bluuebunny/crossref_metadata_2025 huggingface dataset:
Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island.
Authors: Macleod D , Charlton J , Mullins J , Bird A P
Published: June 2007

In an attempt to find the mechanism by which CpG islands remain free of methylation we have undertaken a detailed examination of the mouse adenine phosphoribosyltransferase (aprt) gene. This housekeeping gene has a CpG island that extends over the gene promoter and includes the first two exons. We show that the island is free of methylation at all CpGs, whereas the flanks are methyated. Detailed patterns of methylation beyond the boundaries of the CpG island vary between cells. In vivo footprinting across the island region shows that three GC boxes clustered at the 5' edge of the CpG island are occupied, most probably by Sp1. No other footprints are detected within the island region. Deletion or mutagenesis of the Sp1 sites causes de novo methylation of the CpG island in a transgenic mouse assay. Thus, the peripherally located Sp1 sites are necessary to keep the aprt island methylation free.
Drosophila TFIIA directs cooperative DNA binding with TBP and mediates transcriptional activation.
Authors: Yokomori K , Zeidler M P , Chen J L , Verrijzer C P , Mlodzik M , Tjian R
Published: June 2007

Drosophila transcription factor IIA (TFIIA) is composed of three subunits (30, 20, and 14 kD) that function during initiation of transcription. We reported previously the characterization of cDNAs that encode a precursor (dTFIIA-L) of the Drosophila TFIIA 30- and 20-kD subunits. In the absence of the smallest subunit, dTFIIA-S (14 kD), the unprocessed large subunit failed to exhibit any detectable promoter binding or transcriptional activity. Here, we report the molecular cloning and expression of dTFIIA-S, which has allowed the assembly of holo-dTFIIA (dTFIIA-L/S). Subunit interaction studies indicate that dTFIIA-S binds to an amino-terminal domain of dTFIIA-L, which likely corresponds to the endogenous 30-kD processed species. In addition, both dTFIIA-S and the carboxy-terminal domain of dTFIIA-L, which corresponds to the 20-kD species, independently interact weakly with the TATA-binding protein (TBP). In contrast, the holo-dTFIIA (L/S) binds TBP with high affinity. The dTFIIA-L/S complex also binds cooperatively with TBP to TATA box DNA sequences, generating an extended DNase footprint pattern. The reconstituted holo-dTFIIA is able to stimulate basal transcription of several core promoter templates. Interestingly, dTFIIA-L/S is also able to significantly enhance transcriptional activation by upstream transcription factors including Sp1, VP16, and NTF-1. These results suggest that dTFIIA is a multifunctional transcription factor capable of influencing DNA binding as well as interactions with the basal machinery, thereby enhancing activator-dependent transcription.
Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription.
Authors: Sun X , Ma D , Sheldon M , Yeung K , Reinberg D
Published: June 2007

Human TFIIA activity is composed of three subunits (alpha, beta, gamma). Here we report the isolation of a human cDNA clone encoding the gamma-subunit and the reconstitution of TFIIA activity from recombinant polypeptides (holo-TFIIA). Protein-protein interaction analysis established that the beta and gamma subunits of TFIIA interact with the TBP component of TFIID. The alpha-subunit is recruited into the complex by association with the gamma-subunit. Functional studies indicate that recombinant TFIIA stimulates basal TFIID-dependent transcription but is without effect on TBP-dependent transcription. Our studies indicate that TFIIA not only functions by physically removing negative components present in TFIID (antirepression), as demonstrated previously, but that it can stimulate basal transcription through components of the TFIID complex. Holo-TFIIA also stimulated activation of transcription in vitro as well as in vivo in transfected HeLa cells.
A stem/loop in U6 RNA defines a conformational switch required for pre-mRNA splicing.
Authors: Fortner D M , Troy R G , Brow D A
Published: June 2007

