bluuebunny/crossref_metadata_2025 huggingface dataset:
PorA types in Neisseria meningitidis serogroup B isolated in Argentina from 2001 to 2003: implications for the design of an outer membrane protein-based vaccine
Authors: Sorhouet Pereira Cecilia , Regueira Mabel , Mollerach Marta
Published: February 2008

Identification ofNeisseria meningitidisPorA types remains important, as the PorA protein is a major immunogenic component of several meningococcal vaccines under development. In this study, 191N. meningitidisserogroup B isolates collected in Argentina through active laboratory-based surveillance from 2001 to 2003 were serosubtyped. Nucleotide sequences of theporAvariable region 1 (VR1) and VR2 regions were determined in 52 non-serosubtypeable isolates. A substantial number of distinct VR types were identified, and a new VR2 variant from the P1.16 family was described. This is the first report describing PorA types inN. meningitidisserogroup B isolates in Argentina. Furthermore, the wide diversity of subtypes detected by serosubtyping and genosubtyping reveals the difficulty in designing a useful outer-membrane vaccine applicable in this country. A possible mechanism responsible for altered PorA expression was analysed in two PorA types.
Molecular typing of nasal carriage isolates of Staphylococcus aureus from an Irish university student population based on toxin gene PCR, agr locus types and multiple locus, variable number tandem repeat analysis
Authors: Collery Mark M. , Smyth Davida S. , Twohig Jane M. , Shore Anna C. , Coleman David C. , Smyth Cyril J.
Published: February 2008

Forty-eight Staphylococcus aureus isolates collected from a young, healthy, Irish university student population from 1995 to 2004 were screened for 16 enterotoxin (SE) and enterotoxin-like (SEl) genes (sea–see, seg–sei, selj–selo, selq, selu), and for the toxic shock toxin syndrome toxin-1 gene, tst. All of the isolates harboured at least one SE or SEl gene and 66.7 % possessed a classical SE gene (sea, seb, sec), the commonest being the seb gene. Most of the isolates (85.4 %) had a complete egc locus (selo, selm, sei, seln, seg). The intergenic sei–seln region of the egc locus was typed by PCR-RFLP in 34 isolates, 15 possessing pseudogenes ψent1 and ψent2 and 19 having the selu gene. The seh and sell genes, the selk–selq gene combination, and the tst gene were each found in <15 % of isolates. The agr genotype distribution was agr type III, 37.5 %; agr type I, 35.4 %; agr type II, 25 %; and agr type IV, 2.1 %. There was no association between SE–SEl genotype and agr type. All tst gene-positive isolates were of agr type III and harboured a classical SE gene. Multiple locus, variable number tandem repeat analysis (MLVA) produced 47 different patterns. While the sdr locus was present in all isolates, half of them lacked one or two of the sdr gene amplimers. Twenty isolates harboured the bbp gene, its presence being associated with agr type III, but not with the SE–SEl gene profile. The agr types of isolates were associated with MLVA subclusters. Selective MLST analysis revealed seven novel sequence types and a new aroE allele. Five clonal clusters (CCs), including CCs comprising major pandemic clones CC30, CC5 and CC22 and minor lineages CC6 and CC9, and three singletons were identified.
Repression of p53-mediated transcription by MDM2: a dual mechanism
Authors: Thut Catherine J. , Goodrich James A. , Tjian Robert
Published: February 2008

The oncoprotein MDM2 binds to the activation domain of the tumor suppressor p53 and inhibits its ability to stimulate transcription. This same region of p53 is able to bind several basal transcription factors that appear to be important for the transactivation function of p53. It has therefore been suggested that MDM2 acts to inhibit p53 by concealing its activation domain from the basal machinery. Here we present data suggesting that MDM2 possesses an additional inhibitory function. Our experiments reveal that in addition to a p53-binding domain, MDM2 also contains an inhibitory domain that can directly repress basal transcription in the absence of p53. By fusing portions of MDM2 to a heterologous DNA-binding domain to allow p53-independent promoter recruitment, we have localized this inhibitory domain to a region encompassing amino acids 50–222 of MDM2. Furthermore, the function of this inhibitory domain does not require the presence of either TFIIA or the TAFs. Of the remaining basal factors, both the small subunit of TFIIE and monomeric TBP are bound by the MDM2 inhibitory domain. It is possible that MDM2 inhibits the ability of the preinitiation complex to synthesize RNA through one of these interactions. Our results are consistent with a model in which MDM2 represses p53-dependent transcription by a dual mechanism: a masking of the activation domain of p53 through a protein–protein interaction that additionally serves to recruit MDM2 to the promoter where it directly interferes with the basal transcription machinery.
Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog
Authors: Blagden Chris S. , Currie Peter D. , Ingham Philip W. , Hughes Simon M.
Published: February 2008

The patterning of vertebrate somitic muscle is regulated by signals from neighboring tissues. We examined the generation of slow and fast muscle in zebrafish embryos and show that Sonic hedgehog (Shh) secreted from the notochord can induce slow muscle from medial cells of the somite. Slow muscle derives from medial adaxial myoblasts that differentiate early, whereas fast muscle arises later from a separate myoblast pool. Mutant fish lacking shh expression fail to form slow muscle but do form fast muscle. Ectopic expression of shh,either in wild-type or mutant embryos, leads to ectopic slow muscle at the expense of fast. We suggest that Shh acts to induce myoblasts committed to slow muscle differentiation from uncommitted presomitic mesoderm.
An RNA enhancer in a phage transcriptional antitermination complex functions as a structural switch
Authors: Su Leila , Radek James T. , Labeots Laura A. , Hallenga Klaas , Hermanto Patrick , Chen Huifen , Nakagawa Satoe , Zhao Ming , Kates Steve , Weiss Michael A.
Published: February 2008

