact with histone deacetylase-1 . Although Grg-5 contains the GP homology domain that mediates HDAC binding in long Grg proteins, Grg-5 fails to bind this co-repressor, explaining how it can de-repress transcription.

J Cell Biol, 2001 Jan 22, 152(2), 401 - 10
Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability; Hobbs AE et al.; In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell . Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles . To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells . We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids . We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature . Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics . Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains . We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.

J Cell Biol, 2001 Jan 22, 152(2), 289 - 300
Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex; Krimmer T et al.; Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane . The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial . We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin . Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria . A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore . Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane {TOM} core complex) . The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40 . Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway . We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

Nat Rev Mol Cell Biol, 2001 Mar, 2(3), 211 - 6
Molecular dissection of autophagy: two ubiquitin-like systems; Ohsumi Y; Recent analyses of the genes required for autophagy--intracellular bulk protein degradation--in yeast have revealed two ubiquitin-like systems, both of which are involved in the membrane dynamics of the process . Molecular dissection of these systems is now revealing some surprises.

Nat Rev Mol Cell Biol, 2001 Mar, 2(3), 202 - 10
SUMO, ubiquitin's mysterious cousin; Muller S et al.; Covalent modification of cellular proteins by the ubiquitin-like modifier SUMO regulates various cellular processes, such as nuclear transport, signal transduction, stress response and cell-cycle progression . But, in contrast to ubiquitylation, sumoylation does not tag proteins for degradation, but seems to enhance their stability or modulate their subcellular compartmentalization.

Nat Rev Mol Cell Biol, 2001 Mar, 2(3), 195 - 201
Protein regulation by monoubiquitin; Hicke L; Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitylated proteins by the proteasome, but other functions of ubiquitin have been discovered that do not involve the proteasome . Some proteins are modified by a single ubiquitin or short ubiquitin chains . Instead of sending proteins to their death through the proteasome, monoubiquitylation regulates processes that range from membrane transport to transcriptional regulation.

Acc Chem Res, 2001 Feb, 34(2), 119 - 28
Copper delivery by metallochaperone proteins; Rosenzweig AC; Copper is an essential element in all living organisms, serving as a cofactor for many important proteins and enzymes . Metallochaperone proteins deliver copper ions to specific physiological partners by direct protein-protein interactions . The Atx1-like chaperones transfer copper to intracellular copper transporters, and the CCS chaperones shuttle copper to copper,zinc superoxide dismutase . Crystallographic studies of these two copper chaperone families have provided insights into metal binding and target recognition by metallochaperones and have led to detailed molecular models for the copper transfer mechanism.

Pac Symp Biocomput . 2001;:115-26.
SAMIE: statistical algorithm for modeling interaction energies; Benos PV et al.; We are investigating the rules that govern protein-DNA interactions, using a statistical mechanics based formalism that is related to the Boltzmann Machine of the neural net literature . Our approach is data-driven, in which probabilistic algorithms are used to model protein-DNA interactions, given SELEX and/or phage data as input . In the current report, we trained the network using SELEX data, under the "one-to-one" model of interactions (i.e . one amino acid contacts one base) . The trained network was able to successfully identify the wild-type binding sites of EGR and MIG protein families . The predictions using our method are the same or better than that of methods existing in the literature . However our methodology offers the potential to capitalise in quantitative detail, as well as to be used to explore more general model of interactions, given availability of data.

J Biol Chem, 2001 May 18, 276(20), 16597 - 600 Epub 2001 Mar 21.
Tip60 and HDAC7 interact with the endothelin receptor a and may be involved in downstream signaling; Lee HJ et al.; Endothelins exert their biological effects through G protein-coupled receptors . However, the precise mechanism of downstream signaling and trafficking of the receptors is largely unknown . Here we report that the histone acetyltransferase Tip60 and the histone deacetylase HDAC7 interact with one of the ET receptors, ETA, as determined by yeast two-hybrid analysis, glutathione S-transferase pull-down assays, and co-immunoprecipitation from transfected COS-7 cells . In the absence of ET-1, Tip60 and HDAC7 were localized mainly in the cell nucleus while ETA was predominantly confined to the plasma membrane . Stimulation with ET-1 resulted in the internalization of ETA to the perinuclear compartment and simultaneously in the efflux of Tip60 and HDAC7 from the nucleus to the same perinuclear compartment where each protein co-localized with the receptor . Upon co-transfection with ETA into COS-7 cells, Tip60 strongly increased ET-1-induced ERK1/2 phosphorylation, whereas HDAC7 had no significant effect . We thus suggest that protein acetylase and deacetylase interact with ETA in a ligand-dependent fashion and may participate in ET signal transduction.

J Agric Food Chem, 2001 Feb, 49(2), 633 - 40
Determination of estrogenic activity in beer by biological and chemical means; Promberger A et al.; It has been suspected that beer drinking may change the hormonal status of men caused by phytoestrogens . Five different Austrian lager beers have been investigated for estrogenic activity by a yeast two-plasmid system harboring the human estrogen receptor alpha, after concentration by solid phase extraction . The beer concentrate was further fractionated by reversed phase HPLC, and then the fractions were characterized by the biological assay and GC-MS . The most potent fraction did not contain a known phytoestrogen . The total activity corresponded to an average of 43 ng of 17beta-estradiol/L of beer . It was concluded that the human health hazard of beer drinking originating from compounds activity on the estrogen receptor alpha is negligible.

Mol Microbiol, 2001 Mar, 39(6), 1638 - 50
Mammalian 14-3-3beta associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG; Scidmore MA et al.; Chlamydiae replicate intracellularly within a vacuole that is modified early in infection to become fusogenic with a subset of exocytic vesicles . We have recently identified four chlamydial inclusion membrane proteins, IncD-G, whose expression is detected within the first 2 h after internalization . To gain a better understanding of how these Inc proteins function, a yeast two-hybrid screen was employed to identify interacting host proteins . One protein, 14-3-3beta, was identified that interacted specifically with IncG . The interaction between 14-3-3beta and IncG was confirmed in infected HeLa cells by indirect immunofluorescence microscopy and interaction with a GFP-14-3-3beta fusion protein . 14-3-3 proteins are phosphoserine-binding proteins . Immunoprecipitation studies with {32P}-orthophosphate-labelled cells demonstrated that IncG is phosphorylated in both chlamydia-infected HeLa cells and in yeast cells expressing IncG . Site-directed mutagenesis of predicted 14-3-3 phosphorylation sites demonstrated that IncG binds to 14-3-3beta via a conserved 14-3-3-binding motif (RS164RS166F) . Finally, indirect immunofluorescence demonstrated that 14-3-3beta interacts with Chlamydia trachomatis inclusions but not C . psittaci or C . pneumoniae inclusions . 14-3-3beta is the first eukaryotic protein found to interact with the chlamydial inclusion; however, its unique role in C . trachomatis pathogenesis remains to be determined.

Mol Microbiol, 2001 Mar, 39(6), 1482 - 93
RcoA has pleiotropic effects on Aspergillus nidulans cellular development; Hicks J et al.; Aspergillus nidulans rcoA encodes a member of the WD repeat family of proteins . The RcoA protein shares sequence similarity with other members of this protein family, including the Saccharomyces cerevisiae Tup1p and Neurospora crassa RCO1 . Tup1p is involved in negative regulation of an array of functions including carbon catabolite repression . RCO1 functions in regulating pleiotropic developmental processes, but not carbon catabolite repression . In A . nidulans, deletion of rcoA (DeltarcoA), a recessive mutation, resulted in gross defects in vegetative growth, asexual spore production and sterigmatocystin (ST) biosynthesis . Expression of the asexual and ST pathway-specific regulatory genes, brlA and aflR, respectively, but not the signal transduction genes (i.e . flbA, fluG or fadA) regulating brlA and aflR expression was delayed (brlA) or eliminated (aflR) in a DeltarcoA strain . Overexpression of aflR in a DeltarcoA strain could not rescue normal expression of downstream targets of AflR . CreA-dependent carbon catabolite repression of starch and ethanol utilization was only weakly affected in a DeltarcoA strain . The strong role of RcoA in development, vegetative growth and ST production, compared with a relatively weak role in carbon catabolite repression, is similar to the role of RCO1 in N . crassa.