U6 small nuclear RNA (snRNA) is an essential component of the spliceosome, the ribonucleoprotein complex that carries out the splicing of pre-mRNAs. The precise function of U6 RNA is unknown, but it has been proposed to participate directly in catalysis of the splicing reaction. We present biochemical and genetic evidence for an intramolecular stem/loop structure in the 3' half of U6 RNA of the yeast Saccharomyces cerevisiae that is mutually exclusive with the intermolecular base-pairing between U6 RNA and U4 snRNA. Strains with mutations that stabilize the U6 RNA 3'-intramolecular stem exhibit cold-sensitive growth and accumulate free U4 RNA, indicative of a block in U4/U6 snRNP assembly. The cold sensitivity can be partially suppressed by overexpression of U4 RNA. Mutations that disrupt base-pairing in the intramolecular 3' stem and mutations elsewhere in U6 RNA also suppress the growth defect. We conclude that a large conformational switch, involving melting of the U6 RNA 3' stem, is required for U4/U6 snRNP assembly. We hypothesize that formation of the U6 RNA intramolecular 3' stem after U4 RNA leaves the assembled spliceosome serves to activate U6 RNA for splicing by juxtaposing regions in U6 RNA that interact with U2 small nuclear RNA.
Subcellular localization of proteins involved in the assembly of the spore coat of Bacillus subtilis.
Authors: Driks A , Roels S , Beall B , Moran C P , Losick R
Published: June 2007

Spores of the bacterium Bacillus subtilis are encased in a two-layered protein shell, which consists of an electron-translucent, lamellar inner coat, and an electron-dense outer coat. The coat protein CotE is both a structural component of the coat and a morphogenetic protein that is required for the assembly of the outer coat. We now show that CotE is located in the outer coat of the mature spore and that at an intermediate stage of sporulation, when the developing spore (the forespore) is present as a free protoplast within the sporangium, CotE is localized in a ring that surrounds the forespore but is separated from it by a small gap. We propose that the ring is the site of assembly of the outer coat and that the gap is the site of formation of the inner coat. Assembly of the ring depends on the sporulation protein SpoIVA, which sits close to or on the surface of the outer membrane that encircles the forespore. We propose that SpoIVA creates a basement layer around the forespore on which coat assembly takes place. The subcellular localization and assembly of CotE and other coat proteins are therefore determined by the capacity of SpoIVA to recognize and adhere to a specific surface within the sporangium, the outer membrane of the forespore.
The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast.
Authors: Allen J B , Zhou Z , Siede W , Friedberg E C , Elledge S J
Published: June 2007

Inhibition of DNA synthesis prevents mitotic entry through the action of the S-phase checkpoint. We have isolated S-phase arrest-defective (sad) mutants that show lethality in the presence of the DNA synthesis inhibitor hydroxyurea (HU). Several of these mutants show phenotypes consistent with inappropriate mitotic entry in the presence of unreplicated DNA, indicating a defect in the S-phase checkpoint. sad1 mutants are additionally defective for the G1 and G2 DNA damage checkpoints, and for DNA damage-induced transcription of RNR2 and RNR3. The transcriptional response to DNA damage requires activation of the Dun1 protein kinase. Activation of Dun1 in response to replication blocks or DNA damage is blocked in sad1 mutants. The HU sensitivity of sad1 mutants is suppressed by mutations in CKS1, a subunit of the p34CDC28 kinase, further establishing a link between cell cycle progression and lethality. sad1 mutants are allelic to rad53, a radiation-sensitive mutant. SAD1 encodes an essential protein kinase. The observation that SAD1 controls three distinct checkpoints suggests a common mechanism for cell cycle arrest at these points. Together, these observations implicate protein phosphorylation in the cellular response to DNA damage and replication blocks.
Regulated expression of the beta-globin gene locus in synthetic nuclei.
Authors: Barton M C , Emerson B M
Published: June 2007

Regulated gene expression within a complex chromosomal locus requires multiple nuclear processes. We have analyzed the transcriptional properties of the cloned chick beta-globin gene family when assembled into synthetic nuclei made by use of Xenopus egg extracts. Assembly in an erythroid protein environment correctly recapitulates tissue-specific chromatin structure and long-range promoter-enhancer interaction within the chromosomal locus, resulting in beta-globin gene activation. Nucleosome-repressed beta-globin templates can be transcriptionally activated by double-stranded DNA replication in the presence of staged erythroid proteins by remodeling of the chromatin structure within the promoter region and establishment of distal promoter-enhancer communication. The programmed transcriptional state of a gene, as encoded by its chromatin structure and long-range promoter-enhancer interactions, is stable to nuclear decondensation and DNA replication unless active remodeling occurs in the presence of specific DNA-binding proteins.
Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos.
Authors: Chen W S , Manova K , Weinstein D C , Duncan S A , Plump A S , Prezioso V R , Bachvarova R F , Darnell J E
Published: June 2007