Antitermination protein N regulates the transcriptional program of phage λ through recognition of RNA enhancer elements. Binding of an arginine-rich peptide to one face of an RNA hairpin organizes the other, which in turn binds to the host antitermination complex. The induced RNA structure mimics a GNRA hairpin, an organizational element of rRNA and ribozymes. The two faces of the RNA, bridged by a sheared GA base pair, exhibit a specific pattern of base stacking and base flipping. This pattern is extended by stacking of an aromatic amino acid side chain with an unpaired adenine at the N-binding surface. Such extended stacking is coupled to induction of a specific internal RNA architecture and is blocked by RNA mutations associated in vivo with loss of transcriptional antitermination activity. Mimicry of a motif of RNA assembly by an RNA–protein complex permits its engagement within the antitermination machinery.
mre11S—a yeast mutation that blocks double-strand-break processing and permits nonhomologous synapsis in meiosis
Authors: Nairz Knud , Klein Franz
Published: February 2008

During meiotic prophase the repair of self-inflicted DNA double-strand break (DSB) damage leads to meiotic recombination in yeast. We employed a genetic screen to specifically characterize cellular functions that become essential after this DSB formation. As a result a new allele of MRE11, termed mre11S (forSeparation of functions) was isolated that allows initiation but not processing and repair of meiotic DSBs similar to the well-characterized rad50S allele. In contrast, themre11-1 allele blocks initiation of meiotic DSBs as reported previously by others. The mre11S allele, which is mutated in the 5′ part of the gene, can partially complement mre11alleles disrupted close to the 3′ end that cannot initiate DSBs when homozygous. This suggests homodimerization of the Mre11 protein and the presence of separate domains for DSB initiation and 5′ resection. The fact that two genes, RAD50 and MRE11,required for DSB processing are also essential for DSB initiation dictates a model in which a bifunctional initiation/repair complex is required to initiate meiotic recombination. A subset of mre11S nuclei was shown to perform extensive but partially nonhomologous synapsis. We propose that the unprocessed DSBs present in mre11S allow for synapsis, but that homologous synapsis is only ensured at a later stage of recombination.
A β-catenin/XTcf-3 complex binds to thesiamois promoter to regulate dorsal axis specification in Xenopus
Authors: Brannon Mark , Gomperts Miranda , Sumoy Lauro , Moon Randall T. , Kimelman David
Published: February 2008

The Wnt pathway regulates the early dorsal–ventral axis in Xenopus through a complex of β-catenin and HMG box transcription factors of the Lef/Tcf family. We show that the promoter of the dorsalizing homeo box gene siamois is a direct target for the β-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injectingsiamois reporter constructs into the animal pole ofXenopus embryos, we show that a 0.8-kb fragment of thesiamois promoter is strongly activated by β-catenin. The proximal 0.5 kb, which is also activated by β-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the β-catenin-mediated activation of siamois and show thatsiamois is regulated by the β-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamoispromoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of β-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Furthermore, elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of β-catenin. Finally, we find that the endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. We propose that the dorsal activation of siamois by the β-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side ofXenopus embryos.
Identification of an autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is selectively inactivated during protein synthesis inhibition
Authors: Wang Zhengxin , Luo Ting , Roeder Robert G.
Published: February 2008

Transcription by RNA polymerase III (Pol III) requires multiple general initiation factors that, in isolated form, assemble onto the promoter in an ordered fashion. Here, it is shown that all components required for transcription of the VA1 and tRNA genes, including TFIIIB, TFIIIC, and RNA Pol III, can be coimmunopurified from a HeLa cell line that constantly expresses a FLAG epitope-tagged subunit of human RNA Pol III. This finding of an RNA Pol III “holoenzyme” suggests similarities between transcription initiation by RNA Pol II and RNA Pol III and has led to the identification of a novel general initiation factor (TDF, translation dependentfactor) that is present within the holoenzyme. TDF is selectively inactivated during protein synthesis inhibition by cycloheximide and at a late stage of adenovirus infection, thus accounting for the loss of RNA Pol III-mediated transcription of the tRNA and VA RNA genes under these conditions. On the basis of these observations, possible mechanisms for the global regulation of transcription by RNA Pol III and for disassembly of RNA Pol III initiation complexes are proposed.
The PAG gene product, a stress-induced protein with antioxidant properties, is an Abl SH3-binding protein and a physiological inhibitor of c-Abl tyrosine kinase activity
Authors: Wen Shih-Te , Van Etten Richard A.
Published: February 2008