Genes Cells, 2001 Mar, 6(3), 215 - 24
The Drosophila UBC9 homologue lesswright mediates the disjunction of homologues in meiosis I; Apionishev S et al.; BACKGROUND: In Saccharomyces cerevisiae and other organisms, the UBC9 (ubiquitin-conjugating 9) protein modifies the function of many different target proteins through covalent attachment of the ubiquitin-like protein SMT-3/SUMO . RESULTS: Using a second-site suppression screen of a mutation in the nod locus with a variable meiotic phenotype, we have identified mutations in the Drosophila melanogaster UBC9 homologue, encoded by the gene lesswright (lwr) . lwr mutations dominantly suppress the nondisjunction and cytological defects of female meiotic mutations that affect spindle formation . The lwr lethal phenotype is rescued by a Drosophila UBC9/lwr transgene . CONCLUSIONS: We suggest that LWR mediates the dissociation of heterochromatic regions of homologues at the end of meiotic prophase I . Our model proposes that when there is less LWR protein, homologues remain together longer, allowing for more normal spindle formation in mutant backgrounds and therefore more accurate meiotic chromosome segregation.

Genes Cells, 2001 Feb, 6(2), 107 - 19
Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C; Noda Y et al.; BACKGROUND: Asymmetric cell division in the Caenorhabditis elegans embryos requires products of par (partitioning defective) genes 1-6 and atypical protein kinase C (aPKC), whereas Cdc42 and Rac, members of the Rho family GTPases, play an essential role in cell polarity establishment in yeast and mammalian cells . However, little is known about a link between PAR proteins and the GTPases in cell polarization . RESULTS: Here we have cloned cDNAs for three human homologues of PAR6, designated PAR6alpha, beta and gamma, comprising 345, 372 and 376 amino acids, respectively . The PAR6 proteins harbour a PDZ domain and a CRIB-like motif, and directly interact with GTP-bound Rac and Cdc42 via this motif and with the aPKC isoforms PKCiota/lambda and PKCzeta via the N-terminal head-to-head association . These interactions are not mutually exclusive, thereby allowing the PAR6 proteins to form a ternary complex with the GTPases and aPKC, both in vitro and in vivo . When PAR6 and aPKC are expressed with a constitutively active form of Rac in HeLa or COS-7 cells, these proteins co-localize to membrane ruffles, which are known to occur at the leading edge of polarized cells during cell movement . CONCLUSION: Human PAR6 homologues most likely play an important role in the cell polarization of mammalian cells, by functioning as an adaptor protein that links activated Rac and Cdc42 to aPKC signalling.

Genes Cells, 2001 Feb, 6(2), 79 - 86
Histone acetylases--versatile players; Nakatani Y; Genomic DNA in eukaryotes is tightly packed in the form of a highly ordered chromatin structure . In view of this tight packing, one of the most important questions in biology is how the transcriptional machinery regulates target genes in chromatin . Reversible modification of histones by acetylation is involved in transcriptional activation as well as repression in chromatin contexts . Recent studies with highly purified histone acetylases have provided insights into the mechanisms whereby acetylases contribute to transcriptional control . Furthermore, they suggest the possibility that histone acetylases could play roles in various forms of DNA metabolism as well as in transcription in chromatin contexts.

Clin Genet, 2001 Feb, 59(2), 122 - 7
The "flap" endonuclease gene FEN1 is excluded as a candidate gene implicated in the CAG repeat expansion underlying Huntington disease; Otto CJ et al.; At least 12 disorders including Huntington disease (HD) are associated with expansion of a trinucleotide repeat (TNR) . Factors contributing to the risk of expansion of TNRs and the mechanism of expansion have not been elucidated . Data from Saccharomyces cerevisiae suggest that the flap endonuclease FEN1 plays a role in expansion of repetitive DNA tracts . It has been hypothesized that insufficiency of FEN1 or a mutant FEN1 might contribute to the occurrence of expansion events of long repetitive DNA tracts after polymerase slippage events during lagging strand synthesis . The expression pattern of FEN1 was determined, and ubiquitous tissue expression, including germ cells, suggested that FEN1 has the potential to be involved in HD . Fifteen HD parent/child pairs that demonstrated intergenerational increases in CAG length of greater than 10 repeats were examined for possible mutations or polymorphisms within the FEN1 gene that could underlie the saltatory repeat expansions seen in these individuals . No alterations were observed compared to 50 controls, excluding FEN1 as a trans-acting factor underlying TNR expansion . The identification of a candidate gene(s) in HD or other CAG-expansion disorders implicated in TNR instability will elucidate the mechanism of expansion for this growing family of neurological disorders.

Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3738 - 43 Epub 2001 Mar 20.
SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP-N- acetylgalactosamine, and UDP-galactose; Berninsone P et al.; Caenorhabditis elegans sqv mutants are defective in vulval epithelial invagination and have a severe reduction in hermaphrodite fertility . The gene sqv-7 encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi membrane . A Golgi vesicle enriched fraction of Saccharomyces cerevisiae expressing SQV-7 transported UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose (Gal) in a temperature-dependent and saturable manner . These nucleotide sugars are competitive, alternate, noncooperative substrates . The two mutant sqv-7 missense alleles resulted in a severe reduction of these three transport activities . SQV-7 did not transport CMP-sialic acid, GDP-fucose, UDP-N-acetylglucosamine, UDP-glucose, or GDP-mannose . SQV-7 is able to transport UDP-Gal in vivo, as shown by its ability to complement the phenotype of Madin-Darby canine kidney ricin resistant cells, a mammalian cell line deficient in UDP-Gal transport into the Golgi . These results demonstrate that unlike most nucleotide sugar transporters, SQV-7 can transport multiple distinct nucleotide sugars . We propose that SQV-7 translocates multiple nucleotide sugars into the Golgi lumen for the biosynthesis of glycoconjugates that play a pivotal role in development.

Mol Cell Biol, 2001 Apr, 21(7), 2545 - 54
NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells; Guzik BW et al.; TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells . We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells . To do this, the TAP gene was fused in frame to either RevM10 or RevDelta78-79 . These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm . However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function . Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export . Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped to the C-terminal region of TAP and required the domain that interacts with NXT1, as well as the region that has been shown to interact with nucleoporins . RevM10-TAP function was leptomycin B insensitive . In contrast, the function of this protein was inhibited by DeltaCAN, a protein consisting of part of the FG repeat domain of CAN/Nup214 . These results show that TAP can complement Rev nuclear export signal function and redirect the export of intron-containing RNA to a CRM1-independent pathway . These experiments support the role of TAP as an RNA export factor in mammalian cells . In addition, they indicate that NXT1 serves as a crucial cellular cofactor in this process.

Mol Cell Biol, 2001 Apr, 21(7), 2506 - 20
F-box protein Grr1 interacts with phosphorylated targets via the cationic surface of its leucine-rich repeat; Hsiung YG et al.; The flexibility and specificity of ubiquitin-dependent proteolysis are mediated, in part, by the E3 ubiquitin ligases . One class of E3 enzymes, SKp1/cullin/F-box protein (SCF), derives its specificity from F-box proteins, a heterogeneous family of adapters for target protein recognition . Grr1, the F-box component of SCF(Grr1), mediates the interaction with phosphorylated forms of the G(1) cyclins Cln1 and Cln2 . We show that binding of Cln2 by SCF(Grr1) was dependent upon its leucine-rich repeat (LRR) domain and its carboxy terminus . Our structural model for the Grr1 LRR predicted a high density of positive charge on the concave surface of the characteristic horseshoe structure . We hypothesized that specific basic residues on the predicted concave surface are important for recognition of phosphorylated Cln2 . We show that point mutations that converted the basic residues on the concave surface but not those on the convex surface to neutral or acidic residues interfered with the capacity of Grr1 to bind to Cln2 . The same mutations resulted in the stabilization of Cln2 and Gic2 and also in a spectrum of phenotypes characteristic of inactivation of GRR1, including hyperpolarization and enhancement of pseudohyphal growth . It was surprising that the same residues were not important for the role of Grr1 in nutrient-regulated transcription of HXT1 or AGP1 . We concluded that the cationic nature of the concave surface of the Grr1 LRR is critical for the recognition of phosphorylated targets of SCF(Grr1) but that other properties of Grr1 are required for its other functions.