Expression of HNF-4, a transcription factor in the steroid hormone receptor superfamily, is detected only in the visceral endoderm of mouse embryos during gastrulation and is expressed in certain embryonic tissues from 8.5 days of gestation. To examine the role of HNF-4 during embryonic development, we disrupted the gene in embryonic stem cells and found that the homozygous loss of functional HNF-4 protein was an embryonic lethal. Cell death was evident in the embryonic ectoderm at 6.5 days when these cells normally initiate gastrulation. As assessed by expression of Brachyury and HNF-3 beta, primitive streak formation and initial differentiation of mesoderm do occur, but with a delay of approximately 24 h. Development of embryonic structures is severely impaired. These results demonstrate that the expression of HNF-4 in the visceral endoderm is essential for embryonic ectoderm survival and normal gastrulation.
Crystal structure of a PPR1-DNA complex: DNA recognition by proteins containing a Zn2Cys6 binuclear cluster.
Authors: Marmorstein R , Harrison S C
Published: June 2007

PPR1 is a yeast transcription factor that contains a six-cysteine, two-zinc (Zn) domain, homologous to a similar structure in GAL4. Like GAL4, it binds to DNA sites with conserved CGG triplets symmetrically placed near each end. Whereas the GAL4 site has 11 intervening base pairs, the PPR1 site has 6. The crystal structure of a 95-residue fragment of PPR1 in specific complex with DNA shows that the protein binds to a symmetrical 14-bp recognition site as a nonsymmetrical homodimer. An amino-terminal Zn domain interacts with a conserved CGG triplet near each end of the site through major groove contacts, and the carboxy-terminal residues mediate dimerization through a coiled-coil element and an extended strand. A linker region, connecting the Zn domain and the coiled-coil, folds into a beta-hairpin. This hairpin packs differently on the two subunits and leads to a striking asymmetry, which is largely restricted to the dimerization and linker regions of the protein. Comparison with the GAL4-DNA structure shows that their specificities for sites of different length are determined by the preferred folds of their respective linker segments and by residues at the amino-terminal ends of their coiled-coils. None of these residues contact DNA in PPR1, and they contact only the sugar phosphate backbone in GAL4. We propose that this novel mode of DNA site selection is employed by other proteins that contain a Zn2Cys6 binuclear cluster.
A novel effector domain from the RNA-binding protein TLS or EWS is required for oncogenic transformation by CHOP.
Authors: Zinszner H , Albalat R , Ron D
Published: June 2007

In human myxoid liposarcoma, a chromosomal rearrangement leads to fusion of the growth-arresting and DNA-damage-inducible transcription factor CHOP (GADD153) to a peptide fragment encoded by the TLS gene. We have found that wild-type TLS and a closely related sarcoma-associated protein, EWS, are both abundant nuclear proteins that associate in vivo with products of RNA polymerase II transcription. This association leads to the formation of a ternary complex with other heterogeneous RNA-binding proteins (hnRNPs), such as A1 and C1/C2. An NIH-3T3-based transformation assay was used to study the oncogenic role of the sarcoma-associated domain of these RNA-binding proteins. Transduction of the TLS-CHOP oncogene into cells by means of a retroviral expression vector leads to loss of contact inhibition, acquisition of the ability to grow as colonies in soft agar, and tumor formation in nude mice. Mutations that interfere with the function of the leucine zipper dimerization domain or the adjacent basic region of CHOP abolish transformation. The essential role of the TLS component was revealed by the inability of truncated forms to fully transform cells. Domain swap between TLS- and EWS-associated oncogenes demonstrated that the component contributed by the RNA-binding proteins are functionally interchangeable, whereas the transcription factor component specifies tumor phenotype. The sarcoma-associated component of TLS and EWS contribute a strong transcriptional activation domain to the fusion proteins; however, transforming activity cannot be fully substituted by fusion of CHOP to other strong trans-activators. The juxtaposition of a novel effector domain from sarcoma-associated RNA-binding proteins to the targeting domain of transcription factors such as CHOP leads to the creation of a potent oncogene.
Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity.
Authors: Sun P , Enslen H , Myung P S , Maurer R A
Published: June 2007

The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases.
A human keratin 14 "knockout": the absence of K14 leads to severe epidermolysis bullosa simplex and a function for an intermediate filament protein.
Authors: Chan Y , Anton-Lamprecht I , Yu Q C , Jäckel A , Zabel B , Ernst J P , Fuchs E
Published: June 2007