Biochemical and genetic evidence suggests that the tyrosine kinase activity of c-Abl is tightly regulated in vivo by a cellular factor binding to the Src homology 3 (SH3) domain of Abl. We used the yeast two-hybrid system to identify a gene, PAG, whose protein product (Pag) interacts specifically with the Abl SH3 domain. Pag, also known as macrophage 23-kD stress protein (MSP23), is a member of a novel family of proteins with antioxidant activity implicated in the cellular response to oxidative stress and in control of cell proliferation and differentiation. In a co-expression assay, Pag associates with c-Abl in vivo and inhibits tyrosine phosphorylation induced by overexpression of c-Abl. Inhibition requires the Abl SH3 and kinase domains and is not observed with other Abl SH3-binding proteins. Expression of Pag also inhibits the in vitro kinase activity of c-Abl, but not SH3-mutated Abl or v-Abl. When transfected in NIH-3T3 cells, Pag is localized to nucleus and cytoplasm and rescues the cytostatic effect induced by c-Abl. These observations suggest Pag is a physiological inhibitor of c-Abl in vivo.
Inhibition of patterned cell shape change and cell invasion by Discs large during Drosophila oogenesis
Authors: Goode Scott , Perrimon Norbert
Published: February 2008

Drosophila Discs large (Dlg) is a tumor suppressor gene whose loss in epithelial tissues causes disrupted cell polarity and increased cell proliferation. A human Dlg homolog, hDlg, has been implicated in tumorigenic processes via its association with the product of the Adenomatous Polyposis Coli (APC) gene. We show for the first time that Drosophila Dlg is required to block cell invasion. Loss of dlg activity during oogenesis causes follicle cells to change shape and invade in a pattern similar to border cells, a small population of cells that break from the post-mitotic follicular epithelium during wild-type oogenesis, yet dlg mutant cells have not adopted a border cell fate. Both functional and morphological evidence indicates that cooperation between germ cell and follicle cell Dlg, probably mediated by Dlg PDZ domains, is crucial for regulating cell mixing, suggesting a novel developmental mechanism and mode of action for the Dlg family of molecules. These findings suggest that Dlg does not simply inhibit individual cell behaviors during oogenesis, but rather acts in a developmental pathway essential for blocking cell proliferation and migration in a spatio-temporally defined manner. A model for Dlg action in blocking cell invasion is presented.
P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro
Authors: Mancebo Helena S.Y. , Lee Gary , Flygare John , Tomassini Joanne , Luu Percy , Zhu Yuerong , Peng Junmin , Blau Carol , Hazuda Daria , Price David , Flores Osvaldo
Published: February 2008

To identify novel inhibitors of transcriptional activation by the HIV Tat protein, we used a combination of in vitro and in vivo Tat-dependent transcription assays to screen >100,000 compounds. All compounds identified blocked Tat-dependent stimulation of transcriptional elongation. Analysis of a panel of structurally diverse inhibitors indicated that their target is the human homolog ofDrosophila positive transcription elongation factor b (P-TEFb). Loss of Tat transactivation in extracts depleted of the kinase subunit of human P-TEFb, PITALRE, was reversed by addition of partially purified human P-TEFb. Transfection experiments with wild-type or kinase knockout PITALRE demonstrated that P-TEFb is required for Tat function. Our results suggest that P-TEFb represents an attractive target for the development of novel HIV therapeutics.
The DAF-3 Smad protein antagonizes TGF-β-related receptor signaling in the Caenorhabditis elegansdauer pathway
Authors: Patterson Garth I. , Koweek Allison , Wong Arthur , Liu Yanxia , Ruvkun Gary
Published: February 2008

Signals from TGF-β superfamily receptors are transduced to the nucleus by Smad proteins, which transcriptionally activate target genes. In Caenorhabditis elegans, defects in a TGF-β-related pathway cause a reversible developmental arrest and metabolic shift at the dauer larval stage. Null mutations in daf-3 suppress mutations in genes encoding this TGF-β signal, its receptors, and associated Smad signal transduction proteins. daf-3 encodes a Smad protein that is most closely related to mammalian DPC4, and is expressed throughout development in many of the tissues that are remodeled during dauer development. DAF-4, the type II TGF-β receptor in this pathway, is also expressed in remodeled tissues. These data suggest that the DAF-7 signal from sensory neurons acts as a neuroendocrine signal throughout the body to directly regulate developmental and metabolic shifts in tissues that are remodeled during dauer formation. A full-length functional DAF-3/GFP fusion protein is predominantly cytoplasmic, and this localization is independent of activity of the upstream TGF-β-related pathway. However, this fusion protein is associated with chromosomes in mitotic cells, suggesting that DAF-3 binds DNA directly or indirectly. DAF-3 transgenes also interfere with dauer formation, perhaps attributable to a dosage effect. A truncated DAF-3/GFP fusion protein that is predominantly nuclear interferes with dauer formation, implying a role for DAF-3 in the nucleus. These data suggest that DAF-7 signal transduction antagonizes or modifies DAF-3 Smad activity in the nucleus to induce reproductive development; when DAF-7 signals are disabled, unmodified DAF-3 Smad activity mediates dauer arrest and its associated metabolic shift. Therefore, daf-3 is unique in that it is antagonized, rather than activated, by a TGF-β pathway.
The Drosophila neuregulin homolog Vein mediates inductive interactions between myotubes and their epidermal attachment cells
Authors: Yarnitzky Talia , Min Li , Volk Talila
Published: February 2008