Mol Cell Biol, 2001 Apr, 21(7), 2435 - 48
Heterozygous disruption of the TATA-binding protein gene in DT40 cells causes reduced cdc25B phosphatase expression and delayed mitosis; Um M et al.; TATA-binding protein (TBP) is a key general transcription factor required for transcription by all three nuclear RNA polymerases . Although it has been intensively analyzed in vitro and in Saccharomyces cerevisiae, in vivo studies of vertebrate TBP have been limited . We applied gene-targeting techniques using chicken DT40 cells to generate heterozygous cells with one copy of the TBP gene disrupted . Such TBP-heterozygous (TBP-Het) cells showed unexpected phenotypic abnormalities, resembling those of cells with delayed mitosis: a significantly lower growth rate, larger size, more G2/-M- than G1-phase cells, and a high proportion of sub-G1, presumably apoptotic, cells . Further evidence for delayed mitosis in TBP-Het cells was provided by the differential effects of several cell cycle-arresting drugs . To determine the cause of these defects, we first examined the status of cdc2 kinase, which regulates the G2/M transition, and unexpectedly observed more hyperphosphorylated, inactive cdc2 in TBP-Het cells . Providing an explanation for this, mRNA and protein levels of cdc25B, the trigger cdc2 phosphatase, were significantly and specifically reduced . These properties were all due to decreased TBP levels, as they could be rescued by expression of exogeneous TBP, including, in most but not all cases, a mutant form lacking the species-specific N-terminal domain . Our results indicate that small changes in TBP concentration can have profound effects on cell growth in vertebrate cells.

Mol Cell Biol, 2001 Apr, 21(7), 2349 - 58
Novel function of Rad27 (FEN-1) in restricting short-sequence recombination; Negritto MC et al.; Saccharomyces cerevisiae mutants lacking the structure-specific nuclease Rad27 display an enhancement in recombination that increases as sequence length decreases, suggesting that Rad27 preferentially restricts recombination between short sequences . Since wild-type alleles of both RAD27 and its human homologue FEN1 complement the elevated short-sequence recombination (SSR) phenotype of a rad27-null mutant, this function may be conserved from yeast to humans . Furthermore, mutant Rad27 and FEN-1 enzymes with partial flap endonuclease activity but without nick-specific exonuclease activity partially complement the SSR phenotype of the rad27-null mutant . This suggests that the endonuclease activity of Rad27 (FEN-1) plays a role in limiting recombination between short sequences in eukaryotic cells.

Mol Cell Biol, 2001 Apr, 21(7), 2337 - 48
Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors; Meisinger C et al.; The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a approximately 400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5 . We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH . Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex . A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex . Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex . Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex . Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins . The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

Mol Cell Biol, 2001 Apr, 21(7), 2281 - 91
Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome; Araujo SJ et al.; In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF . Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae . Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed . However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions . In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads . Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins . We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form . There was no evidence for a repair complex that contained all of the NER components . At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA . At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors . The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair . A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.

Mol Cell Biol, 2001 Apr, 21(7), 2249 - 58
MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein; Ernst P et al.; A fragment of the mixed-lineage leukemia (MLL) gene (Mll, HRX, ALL-1) was identified in a yeast genetic screen designed to isolate proteins that interact with the CREB-CREB-binding protein (CBP) complex . When tested for binding to CREB or CBP individually, this MLL fragment interacted directly with CBP, but not with CREB . In vitro binding experiments refined the minimal region of interaction to amino acids 2829 to 2883 of MLL, a potent transcriptional activation domain, and amino acids 581 to 687 of CBP (the CREB-binding or KIX domain) . The transactivation activity of MLL was dependent on CBP, as either adenovirus E1A expression, which inhibits CBP activity, or alteration of MLL residues important for CBP interaction proved effective at inhibiting MLL-mediated transactivation . Single amino acid substitutions within the MLL activation domain revealed that five hydrophobic residues, potentially forming a hydrophobic face of an amphipathic helix, were critical for the interaction of MLL with CBP . Using purified components, we found that the MLL activation domain facilitated the binding of CBP to phosphorylated CREB . In contrast with paradigms in which factors compete for limiting quantities of CBP, these results reveal that two distinct transcription factor activation domains can cooperatively target the same motif on CBP.

J Pharmacol Exp Ther, 2001 Apr, 297(1), 114 - 20
Discovery of a novel antitumor benzolactone enamide class that selectively inhibits mammalian vacuolar-type (H+)-atpases; Boyd MR et al.; A series of naturally occurring compounds reported recently by multiple laboratories defines a new small-molecule class sharing a unique benzolactone enamide core structure and diverse biological actions, including inhibition of growth of tumor cells and oncogene-transformed cell lines . Here we show that representative members of this class, including salicylihalamide A, lobatamides A-F, and oximidines I and II inhibit mammalian vacuolar-type (H+)-ATPases (V-ATPases) with unprecedented selectivity . Data derived from the NCI 60-cell antitumor screen critically predicted the V-ATPase molecular target, while specific biochemical assays provided confirmation and further illumination . The compounds potently blocked representative V-ATPases from human kidney, liver, and osteoclastic giant-cell tumor of bone but were essentially inactive against V-ATPases of Neurospora crassa and Saccharomyces cerevisiae and other membrane ATPases . Essential regulation of pH in cytoplasmic, intraorganellar, and local extracellular spaces is provided by V-ATPases, which are ubiquitously distributed among eukaryotic cells and tissues . The most potent and selective V-ATPase inhibitors heretofore known were the bafilomycins and concanamycins, which do not discriminate between mammalian and nonmammalian V-ATPases . Numerous physiological processes are mediated by V-ATPases, and aberrant V-ATPase functions are implicated in many different human diseases . Previous efforts to develop therapeutic pharmacological modulators of V-ATPases have been frustrated by a lack of synthetically tractable and biologically selective leads . Therefore, availability of the unique benzolactone enamide inhibitor class may enable further elucidation of functional and architectural features of mammalian versus nonmammalian V-ATPase isoforms and provide new opportunities for targeting V-ATPase-mediated processes implicated in diverse pathophysiological phenomena, including cancer.

J Biol Chem, 2001 Jun 8, 276(23), 20536 - 43 Epub 2001 Mar 19.
Overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates nuclear mRNA export; Braun IC et al.; Human TAP and its yeast orthologue Mex67p are members of the multigene family of NXF proteins . A conserved feature of NXFs is a leucine-rich repeat domain (LRR) followed by a region related to the nuclear transport factor 2 (the NTF2-like domain) . The NTF2-like domain of metazoan NXFs heterodimerizes with a protein known as p15 or NXT . A C-terminal region related to ubiquitin-associated domains (the UBA-like domain) is present in most, but not all NXF proteins . Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans NXF1 are essential for the export of messenger RNA from the nucleus . Human TAP mediates the export of simian type D retroviral RNAs bearing the constitutive transport element, but the precise role of TAP and p15 in mRNA nuclear export has not yet been established . Here we show that overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates the export of mRNAs that are otherwise exported inefficiently . This stimulation of mRNA export is strongly reduced by removing the UBA-like domain of TAP and abolished by deleting the LRR domain or the NTF2-like domain . Similar results are obtained when TAP/p15 heterodimers are directly tethered to the RNA export cargo . Our data indicate that formation of TAP/p15 heterodimers is required for TAP-mediated export of mRNA and show that the LRR domain of TAP plays an essential role in this process.