Since their discovery, the function of intermediate filaments (IFs) has remained obscure. In skin, epidermal cells have extensive cytoskeletal architectures of IFs, composed of type I and type II keratin heterodimers. Clues to possible functions of these proteins have come from recent studies showing that several autosomal-dominant, blistering skin disorders are caused by defects in genes that encode epidermal keratins. These diseases all exhibit cell degeneration and keratin network perturbations in cells that express the particular mutant keratin gene. However, it is not clear from these studies whether cytolysis arises from the presence of large insoluble keratin aggregates that compromise cellular physiology or from the absence of an extensive keratin filament network, which jeopardizes mechanical integrity. We report here the analysis of an extremely rare case of severe recessive epidermolysis bullosa simplex (EBS), where the patient lacks a discernible keratin filament network in basal epidermal cells. Genetic analyses revealed a homozygous point mutation that yielded a premature termination codon in the major basal type I keratin gene and caused complete ablation of K14. The consanguineous parents were normal, each harboring one copy of the null K14 mutation. Analysis of cultured keratinocytes enabled us to document that the loss of K14 is not compensated for by the up-regulation of any other type I keratin. When taken together with the in vivo studies showing the presence of cell fragility generated from the lack of an extensive basal keratin network, these findings provide the first clear demonstration of loss of function associated with the absence of an IF protein in vivo.
Dorsal-ventral patterning of the Drosophila embryo depends on a putative negative growth factor encoded by the short gastrulation gene.
Authors: Francois V , Solloway M , O'Neill J W , Emery J , Bier E
Published: June 2007

Pattern formation in the dorsal region of the Drosophila embryo depends on the activity of a small group of zygotically acting genes. dpp, a key gene in this group, encodes a TGF-beta-like product (Dpp) that has been proposed to function as a morphogen with peak levels of Dpp-specifying amnioserosa, the dorsal-most cell type, and lower Dpp levels specifying dorsal ectoderm. The short gastrulation gene also contributes to patterning the dorsal region, but unlike the other genes involved in this process, sog activity is only required in ventral cells. Genetic evidence indicates that sog functions to antagonize dpp activity. In this report we present further phenotypic characterization of sog mutant embryos in dorsal and lateral regions and describe the cloning of the sog locus. sog is expressed in a broad lateral stripe of cells that abuts the dorsal territory of dpp-expressing cells. sog is predicted to encode a protein with an internal signal sequence and a large extracellular domain containing four repeats of a novel motif defined by the spacing of 10 cysteine residues that is distantly related to domains present in thrombospondin and procollagen. We propose that one or more of these cysteine repeats can be liberated by proteolytic cleavage of the primary Sog protein. These putative soluble Sog peptides may then diffuse into the dorsal region to antagonize the activity of Dpp, leading to the subdivision of the dorsal territory into amnioserosa and dorsal ectoderm.
Characterization of a unique protein component of yeast RNase MRP: an RNA-binding protein with a zinc-cluster domain.
Authors: Schmitt M E , Clayton D A
Published: June 2007

RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to cleave mitochondrial primer RNA sequences from a variety of sources. Most of the RNase MRP activity is found in the nucleus where it plays a role in the processing of 5.8S rRNA. A temperature-conditional point mutation in the yeast RNA component of the enzyme has been identified. This mutation results in a loss of normal rRNA processing at the nonpermissive temperature while cellular levels of the RNA component of RNase MRP remain stable. High-copy suppressor analysis of this point mutation was employed to identify interacting proteins. A unique suppressor, termed SNM1 (suppressor of nuclear mitochondrial endoribonuclease 1), was identified repeatedly. The SNM1 gene was localized to the right arm of chromosome IV, directly adjacent to the SNF1 gene, and it contains an open reading frame encoding a protein of 198 amino acids. The protein contains a leucine zipper motif, a zinc-cluster motif, and a serine/lysine-rich tail. The gene was found to be essential for viability in a yeast cell, consistent with it being a protein component of the RNase MRP ribonucleoprotein complex. Recombinant SNM1 protein binds RNA in both gel retardation and Northwestern assays. Antibodies raised against bacterially expressed proteins identified four separate species in yeast whole cell extracts. Antibodies directed against the SNM1 protein immunoprecipitated RNase MRP RNA from whole-cell extracts without precipitating the structurally and functionally related RNase P RNA. We propose that the SNM1 protein is an essential and specific component of the RNase MRP ribonucleoprotein complex, the first unique protein of this complex to be identified.
Influence of a steroid receptor DNA-binding domain on transcriptional regulatory functions.
Authors: Lefstin J A , Thomas J R , Yamamoto K R
Published: June 2007