Inductive interactions between cells of distinct fates underlie the basis for morphogenesis and organogenesis across species. In theDrosophila embryo, somatic myotubes form specific interactions with their epidermal muscle attachment (EMA) cells. The establishment of these interactions is a first step toward further differentiation of the EMA cells into elongated tendon cells containing an organized array of microtubules and microfilaments. Here we show that the molecular signal for terminal differentiation of tendon cells is the secretedDrosophila neuregulin-like growth factor Vein produced by the myotubes. Although vein mRNA is produced by all of the myotubes, Vein protein is secreted and accumulates specifically at the muscle–tendon cell junctional site. In loss-of-function veinmutant embryos, muscle-dependent differentiation of tendon cells, measured by the level of expression of specific markers (Delilah and β1 tubulin) is blocked. When Vein is expressed in ectopic ectodermal cells, it induces the ectopic expression of these genes. Our results favor the possibility that the Drosophila EGF receptor DER/Egfr expressed by the EMA cells functions as a receptor for Vein. We show that Vein/Egfr binding activates the Ras pathway in the EMA cells leading to the transcription of the tendon-specific genes, stripe, delilah, andβ1 tubulin. In Egfr1F26mutant embryos that lack functional Egfr expression, the levels of Delilah and β1 tubulin are very low. In addition, the ability of ectopic Vein to induce the expression of Delilah and β1 tubulin depends on the presence of functional Egfrs. Finally, activation of the Egfr signaling pathway by either ectopically secreted Spitz, or activated Ras, leads to the ectopic expression of Delilah. These results suggest that inductive interactions between myotubes and their epidermal muscle attachment cells are initiated by the binding of Vein, to the Egfr on the surface of EMA cells.
Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation
Authors: Jallepalli Prasad V. , Brown Grant W. , Muzi-Falconi Marco , Tien Deborah , Kelly Thomas J.
Published: February 2008

Cyclin-dependent kinases (CDKs) promote the initiation of DNA replication and prevent reinitiation before mitosis, presumably through phosphorylation of key substrates at origins of replication. In fission yeast, the p65cdc18 protein is required to initiate DNA replication and interacts with the origin recognition complex (ORC) and the p34cdc2 CDK. Here we report that p65cdc18 becomes highly phosphorylated as cells undergo the G1 → S phase transition. This modification is dependent on p34cdc2 protein kinase activity, as well as six consensus CDK phosphorylation sites within the p65cdc18 polypeptide. Genetic interactions betweencdc18+ and the S-phase cyclin cig2+ suggest that CDK-dependent phosphorylation antagonizes cdc18+ function in vivo. Using site-directed mutagenesis, we show that phosphorylation at CDK consensus sites directly targets p65cdc18 for rapid degradation and inhibits its replication activity, as strong expression of a constitutively hypophosphorylated mutant form of p65cdc18 results in large amounts of DNA over-replication in vivo. Furthermore, the over-replication phenotype produced by this mutant p65cdc18 is resistant to increased mitotic cyclin/CDK activity, a known inhibitor of over-replication. Therefore, p65cdc18 is the first example of a cellular initiation factor directly regulated in vivo by CDK-dependent phosphorylation and proteolysis. Regulation of p65cdc18 by CDK phosphorylation is likely to contribute to the CDK-driven “replication switch” that restricts initiation at eukaryotic origins to once per cell cycle.
Functionally interacting telomerase RNAs in the yeast telomerase complex
Authors: Prescott John , Blackburn Elizabeth H.
Published: February 2008

The ribonucleoprotein (RNP) enzyme telomerase fromSaccharomyces cerevisiae adds telomeric DNA to chromosomal ends in short increments both in vivo and in vitro. Whether or not telomerase functions as a multimer has not been addressed previously. Here we show, first, that following polymerization, the telomerase RNP remains stably bound to its telomeric oligonucleotide reaction product. We then exploit this finding and a previously reported mutant telomerase RNA to demonstrate that, unexpectedly, the S. cerevisiae telomerase complex contains at least two functionally interacting RNA molecules that both act as templates for DNA polymerization. Here, functional telomerase contains at least two active sites.
CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival
Authors: Duckett Colin S. , Thompson Craig B.
Published: February 2008

CD30 is a cell-surface receptor that can augment lymphocyte activation and survival through its ability to induce the transcription factor NF-κB. CD30, however, has also been implicated in the induction of apoptotic cell death of lymphocytes. Here we show that one of the effects of CD30 signal transduction is to render cells sensitive to apoptosis induced by the type 1 tumor necrosis factor receptor (TNFR1). This sensitization is dependent on the TRAF-binding sites within the CD30 cytoplasmic domain. One of the proteins that binds to these sites is TRAF2, a signal transduction molecule that is also utilized by TNFR1 to mediate the activation of several downstream kinases and transcription factors. During CD30 signal transduction, we found that binding of TRAF2 to the cytoplasmic domain of CD30 results in the rapid depletion of TRAF2 and the associated protein TRAF1 by proteolysis. These data suggest a model in which CD30 limits its own ability to transduce cell survival signals through signal-coupled depletion of TRAF2. Depletion of intracellular TRAF2 and its coassociated proteins also increased the sensitivity of the cell to undergoing apoptosis during activation of death-inducing receptors such as TNFR1. Consistent with this hypothesis, expression of a dominant-negative form of TRAF2 was found to potentiate TNFR1-mediated death. These studies provide a potential mechanism through which CD30, as well as other TRAF-binding members of the TNFR superfamily, can negatively regulate cell survival.
Cooperative effects of INK4a and ras in melanoma susceptibility in vivo
Authors: Chin Lynda , Pomerantz Jason , Polsky David , Jacobson Mark , Cohen Carlos , Cordon-Cardo Carlos , Horner James W. , DePinho Ronald A.
Published: February 2008