Biochemistry, 2001 Mar 6, 40(9), 2790 - 6
Stabilization of coiled-coil peptide domains by introduction of trifluoroleucine; Tang Y et al.; Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-p1d increases the thermal stability of the coiled-coil structure . The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13 degrees C at 30 microM peptide concentration . The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5-1.2 kcal/mol larger than that of the hydrogenated form . A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated form, while demonstrating enhanced thermal stability . Molecular dynamics simulation on the fluorinated GCN4-p1d peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination . These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor-ligand interactions.

Electrophoresis, 2001 Feb, 22(3), 552 - 9
A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics; Brancia FL et al.; The identification of individual protein species within an organism's proteome has been optimised by increasing the information produced from mass spectral analysis through the chemical derivatisation of tryptic peptides and the development of new software tools . Peptide fragments are subjected to two forms of derivatisation . First, lysine residues are converted to homoarginine moieties by guanidination . This procedure has two advantages, first, it usually identifies the C-terminal amino acid of the tryptic peptide and also greatly increases the total information content of the mass spectrum by improving the signal response of C-terminal lysine fragments . Second, an Edman-type phenylthiocarbamoyl (PTC) modification is carried out on the N-terminal amino acid . The renders the first peptide bond highly susceptible to cleavage during mass spectrometry (MS) analysis and consequently allows the ready identification of the N-terminal residue . The utility of the procedure has been demonstrated by developing novel bioinformatic tools to exploit the additional mass spectral data in the identification of proteome proteins from the yeast Saccharomyces cerevisiae . With this combination of novel chemistry and bioinformatics, it should be possible to identify unambiguously any yeast protein spot or band from either two-dimensional or one-dimensional electropheretograms.

EMBO Rep, 2001 Feb, 2(2), 124 - 32
An activation-independent role of transcription factors in insulator function; Fourel G et al.; Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter . Here we show that yeast subtelomeric anti-silencing regions behave as boundaries to telomere-driven silencing and also allow discontinuous propagation of silent chromatin . These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA . Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter . These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains.

J Cell Biol, 2001 Mar 19, 152(6), 1267 - 78
Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication; Furstenthal L et al.; Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases . In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA . We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs . Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts . Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts . Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication . During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity . In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis . In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin . Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro . These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.

J Cell Biol, 2001 Mar 19, 152(6), 1123 - 34
UGO1 encodes an outer membrane protein required for mitochondrial fusion; Sesaki H et al.; Membrane fusion plays an important role in controlling the shape, number, and distribution of mitochondria . In the yeast Saccharomyces cerevisiae, the outer membrane protein Fzo1p has been shown to mediate mitochondrial fusion . Using a novel genetic screen, we have isolated new mutants defective in the fusion of their mitochondria . One of these mutants, ugo1, shows several similarities to fzo1 mutants . ugo1 cells contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells . ugo1 mutants lose mitochondrial DNA (mtDNA) . In zygotes formed by mating two ugo1 cells, mitochondria do not fuse and mix their matrix contents . Fragmentation of mitochondria and loss of mtDNA in ugo1 mutants are rescued by disrupting DNM1, a gene required for mitochondrial division . We find that UGO1 encodes a 58-kD protein located in the mitochondrial outer membrane . Ugo1p appears to contain a single transmembrane segment, with its NH(2) terminus facing the cytosol and its COOH terminus in the intermembrane space . Our results suggest that Ugo1p is a new outer membrane component of the mitochondrial fusion machinery.

Antimicrob Agents Chemother, 2001 Apr, 45(4), 1271 - 7
In vitro generation of novel pyrimethamine resistance mutations in the Toxoplasma gondii dihydrofolate reductase; Reynolds MG et al.; Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widely used in the treatment of opportunistic infections caused by the protozoan parasite Toxoplasma gondii . In order to assess the potential role of dhfr sequence polymorphisms in drug treatment failures, we examined the dhfr-ts genes of representative isolates for T . gondii virulence types I, II, and III . These strains exhibit differences in their sensitivities to pyrimethamine but no differences in predicted dhfr-ts protein sequences . To assess the potential for pyrimethamine-resistant dhfr mutants to emerge, three drug-sensitive variants of the T . gondii dhfr-ts gene (the wild-type T . gondii sequence and two mutants engineered to reflect polymorphisms observed in drug-sensitive Plasmodium falciparum) were subjected to random mutagenesis and transfected into either wild-type T . gondii parasites or dhfr-deficient Saccharomyces cerevisiae under pyrimethamine selection . Three resistance mutations were identified, at amino acid residues 25 (Trp-->Arg), 98 (Leu-->Ser), and 134 (Leu-->His).

J Cell Sci, 2001 Apr, 114(Pt 7), 1379 - 86
The Tem1 small GTPase controls actomyosin and septin dynamics during cytokinesis; Lippincott J et al.; Cytokinesis in budding yeast involves an actomyosin-based ring which assembles in a multistepped fashion during the cell cycle and constricts during cytokinesis . In this report, we have investigated the structural and regulatory events that occur at the onset of cytokinesis . The septins, which form an hour-glass like structure during early stages of the cell cycle, undergo dynamic rearrangements prior to cell division: the hourglass structure splits into two separate rings . The contractile ring, localized between the septin double rings, immediately undergoes contraction . Septin ring splitting is independent of actomyosin ring contraction as it still occurs in mutants where contraction fails . We hypothesize that septin ring splitting may remove a structural barrier for actomyosin ring to contract . Because the Tem1 small GTPase (Tem1p) is required for the completion of mitosis, we investigated its role in regulating septin and actomyosin ring dynamics in the background of the net1-1 mutation, which bypasses the anaphase cell cycle arrest in Tem1-deficient cells . We show that Tem1p plays a specific role in cytokinesis in addition to its function in cell cycle progression . Tem1p is not required for the assembly of the actomyosin ring but controls actomyosin and septin dynamics during cytokinesis.

EMBO Rep, 2000 Sep, 1(3), 244 - 52
Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang; Samper E et al.; Ku86 together with Ku70, DNA-PKcs, XRCC4 and DNA ligase IV forms a complex involved in repairing DNA double-strand breaks (DSB) in mammals . Yeast Ku has an essential role at the telomere; in particular, Ku deficiency leads to telomere shortening, loss of telomere clustering, loss of telomeric silencing and deregulation of the telomeric G-overhang . In mammals, Ku proteins associate to telomeric repeats; however, the possible role of Ku in regulating telomere length has not yet been addressed . We have measured telomere length in different cell types from wild-type and Ku86-deficient mice . In contrast to yeast, Ku86 deficiency does not result in telomere shortening or deregulation of the G-strand overhang . Interestingly, Ku86-/- cells show telomeric fusions with long telomeres (>81 kb) at the fusion point . These results indicate that mammalian Ku86 plays a fundamental role at the telomere by preventing telomeric fusions independently of the length of TTAGGG repeats and the integrity of the G-strand overhang.

Mol Biochem Parasitol, 2001 Mar, 113(1), 55 - 65
In-vitro competition analysis of procyclin gene and variant surface glycoprotein gene expression site transcription in Trypanosoma brucei; Laufer G et al.; In Trypanosoma brucei, alpha-amanitin-resistant transcription characteristic of RNA polymerase I is initiated at ribosomal RNA gene (RRNA), procyclin gene (GPEET or EP1), and variant surface glycoprotein gene expression site (VSG ES) promoters . The three promoter types do not share obvious sequence homologies, but contain a proximal domain I and a distal domain II within 80 bp upstream of the transcription initiation site . RRNA, GPEET and EP1, but not the VSG ES promoter, require additional upstream sequences for full activity . In the present study, we competed in-vitro transcription of circular template DNA with linear DNA fragments to identify promoter domains responsible for binding and sequestering essential trans-acting transcription factors . For the GPEET promoter, we found that domain III, located between positions -141 and -92, was most important for the DNA fragment to exert a transcription competition effect, whereas domain I, the only element absolutely required for transcription, was not . Moreover, insertions between promoter domains II and III reduced both transcription from the GPEET promoter and competition with the GPEET promoter fragment, suggesting that these two domains cooperate in the formation of a stable DNA-protein complex . Taken together, these results indicate a promoter structure very similar to that of the Saccharomyces cerevisiae RRNA promoter . In contrast, VSG ES promoter analysis showed that domains I and II are both necessary and sufficient to compete transcription . Despite this structural difference, our analysis provide evidence that GPEET and VSG ES promoters interact with a common factor that is also important for RRNA promoter transcription.

Mol Gen Genet, 2001 Feb, 264(6), 894 - 901
The Neurospora crassa colonial temperature-sensitive 3 (cot-3) gene encodes protein elongation factor 2; Propheta O et al.; At elevated temperatures, the Neurospora crassa mutant colonial, temperature-sensitive 3 (cot-3) forms compact, highly branched colonies . Growth of the cot-3 strain under these conditions also results in the loss of the lower molecular weight (LMW) isoform of the Ser/Thr protein kinase encoded by the unlinked cot-1 gene, whose function is also involved in hyphal elongation . The unique cot-3 gene has been cloned by complementation and shown to encode translation elongation factor 2 (EF-2) . As expected for a gene with a general role in protein synthesis, cot-3 mRNA is abundantly expressed throughout all asexual phases of the N . crassa life cycle . The molecular basis of the cot-3 mutation was determined to be an ATT to AAT transversion, which causes an Ile to Asn substitution at residue 278 . Treatment with fusidic acid (a specific inhibitor of EF-2) inhibits hyphal elongation and induces hyperbranching in a manner which mimics the cot-3 phenotype, and also leads to a decrease in the abundance of the LMW isoform of COT1 . This supports our conclusion that the mutation in cot-3 which results in abnormal hyphal elongation/branching impairs EF-2 function and confirms that the abundance of a LMW isoform of COT1 kinase is dependent on the function of this general translation factor.

Mol Gen Genet, 2001 Feb, 264(6), 883 - 93
Functional asymmetry of the two nucleotide binding domains in the ABC transporter Ste6; Proff C et al.; The yeast a-factor transporter Ste6 is a member of the ABC transporter family and is closely related to human MDR1 . We constructed a set of 26 Ste6 mutants using a random mutagenesis approach . Cell fractionation experiments demonstrated that most of the mutants, with the notable exception of those with alterations in TM1, are transported to the plasma membrane, the presumptive site of action of Ste6 . Trafficking, therefore, does not seem to be affected in most of the mutants . To identify regions in Ste6 that interact with the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N) . Suppressor mutations were identified in TM12 and upstream of TM6 . Surprisingly, these mutations also suppressed the Walker A mutation G397D, which should be defective in ATP-binding and hydrolysis at NBD1 . Photoaffinity labeling experiments with 8-azido-{alpha-32P}ATP showed that ATP binding at NBD2 is reduced by the suppressor mutation in TM12 . The experiments further suggest that the two NBDs of Ste6 are not equivalent and affect each other's ability to bind and hydrolyze ATP.

Biotechniques, 2001 Mar, 30(3), 520 - 3
Oligonucleotide-mediated, PCR-independent cloning by homologous recombination; DeMarini DJ et al.; We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR . In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides . The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert . The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA . Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides . This cloning method, which is performed by co-transforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).

EMBO Rep, 2001 Jan, 2(1), 21 - 6
Physical association between the histone acetyl transferase CBP and a histone methyl transferase; Vandel L et al.; CBP (CREB-binding protein) is involved in transcriptional activation by a great variety of sequence-specific transcription factors . CBP has been shown to activate transcription through its histone acetyl transferase activity . Acetylation is a common post-translational modification of nucleosomal histone N-terminal tails, which generally correlates with transcriptional activation . Histone N-terminal tails are also modified by methylation but its functional consequences are largely unknown . Here we found that immunoprecipitation of CBP, or of the highly related p300, led to the co-immunoprecipitation of a robust histone methyl transferase (HMT) activity, indicating that CBP physically interacts with an HMT in living cells . The CBP-associated HMT is specific for lysines 4 and 9 of histone H3, which are known to be methylated in living cells . These results suggest that histone methylation could be involved in transcriptional activation . Furthermore, they raise the question of the link between histone methylation and acetylation.

Mol Microbiol, 2001 Feb, 39(4), 973 - 81
Plasmodium protein phosphatase 2C dephosphorylates translation elongation factor 1beta and inhibits its PKC-mediated nucleotide exchange activity in vitro; Mamoun CB et al.; The elongation step of protein synthesis involves binding of aminoacyl-tRNA to the ribosomal A site, formation of a peptide bond and translocation of the newly formed peptidyl-tRNA to the P site . The nucleotide exchange factor EF-1beta plays a major role in the regulation of this process by regenerating a GTP-bound EF-1alpha necessary for each elongation cycle . EF-1beta has been shown to be phosphorylated and its phosphorylation is critical for optimal activity . We have previously identified a serine/threonine protein phosphatase 2C (PP2C) from the human malaria parasite Plasmodium falciparum . In the current work, we performed Far-Western analysis to identify PfPP2C substrates . Several components of the translation and transcription machinery were identified, including translation elongation factor 1-beta (PfEF-1beta) . PfEF-1beta is efficiently phosphorylated by protein kinase C and this phosphorylation results in a 400% increase in its nucleotide exchange activity . PKC-phosphorylated PfEF-1beta is readily and selectively dephosphorylated by recombinant and native PfPP2C, which downregulates the nucleotide exchange activity to its basal level . The identification of a translation elongation component as substrate for PP2C suggests an important regulatory function for this enzyme and suggests that it may be a good target for drug design in the fight against malaria.

J Immunol Methods, 2001 Apr, 250(1-2), 45 - 66
Use of serial analysis of gene expression (SAGE) technology; Yamamoto M et al.; Serial analysis of gene expression, or SAGE, is an experimental technique designed to gain a direct and quantitative measure of gene expression . The SAGE method is based on the isolation of unique sequence tags (9-10 bp in length) from individual mRNAs and concatenation of tags serially into long DNA molecules for a lump-sum sequencing . The SAGE method can be applied to the studies exploring virtually any kinds of biological phenomena in which the changes in cellular transcription are responsible . SAGE is a highly competent technology that can not only give a global gene expression profile of a particular type of cell or tissue, but also help us identify a set of specific genes to the cellular conditions by comparing the profiles constructed for a pair of cells that are kept at different conditions . In this review, we present an outline of the original method, several studies achieved by using the method as a major strategic tool, technological difficulties and intrinsic problems that emerged, and improvements and modifications of the method to cope with these drawbacks . We then present our modified SAGE procedure that generates longer sequence tags (14 bp) rather in detail, and the profile (80K profile) derived from HeLa cells that is composed of 80000 tags obtained from a single library . In addition, a series of smaller profiles (2, 4, 10, 20 and 40K) was made by dividing the 80K profile . When we compared these smaller profiles with respect to tag counts for a number of genes, it became apparent that counts of most gene tags increase stably and constantly as the size of profiles increase, while several genes do not . This may be another problem we have to keep in mind, when the profiles are compared for the identification of 'specific genes'.