The familial melanoma gene (INK4a/MTS1/CDKN2) encodes potent tumor suppressor activity. Although mice null for the ink4ahomolog develop a cancer-prone condition, a pathogenetic link to melanoma susceptibility has yet to be established. Here we report that mice with melanocyte-specific expression of activated H-rasG12V on an ink4a-deficient background develop spontaneous cutaneous melanomas after a short latency and with high penetrance. Consistent loss of the wild-type ink4a allele was observed in tumors arising in ink4a heterozygous transgenic mice. No homozygous deletion of the neighboring ink4b gene was detected. Moreover, as in human melanomas, the p53 gene remained in a wild-type configuration with no observed mutation or allelic loss. These results show that loss of ink4a and activation of Ras can cooperate to accelerate the development of melanoma and provide the first in vivo experimental evidence for a causal relationship between ink4a deficiency and the pathogenesis of melanoma. In addition, this mouse model affords a system in which to identify and analyze pathways involved in tumor progression against the backdrop of genetic alterations encountered in human melanomas.
Rescue of osteoclast function by transgenic expression of kinase-deficient Src insrc−/− mutant mice
Authors: Schwartzberg Pamela L. , Xing Lianping , Hoffmann Oskar , Lowell Clifford A. , Garrett Lisa , Boyce Brendan F. , Varmus Harold E.
Published: February 2008

The Src tyrosine kinase has been implicated in a wide variety of signal transduction pathways, yet despite the nearly ubiquitous expression of c-src, src−/− mice show only one major phenotype—osteopetrosis caused by an intrinsic defect in osteoclasts, the cells responsible for resorbing bone. To explore further the role of Src both in osteoclasts and other cell types, we have generated transgenic mice that express the wild-type and mutated versions of the chicken c-src proto-oncogene from the promoter of tartrate resistant acid phosphatase (TRAP), a gene that is expressed highly in osteoclasts. We demonstrate here that expression of a wild-type transgene in only a limited number of tissues can fully rescue the src−/− phenotype. Surprisingly, expression of kinase-defective alleles of c-srcalso reduces osteopetrosis in src−/− animals and partially rescues a defect in cytoskeletal organization observed in src−/− osteoclasts. These results suggest that there are essential kinase-independent functions for Src in vivo. Biochemical examination of osteoclasts from these mice suggest that Src may function in part by recruiting or activating other tyrosine kinases.
Identification of three regions essential for interaction between a ς-like factor and core RNA polymerase
Authors: Cliften Paul F. , Park Jae-Young , Davis Brian P. , Jang Sei-Heon , Jaehning Judith A.
Published: February 2008

The cyclic interactions that occur between the subunits of the yeast mitochondrial RNA polymerase can serve as a simple model for the more complex enzymes in prokaryotes and the eukaryotic nucleus. We have used two-hybrid and fusion protein constructs to analyze the requirements for interaction between the single subunit core polymerase (Rpo41p), and the ς-like promoter specificity factor (Mtf1p). We were unable to define any protein truncations that retained the ability to interact, indicating that multiple regions encompassing the entire length of the proteins are involved in interactions. We found that 9 of 15 nonfunctional (petite) point mutations in Mtf1p isolated in a plasmid shuffle strategy had lost the ability to interact. Some of the noninteracting mutations are temperature-sensitive petite (ts petite); this phenotype correlates with a precipitous drop in mitochondrial transcript abundance when cells are shifted to the nonpermissive temperature. One temperature-sensitive mutant demonstrated a striking pH dependence for core binding in vitro, consistent with the physical properties of the amino acid substitution. The noninteracting mutations fall into three widely spaced clusters of amino acids. Two of the clusters are in regions with amino acid sequence similarity to conserved regions 2 and 3 of ς factors and related proteins; these regions have been implicated in core binding by both prokaryotic and eukaryotic ς-like factors. By modeling the location of the mutations using the partial structure ofEscherichia coliς70, we find that two of the clusters are potentially juxtaposed in the three-dimensional structure. Our results demonstrate that interactions between ς-like specificity factors and core RNA polymerases require multiple regions from both components of the holoenzymes.
The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae
Authors: Chen Guang-Chao , Kim Yung-Jin , Chan Clarence S.M.
Published: February 2008

BEM2 of Saccharomyces cerevisiae encodes a Rho-type GTPase-activating protein that is required for proper bud site selection at 26°C and for bud emergence at elevated temperatures. We show here that the temperature-sensitive growth phenotype ofbem2 mutant cells can be suppressed by increased dosage of theGIC1 gene. The Gic1 protein, together with its structural homolog Gic2, are required for cell size and shape control, bud site selection, bud emergence, actin cytoskeletal organization, mitotic spindle orientation/positioning, and mating projection formation in response to mating pheromone. Each protein contains a CRIB (Cdc42/Rac-interactivebinding) motif and each interacts in the two-hybrid assay with the GTP-bound form of the Rho-type Cdc42 GTPase, a key regulator of polarized growth in yeast. The CRIB motif of Gic1 and the effector domain of Cdc42 are required for this association. Genetic experiments indicate that Gic1 and Gic2 play positive roles in the Cdc42 signal transduction pathway, probably as effectors of Cdc42. Subcellular localization studies with a functional green fluorescent protein–Gic1 fusion protein indicate that this protein is concentrated at the incipient bud site of unbudded cells, at the bud tip and mother-bud neck of budded cells, and at cortical sites on large-budded cells that may delimit future bud sites in the two progeny cells. The ability of Gic1 to associate with Cdc42 is important for its function but is apparently not essential for its subcellular localization.
Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast
Authors: Brown Jeffrey L. , Jaquenoud Malika , Gulli Marie-Pierre , Chant John , Peter Matthias
Published: February 2008