Science, 2001 Mar 16, 291(5511), 2135 - 8
Role of the ABC transporter Mdl1 in peptide export from mitochondria; Young L et al.; ATP-binding cassette (ABC) adenosine triphosphatases actively transport a wide variety of compounds across biological membranes . Here, the ABC protein Mdl1 was identified as an intracellular peptide transporter localized in the inner membrane of yeast mitochondria . Mdl1 was required for mitochondrial export of peptides with molecular masses of approximately 2100 to 600 daltons generated by proteolysis of inner-membrane proteins by the m-AAA protease in the mitochondrial matrix . Proteolysis by the i-AAA protease in the intermembrane space led to the release of similar-sized peptides independent of Mdl1 . Thus, two pathways of peptide efflux from mitochondria exist that may allow communication between mitochondria and their cellular environment.

Plant Cell, 2001 Mar, 13(3), 667 - 79
Gibberellin/abscisic acid antagonism in barley aleurone cells: site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules; Gomez-Cadenas A et al.; The antagonism between gibberellins (GA) and abscisic acid (ABA) is an important factor regulating the developmental transition from embryogenesis to seed germination . In barley aleurone layers, the expression of genes encoding alpha-amylases and proteases is induced by GA but suppressed by ABA . It has been shown that an ABA-induced protein kinase, PKABA1, mediates the ABA suppression of alpha-amylase expression . Using a barley aleurone transient expression system, we have now localized the site of action of PKABA1 relative to other signal transduction components governing the expression of alpha-amylase . The expression of alpha-amylase can be transactivated by the transcription factor GAMyb, which is itself induced by GA . A truncated GAMyb containing the DNA binding domain but lacking the transactivation domain prevents the GA induction of alpha-amylase, further supporting the notion that GAMyb mediates the GA induction of alpha-amylase expression . Although ABA and PKABA1 strongly inhibit the GA induction of alpha-amylase, they have no effect on GAMyb-transactivated alpha-amylase expression . Using a GAMyb promoter--beta-glucuronidase construct, we also show that both ABA and PKABA1 repress the GA induction of GAMyb . In the slender mutant, GAMyb and alpha-amylase are highly expressed, even in the absence of GA . However, this constitutive expression can still be inhibited by ABA, PKABA1, or an inhibitor of cGMP synthesis . On the basis of these observations, we suggest that PKABA1 acts upstream from the formation of functional GAMyb but downstream from the site of action of the Slender gene product . Because PKABA1 inhibits the GA induction of the GAMyb promoter--beta-glucuronidase construct, it appears that at least part of the action of PKABA1 is to downregulate GAMyb at the transcriptional level.

Plant Cell, 2001 Mar, 13(3), 659 - 66
Disruption of individual members of Arabidopsis syntaxin gene families indicates each has essential functions; Sanderfoot AA et al.; Syntaxins are a large group of proteins found in all eukaryotes involved in the fusion of transport vesicles to target membranes . Twenty-four syntaxins grouped into 10 gene families are found in the model plant Arabidopsis thaliana, each group containing one to five paralogous members . The Arabidopsis SYP2 and SYP4 gene families contain three members each that share 60 to 80% protein sequence identity . Gene disruptions of the yeast (Saccharomyces cerevisiae) orthologs of the SYP2 and SYP4 gene families (Pep12p and Tlg2p, respectively) indicate that these syntaxins are not essential for growth in yeast . However, we have isolated and characterized gene disruptions in two genes from each family, finding that disruption of individual syntaxins from these families is lethal in the male gametophyte of Arabidopsis . Complementation of the syp21-1 gene disruption with its cognate transgene indicated that the lethality is linked to the loss of the single syntaxin gene . Thus, it is clear that each syntaxin in the SYP2 and SYP4 families serves an essential nonredundant function.

Mol Biol Cell, 2001 Mar, 12(3), 521 - 38
Selective formation of Sed5p-containing SNARE complexes is mediated by combinatorial binding interactions; Tsui MM et al.; Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo . We describe in vitro binding experiments in which we identify ternary and quaternary Sed5p-containing SNARE complexes . The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective . In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners . Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales . Although SNARE-SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another . Rather our data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles.

Mol Biol Cell, 2001 Mar, 12(3), 511 - 20
Phosphatidylcholine synthesis influences the diacylglycerol homeostasis required for SEC14p-dependent Golgi function and cell growth; Henneberry AL et al.; Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes . In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine . EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo . In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway . Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability . Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death . The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20-40-fold over endogenous long-chain diacylglycerol levels . Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

Endocrinology, 2001 Apr, 142(4), 1606 - 15
Glucocorticoid receptor-interacting protein-1 and receptor-associated coactivator-3 differentially interact with the vitamin D receptor (VDR) and regulate VDR-retinoid X receptor transcriptional cross-talk; Issa LL et al.; The vitamin D(3) receptor (VDR) is a ubiquitously expressed nuclear hormone receptor, and its ligand, calcitriol, has diverse biological effects . The extent to which transcriptional coactivators are involved in modulating tissue-specific functions of the VDR is unclear . Hence, the current studies investigated the role of p160 coactivators in regulating VDR function and interaction with RXR . Two p160 coactivators, glucocorticoid receptor-interacting protein-1 (GRIP1) and receptor-associated coactivator-3 (RAC3), which are expressed in an inverse fashion in cell lines representative of calcitriol target tissues, interacted directly with the VDR, both in vitro and in yeast cells, but only in the presence of calcitriol . Deletional analyses of VDR indicated that GRIP1 and RAC3 required an intact VDR activation function (AF-2) domain for efficient interaction as well as additional but distinct regions of the VDR . Coexpression experiments in yeast cells indicated that both GRIP1 and RAC3 coassemble with the VDR to form an active transcriptional complex . They also form ternary complexes with VDR homodimers and VDR:RXRalpha heterodimers . In mammalian cells, GRIP1 augmented VDR activation of the osteocalcin promoter, whereas RAC3 enhanced VDR activation indirectly through RXR . These data suggest different coactivators regulate VDR function via distinct mechanisms and support the hypothesis that the VDR recruits different coactivators depending on specific gene and cellular contexts.

EMBO J, 2001 Mar 15, 20(6), 1353 - 62
Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription; Muth V et al.; Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I . In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter . We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter . Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system . Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription . The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

Curr Opin Genet Dev, 2001 Apr, 11(2), 189 - 98
Telomeric chromatin: replicating and wrapping up chromosome ends; Shore D; Recent advances in our understanding of the specialized chromatin structure at telomeres, the ends of eukaryotic chromosomes, have focused on three separate areas: replication of telomeres through the coordinated action of conventional DNA polymerases and the telomerase enzyme, protection of the chromosome end from DNA damage checkpoint sensors and DNA-repair processes, and the discovery of a novel deacetylase enzyme (Sir2p) required for the establishment and maintenance of telomeric heterochromatin . Although the number of proteins and the complexity of their interactions at telomeres continues to grow, a picture of at least some of the major players and mechanisms underlying telomere replication, end 'capping' and chromatin assembly is beginning to emerge.

Curr Opin Genet Dev, 2001 Apr, 11(2), 155 - 61
Histone acetyltransferases: function, structure, and catalysis; Marmorstein R et al.; Histone acetyltransferases (HATs) directly link chromatin modification to gene activation . Recent structure/function studies provide insights into HAT catalysis and histone binding, and genetic studies suggest cross-talk between acetylation and other histone modifications . Developmental aberrations in mice and certain human cancers are associated with HAT mutations, further highlighting the importance of these enzymes to normal cell growth and differentiation.

Curr Opin Cell Biol, 2001 Apr, 13(2), 232 - 8
Enzymatic activities of Sir2 and chromatin silencing; Moazed D; Heritable domains of generalized repression are a common feature of eukaryotic chromosomes and involve the assembly of DNA into a silenced chromatin structure . Sir2, a conserved protein required for silencing in yeast, has recently been shown to couple histone deacetylation to cleavage of a high-energy bond in nicotinamide adenine dinucleotide (NAD) and the synthesis of a novel product, O-acetyl-ADP-ribose . The deacetylase activity provides a direct link between Sir2 and the hypoacetylated state of silent chromatin . However, the unusual coupling of deacetylation to cleavage and synthesis of other bonds raises the possibility that deacetylation is not the only crucial function of Sir2.

Curr Opin Cell Biol, 2001 Apr, 13(2), 218 - 24
HATs on and beyond chromatin; Chen H et al.; The role of histone acetylation as a key mechanism of transcriptional regulation has been well established . Recent advances suggest that histone acetyltransferases also play important roles in histone-modulated processes such as DNA replication, recombination and repair . In addition, acetylation of transcriptional cofactors and other proteins is an efficient means of regulating a diverse range of molecular interactions . As new histone acetyltransferases and substrates are rapidly emerging, it is becoming apparent that protein acetylation may rival phosphorylation as a mechanism to transduce cellular regulatory signals.

FEBS Lett, 2001 Mar 9, 492(1-2), 139 - 45
The Ras GDP/GTP cycle is regulated by oxidizing agents at the level of Ras regulators and effectors; Accorsi K et al.; Reactive oxygen species (ROS) have been found to play important roles in regulating cellular functions . Their action in vivo has been related to specific effects on signal transduction pathways, such as Ras pathway . In order to characterize which elements of Ras pathway are affected by ROS, we have analyzed the action of different oxidizing agents on the ability of GTPase activating protein GAP and nucleotide exchange factor GEF to enhance the intrinsic activities of Ras . The action of these agents on the binding between H-Ras and its effector c-Raf-1 was also investigated . No effects were observed on the intrinsic activities of H-Ras or Ras2p . On the other hand, reversible inhibitions of GEF and GAP actions on Ras were found, whose extent was dependent on the agent used . As tested with the scintillation proximity assay, these agents also inhibited the binding of c-Raf-1 to H-Ras . Our data reveal new potential targets for the action of ROS on Ras pathway and suggest that they can influence the Ras activation state indirectly via regulators and effectors.

Toxicol Sci, 2001 Apr, 60(2), 296 - 304
Defining the impact of weakly estrogenic chemicals on the action of steroidal estrogens; Rajapakse N et al.; We tested whether bisphenol A (BPA) or o,p'-DDT, when combined with 17beta-estradiol (E2), would contribute to the overall mixture effect using a yeast reporter gene assay, the yeast estrogen screen . Following comprehensive concentration-response analyses of the single agents, the pharmacologically well-founded models of concentration addition and independent action were used to predict entire concentration-response relationships for mixtures of the agents with a variety of fixed mixture ratios, assuming additivity . For molar mixture ratios proportional to the levels normally found in human tissues (i.e., below 1:5000, E2:BPA or o,p'-DDT), these predictions suggest that the effects of individual xenoestrogens are too weak to create an impact on the actions of steroidal hormones . However, at mixture ratios more in favor of the xenoestrogens, a significant contribution to the overall mixture effect was predicted . The predictions were tested experimentally . The observed combined effects of mixtures of E2 with either BPA or o,p'-DDT did not deviate from the additivity expectation . On combining E2 with either BPA or o,p'-DDT at approximately equieffective concentrations corresponding to molar mixture ratios between 1:20,000 and 1:100,000 (E2:BPA or o,p'-DDT), substantial modulations of the effects of E2 became discernible . The assumption that weak xenoestrogens are generally unable to create an impact upon the already strong effects of endogenous steroidal estrogens is not supported by our observations . Our studies indicate that the potential health implication of additive combination effects between xenoestrogens and steroidal estrogens deserve serious consideration.

Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3243 - 8 Epub 2001 Mar 06.
Genetic interactions between ATM and the nonhomologous end-joining factors in genomic stability and development; Sekiguchi J et al.; DNA ligase IV (Lig4) and the DNA-dependent protein kinase (DNA-PK) function in nonhomologous end joining (NHEJ) . However, although Lig4 deficiency causes late embryonic lethality, deficiency in DNA-PK subunits (Ku70, Ku80, and DNA-PKcs) does not . Here we demonstrate that, similar to p53 deficiency, ataxia-telangiectasia-mutated (ATM) gene deficiency rescues the embryonic lethality and neuronal apoptosis, but not impaired lymphocyte development, associated with Lig4 deficiency . However, in contrast to p53 deficiency, ATM deficiency enhances deleterious effects of Lig4 deficiency on growth potential of embryonic fibroblasts (MEFs) and genomic instability in both MEFs and cultured progenitor lymphocytes, demonstrating significant differences in the interplay of p53 vs . ATM with respect to NHEJ . Finally, in dramatic contrast to effects on Lig4 deficiency, ATM deficiency causes early embryonic lethality in Ku- or DNA-PKcs-deficient mice, providing evidence for an NHEJ-independent role for the DNA-PK holoenzyme.

Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3174 - 9 Epub 2001 Mar 06.
Recombination-mediated lengthening of terminal telomeric repeats requires the Sgs1 DNA helicase; Cohen H et al.; The Saccharomyces cerevisiae SGS1 gene encodes a RecQ-like DNA helicase, human homologues of which are implicated in the genetic instability disorders, Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), and Werner syndrome (WS) . Telomerase-negative yeast cells can recover from senescence via two recombinational telomere elongation pathways . The "type I" pathway generates telomeres with large blocks of telomeric and subtelomeric sequences and short terminal repeat tracts . The "type II" pathway generates telomeres with extremely long heterogeneous terminal repeat tracts, reminiscent of the long telomeres observed in telomerase-deficient human tumors and tumor-derived cell lines . Here, we report that telomerase-negative (est2) yeast cells lacking SGS1 senesced more rapidly, experienced a higher rate of telomere erosion, and were delayed in the generation of survivors . The est2 sgs1 survivors that were generated grew poorly, arrested in G(2)/M and possessed exclusively type I telomeres, implying that SGS1 is critical for the type II pathway . The mouse WS gene suppressed the slow growth and G(2)/M arrest phenotype of est2 sgs1 survivors, arguing that the telomeric function of SGS1 is conserved . Reintroduction of SGS1 into est2 sgs1 survivors restored growth rate and extended terminal tracts by approximately 300 bp . Both phenotypes were absolutely dependent on Sgs1 helicase activity . Introduction of an sgs1 carboxyl-terminal truncation allele with helicase activity restored growth rate without extending telomeres in most cases, demonstrating that type II telomeres are not necessary for normal growth in the absence of telomerase.

Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3115 - 20
Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF; Kamada K et al.; The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-A resolution . The alpha/beta structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3gamma (HNF-3gamma), making it a winged-helix protein . The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3gamma and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the beta subunit of TFIIE . RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3056 - 61
RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit; Xie Y et al.; The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis . RPN4 is a transcriptional activator of genes encoding proteasomal subunits . Here we show that RPN4 is required for normal levels of these subunits . Further, we demonstrate that RPN4 is extremely short-lived (t(1/2) approximately 2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4Delta cells are perturbed in their cell cycle . The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription-activation domains of RPN4 . The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome.

Genomics, 2001 Feb 15, 72(1), 78 - 87
Disruption of Apc10/Doc1 in three alleles of oligosyndactylism; Pravtcheva DD et al.; Oligosyndactylism (Os) is a radiation-induced mouse mutation associated with recessive lethality and a dominant effect on limb and kidney development . The lethal effect of the mutation is due to a cell-autonomous block in the transition from metaphase to anaphase . We have previously characterized two transgene-induced mutations, 94-A and 94-K, which are allelic with Os . These mutations facilitated the identification of genomic segments and transcribed sequences in the affected region . One of the transcripts in this region corresponds to the mouse homolog of the anaphase-promoting complex component APC10/DOC1 . The disruption of this gene can explain the mitotic arrest phenotype of all three alleles of Os .

Plant Mol Biol, 2001 Jan, 45(1), 107 - 12
Protein phosphatase 2A: identification in Oryza sativa of the gene encoding the regulatory A subunit; Yu S et al.; A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library . Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units . The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene . The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns . The rice genome contains only a single copy of this gene as judged by Southern blot analysis . The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum.