Cdc42p, a Rho-related GTP-binding protein, regulates cytoskeletal polarization and rearrangements in eukaryotic cells, but the effectors mediating this control remain unknown. Through the use of the complete yeast genomic sequence, we have identified two novel Cdc42p targets, Gic1p and Gic2p, which contain consensus Cdc42/Rac interactive–binding (CRIB) domains and bind specifically to Cdc42p–GTP. Gic1p and Gic2p colocalize with Cdc42p as cell polarity is established during the cell cycle and during mating in response to pheromones. Cells deleted for both GIC genes exhibit defects in actin and microtubule polarization similar to those observed incdc42 mutants. Finally, the interaction of the Gic proteins and Cdc42p is essential, as mutations in the CRIB domain of Gic2p that eliminate Cdc42p binding disrupt Gic2p localization and function. Thus, Gic1p and Gic2p define a novel class of Cdc42p targets that are specifically required for cytoskeletal polarization in vivo.
Mechanism of synergy between TATA and initiator: synergistic binding of TFIID following a putative TFIIA-induced isomerization
Authors: Emami Katayoon H. , Jain Anjali , Smale Stephen T.
Published: February 2008

The TFIID complex interacts with at least three types of core promoter elements within protein-coding genes, including TATA, initiator (Inr), and downstream promoter elements. We have begun to explore the mechanism by which the TFIID–Inr interaction leads to functional synergy between TATA and Inr elements during both basal and activated transcription. In DNase I footprinting assays, GAL4–VP16 recruited TFIID–TFIIA to core promoters containing either a TATA box, an Inr, or both TATA and Inr elements, with synergistic interactions apparent on the TATA–Inr promoter. Appropriate spacing between the two elements was essential for the synergistic binding. Despite the sequence-specific TFIID–Inr interactions, gel shift experiments revealed that TFIID alone possesses similar affinities for the TATA–Inr and TATA promoters. Interestingly, however, recombinant TFIIA strongly and selectively enhanced TFIID binding to the TATA–Inr promoter, with little effect on binding to the TATA promoter. Studies of the natural adenovirus major late promoter confirmed these findings, despite the existence of specific but nonfunctional TFIID interactions downstream of the Inr in that promoter. These results suggest that a TFIIA-induced conformational change is essential for the sequence-specific TFIID–Inr interaction to occur with sufficient affinity to support the functional synergism between TATA and Inr elements.
Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity
Authors: Lee Jehee , Tonozuka Takashi , Jayaram Makkuni
Published: February 2008

The Flp site-specific recombinase assembles its active site by recruiting the catalytic tyrosine (Tyr-343) from one Flp monomer into the pro-active site containing a triad of Arg-191, His-305, and Arg-308 from a second monomer. In principle, two active sites may be assembled from a Flp dimer by simultaneous, reciprocal contribution of the shared amino acids by its constituent monomers. In practice, only one of the two active sites is assembled at a time, as would be consistent with a recombination mechanism involving two steps of single-strand exchanges. By using substrates containing strand-specific base bulges, we demonstrate that the relative disposition of their DNA arms can account for this active site exclusion. We also show that the exclusion mechanism operates only at the level of positioning Tyr-343 with respect to the pro-active site, and not at the level of orienting the labile phosphodiester bond within the DNA chain. It is not negative cooperativity of substrate binding but, rather, the substrate-induced negative cooperativity in protein orientation that accomplishes half-of-the-sites activity in the Flp system.
Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed
Authors: Jiménez Gerardo , Paroush Ze’ev , Ish-Horowicz David
Published: February 2008

Relatively little is known about the molecular mechanisms involved in transcriptional repression, despite its importance in development and differentiation. Recent evidence suggests that some transcriptional repressors act by way of adaptor molecules known as corepressors. Here, we use in vivo functional assays to test whether different repressor activities are mediated by the Groucho (Gro) corepressor in theDrosophila embryo. Previously, Gro was proposed to mediate repression by the Hairy-related family of basic helix–loop–helix proteins. Our results indicate not only that repression by Hairy requires Gro, but that a repressor domain from the Engrailed (En) homeodomain protein is also Gro dependent. The latter result correlates with an ability of this En domain to bind to Gro in vitro. In contrast, repressor regions from the Even-skipped, Snail, Krüppel, and Knirps transcription factors are effective in the absence of Gro. These results show that Gro is not generally required for repression, but acts as a specific corepressor for a fraction of negative regulators, including Hairy and En.
Viral mimicry: common mode of association with HCF by VP16 and the cellular protein LZIP
Authors: Freiman Richard N. , Herr Winship
Published: February 2008

Upon infection of human cells, the herpes simplex virus protein VP16 associates with the endogenous cell-proliferation factor HCF. VP16 can also associate with HCFs from invertebrates, suggesting that VP16 mimics a cellular protein whose interaction with HCF has been conserved. Here, we show that VP16 mimics the human basic leucine-zipper protein LZIP, which, through association with HCF, may control cell-cycle progression. VP16 and LZIP share a tetrapeptide motif—D/EHXY—used to associate with human HCF. The LZIP-related Drosophila protein BBF-2/dCREB-A contains this HCF-binding motif, indicating that the LZIP–HCF interaction has been conserved during metazoan evolution.
Dual role of the Smad4/DPC4 tumor suppressor in TGFβ-inducible transcriptional complexes
Authors: Liu Fang , Pouponnot Celio , Massagué Joan
Published: February 2008

Upon ligand binding, the receptors of the TGFβ family phosphorylate Smad proteins, which then move into the nucleus where they activate transcription. To carry out this function, the receptor-activated Smads 1 and 2 require association with the product of deleted in pancreatic carcinoma, locus 4 (DPC4), Smad4. We investigated the step at which Smad4 is required for transcriptional activation. Smad4 is not required for nuclear translocation of Smads 1 or 2, or for association of Smad2 with a DNA binding partner, the winged helix protein FAST-1. Receptor-activated Smad2 takes Smad4 into the nucleus where they form a complex with FAST-1 that requires these three components to activate transcription. Smad4 contributes two functions: Through its amino-terminal domain, Smad4 promotes binding of the Smad2/Smad4/FAST-1 complex to DNA; through its carboxy-terminal domain, Smad4 provides an activation function required for Smad1 or Smad2 to stimulate transcription. The dual function of Smad4 in transcriptional activation underscores its central role in TGFβ signaling.
The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses
Authors: Peng Jinrong , Carol Pierre , Richards Donald E. , King Kathryn E. , Cowling Rachel J. , Murphy George P. , Harberd Nicholas P.
Published: February 2008

The Arabidopsis gai mutant allele confers a reduction in gibberellin (GA) responsiveness. Here we report the molecular cloning of GAI and a closely related gene GRS. The predicted GAI (wild-type) and gai (mutant) proteins differ only by the deletion of a 17-amino-acid segment from within the amino-terminal region. GAI and GRS contain nuclear localization signals, a region of homology to a putative transcription factor, and motifs characteristic of transcriptional coactivators. Genetic analysis indicates that GAI is a repressor of GA responses, that GA can release this repression, and that gai is a mutant repressor that is relatively resistant to the effects of GA. Mutations at SPY and GAR2 suppress thegai phenotype, indicating the involvement of GAI, SPY, and GAR2 in a signaling pathway that regulates GA responses negatively. The existence of this pathway suggests that GA modulates plant growth through derepression rather than through simple stimulation.
Cryptococcus neoformans mating and virulence are regulated by the G-protein α subunit GPA1 and cAMP
Authors: Alspaugh J. Andrew , Perfect John R. , Heitman Joseph
Published: February 2008

This study explores signal transduction pathways that function during mating and infection in the opportunistic, human fungal pathogenCryptococcus neoformans. The gene encoding a G-protein α subunit homolog, GPA1, was disrupted by homologous recombination. The gpa1 mutant strain was viable but exhibited a defect in mating in response to nitrogen starvation. Additionally, the gpa1 mutant strain failed to induce two well-established virulence factors—melanin synthesis, in response to glucose starvation; and capsule production, in response to iron limitation. As a consequence, virulence of the gpa1 mutant strain was significantly attenuated in an animal model of cryptococcal meningitis. Reintroduction of the wild-type GPA1 gene complemented thegpa1 mutant phenotypes and restored mating, melanin and capsule production, and virulence. Similarly, exogenous cAMP also suppressed the gpa1 mutant phenotypes, restoring mating and production of melanin and capsule. These observations support a model in which GPA1 has a role in sensing diverse environmental signals required for mating and virulence by regulating cAMP metabolism in C. neoformans.
Organizing a functional junctional complex requires specific domains of the Drosophila MAGUK Discs large
Authors: Hough Colleen D. , Woods Daniel F. , Park Sangbin , Bryant Peter J.
Published: February 2008

Discs large (Dlg) was the first identified member of an increasingly important class of proteins called membrane-associated guanylate kinase homologs (MAGUKs), which are often concentrated at cell junctions and contain distinct peptide domains named PDZ1-3, SH3, HOOK, and GUK. Dlg is localized at and required for the formation of both septate junctions in epithelial cells and synaptic junctions in neurons. In the absence of Dlg, epithelia lose their organization and overgrow. We tested the functions of each domain of Dlg in vivo by constructing transgenic flies expressing altered forms of the protein. In the first set of experiments each domain was examined for its ability to correctly target an epitope-tagged Dlg to pre-existing septate junctions. Based on these results the Hook domain is necessary for localization of the protein to the cell membrane and the PDZ2 is required for restricting the protein to the septate junction. In the second set of experiments each domain was tested for its role in growth regulation and organization of epithelial structure. These results show that PDZ1 and GUK are apparently dispensable for function, PDZ2 and PDZ3 are required for growth regulation but not for epithelial structure, and SH3 and HOOK are essential for both aspects of function. The results demonstrate the functional modularity of Dlg and clarify the functions of individual MAGUK domains in regulating the structure and growth of epithelial tissue.
mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain
Authors: Cho Eun-Jung , Takagi Toshimitsu , Moore Christine R. , Buratowski Stephen
Published: February 2008