Trends Biochem Sci, 2001 Mar, 26(3), 143 - 4
The archaeal homolog of the Imp4 protein, a eukaryotic U3 snoRNP component; Mayer C et al.; Homologs of the Imp4 protein, a component specific to the eukaryotic U3 snoRNP complex, have been found in all archaeal genomes . The archaeal and eukaryotic Imp4 proteins that are related to four other protein families, the Imp4-like, the SSF1 homologs and two sets of hypothetical proteins, are characterized by the Imp4 signature pattern . These findings, together with the presence of other snoRNPs homologs in Archaea, provide evidence for similar RNA processing and folding in Eukarya and Archaea.

Biochim Biophys Acta, 2001 Apr 2, 1504(2-3), 179 - 95
Fungal respiration: a fusion of standard and alternative components; Joseph-Horne T et al.; In animals, electron transfer from NADH to molecular oxygen proceeds via large respiratory complexes in a linear respiratory chain . In contrast, most fungi utilise branched respiratory chains . These consist of alternative NADH dehydrogenases, which catalyse rotenone insensitive oxidation of matrix NADH or enable cytoplasmic NADH to be used directly . Many also contain an alternative oxidase that probably accepts electrons directly from ubiquinol . A few fungi lack Complex I . Although the alternative components are non-energy conserving, their organisation within the fungal electron transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally coupled to proton translocation through at least one site . The alternative oxidase enables respiration to continue in the presence of inhibitors for ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase . This may be particularly important for fungal pathogens, since host defence mechanisms often involve nitric oxide, which, whilst being a potent inhibitor of cytochrome c oxidase, has no inhibitory effect on alternative oxidase . Alternative NADH dehydrogenases may avoid the active oxygen production associated with Complex I . The expression and activity regulation of alternative components responds to factors ranging from oxidative stress to the stage of fungal development.

Am J Physiol Cell Physiol, 2001 Apr, 280(4), C897 - 911
Hypoxia-activated ligand HAL-1/13 is lupus autoantigen Ku80 and mediates lymphoid cell adhesion in vitro; Lynch EM et al.; Hypoxia is known to induce extravasation of lymphocytes and leukocytes during ischemic injury and increase the metastatic potential of malignant lymphoid cells . We have recently identified a new adhesion molecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates the hypoxia-induced increases in lymphocyte and neutrophil adhesion to endothelium and hypoxia-mediated invasion of endothelial cell monolayers by tumor cells . In this report, we used expression cloning to identify this molecule as the lupus antigen and DNA-dependent protein kinase-associated nuclear protein, Ku80 . The HAL-1/13-Ku80 antigen is present on the surface of leukemic and solid tumor cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells . Transfection and ectopic expression of HAL-1/13-Ku80 on (murine) NIH/3T3 fibroblasts confers the ability of these normally nonadhesive cells to bind to a variety of human lymphoid cell lines . This adhesion can be specifically blocked by HAL-1/13 or Ku80-neutralizing antibodies . Loss of expression variants of these transfectants simultaneously lost their adhesive properties toward human lymphoid cells . Hypoxic exposure of tumor cell lines resulted in upregulation of HAL-1/13-Ku80 expression at the cell surface, mediated by redistribution of the antigen from the nucleus . These studies indicate that the HAL-1/13-Ku80 molecule may mediate, in part, the hypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.

Cancer Res, 2001 Feb 15, 61(4), 1585 - 91
End-joining deficiency and radiosensitization induced by gemcitabine; van Putten JWG et al.; The mechanism of radiosensitization by gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is not exactly known . We investigated the possible role of inhibition of the repair of DNA double-strand breaks by dFdC by measuring the extent of radiosensitization in different cell lines deficient and proficient in components of nonhomologous end-joining and in the parental cell lines . Different cell lines were incubated with 0.5 and 5 microM dFdC for 4 h . Cells deficient in DNA-dependent protein-kinase catalytic subunit (V3) showed sensitization similar to that of wild-type cells (AAS) and complemented cells (V3+YAC) . Ku80-deficient cells (xrs5 and xrs6) showed even more radiosensitization by dFdC as compared with wild-type CHO-K1 . However, Ku80-complemented cell lines (xrs5+huKu80 and xrs6+haKu80) did not show radiosensitization . The differences in dFdC-mediated radiosensitization were not attributable to different changes in deoxynucleotide triphosphate levels and cell cycle distribution . We conclude that a functional nonhomologous end-joining pathway is not required for dFdC-mediated radiosensitization.

Oncogene, 2001 Jan 4, 20(1), 125 - 32
BS69, an adenovirus E1A-associated protein, inhibits the transcriptional activity of c-Myb; Ladendorff NE et al.; The carboxyl terminus of c-Myb contains a negative regulatory domain that is absent in the v-Myb oncoprotein, but conserved among all the known Myb proteins of animals . This domain inhibits transcriptional activation by c-Myb in animal cells, but not in budding yeast, suggesting that additional protein(s) present in animal cells but not yeast are required for this negative regulatory function . A yeast two-hybrid screen identified BS69, an adenovirus E1A-associated protein, as interacting with the carboxy-terminal region of c-Myb . BS69 contains regions of similarity to the PHD finger, the bromodomain, and the MYND domain, all of which are found in other proteins present in high molecular weight complexes that regulate transcription and/or modify chromatin structure . Further study showed that BS69 inhibited the transcriptional activity of c-Myb, that this inhibition was specific, that it mapped to the carboxyl termini of the two proteins and that it was dose-dependent . A direct interaction between these two proteins was observed in vitro . Furthermore, the 289R E1A protein could inhibit the BS69-mediated decrease in transcriptional activation by c-Myb . By analogy with the inhibition of the Rb/E2F regulatory axis by E1A, we propose that a BS69/Myb regulatory circuit may also be a target of disruption during oncogenesis . Oncogene (2001) 20, 125 - 132.

J Bacteriol, 2001 Apr, 183(7), 2331 - 4
TOR modulates GCN4-dependent expression of genes turned on by nitrogen limitation; Valenzuela L et al.; In Saccharomyces cerevisiae, the rapamycin-sensitive TOR signaling pathway plays an essential role in up-regulating translation initiation and cell cycle progression in response to nutrient availability . One of the mechanisms by which TOR regulates cell proliferation is by excluding the GLN3 transcriptional activator from the nucleus and, in consequence, preventing its transcriptional activation therein . We examined the possibility that the TOR cascade could also control the transcriptional activity of Gcn4p, which is known to respond to amino acid availability . The results presented in this paper indicate that GCN4 plays a role in the rapamycin-sensitive signaling pathway, regulating the expression of genes involved in the utilization of poor nitrogen sources, a previously unrecognized role for Gcn4p, and that the TOR pathway controls GCN4 activity by regulating the translation of GCN4 mRNA . This constitutes an additional TOR-dependent mechanism which modulates the action of transcriptional activators.

Nat Genet, 2001 Mar, 27(3), 304 - 8
Chromatin profiling using targeted DNA adenine methyltransferase; van Steensel B et al.; Chromatin is the highly complex structure consisting of DNA and hundreds of associated proteins . Most chromatin proteins exert their regulatory and structural functions by binding to specific chromosomal loci . Knowledge of the identity of these in vivo target loci is essential for the understanding of the functions and mechanisms of action of chromatin proteins . We report here large-scale mapping of in vivo binding sites of chromatin proteins, using a novel approach based on a combination of targeted DNA methylation and microarray technology . We show that three distinct chromatin proteins in Drosophila melanogaster cells each associate with specific sets of genes . HP1 binds predominantly to pericentric genes and transposable elements . GAGA factor associates with euchromatic genes that are enriched in (GA)n motifs . A Drosophila homolog of Saccharomyces cerevisiae Sir2p is associated with several active genes and is excluded from heterochromatin . High-resolution, genome-wide maps of target loci of chromatin proteins ('chromatin profiles') provide new insights into chromatin structure and gene regulation.

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