Capping of mRNA occurs shortly after transcription initiation, preceding other mRNA processing events such as mRNA splicing and polyadenylation. To determine the mechanism of coupling between transcription and capping, we tested for a physical interaction between capping enzyme and the transcription machinery. Capping enzyme is not stably associated with basal transcription factors or the RNA polymerase II (Pol II) holoenzyme. However, capping enzyme can directly and specifically interact with the phosphorylated form of the RNA polymerase carboxy-terminal domain (CTD). This association occurs in the context of the transcription initiation complex and is blocked by the CTD–kinase inhibitor H8. Furthermore, conditional truncation mutants of the Pol II CTD are lethal when combined with a capping enzyme mutant. Our results provide in vitro and in vivo evidence that capping enzyme is recruited to the transcription complex via phosphorylation of the RNA polymerase CTD.
Met receptor signaling is required for sensory nerve development and HGF promotes axonal growth and survival of sensory neurons
Authors: Maina Flavio , Hilton Mark C. , Ponzetto Carola , Davies Alun M. , Klein Rüdiger
Published: February 2008

The development of the nervous system is a dynamic process during which factors act in an instructive fashion to direct the differentiation and survival of neurons, and to induce axonal outgrowth, guidance to, and terminal branching within the target tissue. Here we report that mice expressing signaling mutants of the hepatocyte growth factor (HGF) receptor, the Met tyrosine kinase, show a striking reduction of sensory nerves innervating the skin of the limbs and thorax, implicating the HGF/Met system in sensory neuron development. Using in vitro assays, we find that HGF cooperates with nerve growth factor (NGF) to enhance axonal outgrowth from cultured dorsal root ganglion (DRG) neurons. HGF also enhances the neurotrophic activities of NGF in vitro, and Met receptor signaling is required for the survival of a proportion of DRG neurons in vivo. This synergism is specific for NGF but not for the related neurotrophins BDNF and NT3. By using a mild signaling mutant of Met, we have demonstrated previously that Met requires signaling via the adapter molecule Grb2 to induce proliferation of myoblasts. In contrast, the actions of HGF on sensory neurons are mediated by Met effectors distinct from Grb2. Our findings demonstrate a requirement for Met signaling in neurons during development.
Mcm2 is a target of regulation by Cdc7–Dbf4 during the initiation of DNA synthesis
Authors: Lei Ming , Kawasaki Yasuo , Young Michael R. , Kihara Makoto , Sugino Akio , Tye Bik K.
Published: February 2008

The initiation of DNA synthesis is an important cell cycle event that defines the beginning of S phase. This critical event involves the participation of proteins whose functions are regulated by cyclin dependent protein kinases (Cdks). The Mcm2–7 proteins are a family of six conserved proteins that are essential for the initiation of DNA synthesis in all eukaryotes. InSaccharomyces cerevisiae,members of the Mcm2–7 family undergo cell cycle-specific phosphorylation. Phosphorylation of Mcm proteins at the beginning of S phase coincides with the removal of these proteins from chromatin and the onset of DNA synthesis. In this study, we identifiedDBF4,which encodes the regulatory subunit of a Cdk-like protein kinase Cdc7–Dbf4, in a screen for second site suppressors ofmcm2-1.Thedbf4suppressor mutation restores competence to initiate DNA synthesis to themcm2-1mutant. Cdc7–Dbf4 interacts physically with Mcm2 and phosphorylates Mcm2 and three other members of the Mcm2–7 family in vitro. Blocking the kinase activity of Cdc7–Dbf4 at the G1-to-S phase transition also blocks the phosphorylation of Mcm2 at this defined point of the cell cycle. Taken together, our data suggest that phosphorylation of Mcm2 and probably other members of the Mcm2–7 proteins by Cdc7–Dbf4 at the G1-to-S phase transition is a critical step in the initiation of DNA synthesis at replication origins.
The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences
Authors: Tracy Robert B. , Baumohl Jason K. , Kowalczykowski Stephen C.
Published: February 2008

The Rad51 protein of Saccharomyces cerevisiae is a eukaryotic homolog of the RecA protein, the prototypic DNA strand-exchange protein of Escherichia coli. RAD51 gene function is required for efficient genetic recombination and for DNA double-strand break repair. Recently, we demonstrated that RecA protein has a preferential affinity for GT-rich DNA sequences—several of which exhibit enhanced RecA protein-promoted homologous pairing activity. The fundamental similarity between the RecA and Rad51 proteins suggests that Rad51 might display an analogous bias. Using in vitro selection, here we show that the yeast Rad51 protein shares the same preference for GT-rich sequences as its prokaryotic counterpart. This bias is also manifest as an increased ability of Rad51 protein to promote the invasion of supercoiled DNA by homologous GT-rich single-stranded DNA, an activity not previously described for the eukaryotic pairing protein. We propose that the preferred utilization of GT-rich sequences is a conserved feature among all homologs of RecA protein, and that GT-rich regions are loci for increased genetic exchange in both prokaryotes and eukaryotes.