We have isolated two independent mutations in the DNA-binding domain of the rat glucocorticoid receptor, P493R and S459A, that implicate DNA binding in the control of attached transcriptional activation domains, either that of the receptor itself or of VP16. The mutants are capable of activating transcription normally, but unlike wild-type receptors, they interfere with particular transcriptional activators in yeast and mammalian cells, and inhibit growth when overexpressed in yeast. The mutant residues reside at positions within the three-dimensional structure of the receptor that could, in principle, transduce structural changes from the DNA-binding surface of the receptor to other functional domains. These findings, together with the salt dependence of specific and nonspecific DNA binding by these receptors, suggest that specific DNA acts as an allosteric effector that directs the functional interaction of the receptor with targets of transcriptional activation and that the P493R and S459A mutants mimic the allosteric effect of specific DNA, allowing the receptor to interact with regulatory targets even in the absence of specific DNA binding.
A possible role for the 60-kD Ro autoantigen in a discard pathway for defective 5S rRNA precursors.
Authors: O'Brien C A , Wolin S L
Published: June 2007

The Ro autoantigen is a 60-kD protein that is usually found in small cytoplasmic RNA-protein complexes known as Ro RNPs. Although the Ro RNPs are abundant and conserved components of a variety of vertebrate and invertebrate cells, their function is unknown. We have discovered that the Ro protein is also found complexed with certain variant 5S rRNAs in Xenopus oocytes. These RNAs contain one or more point mutations compared with the major oocyte 5S rRNA sequence as well as additional nucleotides at the 3' end. We demonstrate that the Ro protein binds specifically mutant 5S rRNAs containing 3' terminal extensions. These mutant RNAs are processed inefficiently to mature 5S rRNA and most eventually are degraded. The observation that the Ro autoantigen specifically associates with defective 5S rRNA precursors suggests that this protein may function as part of a novel quality control or discard pathway for 5S rRNA production.
Regulation of pyrBI operon expression in Escherichia coli by UTP-sensitive reiterative RNA synthesis during transcriptional initiation.
Authors: Liu C , Heath L S , Turnbough C L
Published: June 2007

Pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12 occurs through UTP-sensitive transcriptional attenuation and through a second mechanism that functions at the level of transcriptional initiation. In this study we demonstrate that this second control mechanism is based on UTP-sensitive reiterative RNA synthesis within a run of three T-A base pairs in the pyrBI initially transcribed region. Our results show that high UTP levels induce the synthesis in vitro of nascent transcripts with the sequence AAUUUUn (where n = 1 to > 30), which are not extended downstream to include pyrBI sequences. Synthesis of these transcripts, which are initiated at the predominant in vivo transcriptional start site, inhibits the production of full-length pyrBI transcripts. A TTT to GTA mutation in the pyrBI initially transcribed region eliminates reiterative transcription and stimulates productive transcription in vitro. When introduced into the E. coli chromosome, this mutation causes a sevenfold increase in pyrBI expression in cells grown under conditions of pyrimidine excess and nearly abolishes pyrimidine-mediated regulation of pyrBI expression when coupled with a mutation that eliminates attenuation control. Additional experiments indicate that the context of the three T-A base pairs within the pyrBI initially transcribed region is important for reiterative transcription. A possible mechanism for reiterative transcription and the likely involvement of this process in the regulation of other genes are discussed.
Termination-altering amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation.
Authors: Weilbaecher R , Hebron C , Feng G , Landick R
Published: June 2007

To identify regions of the largest subunit of RNA polymerase that are potentially involved in transcript elongation and termination, we have characterized amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase that alter expression of reporter genes preceded by terminators in vivo. Termination-altering substitutions occurred in discrete segments of beta', designated 2, 3a, 3b, 4a, 4b, 4c, and 5, many of which are highly conserved in eukaryotic homologs of beta'. Region 2 substitutions (residues 311-386) are tightly clustered around a short sequence that is similar to a portion of the DNA-binding cleft in E. coli DNA polymerase I. Region 3b (residues 718-798) corresponds to the segment of the largest subunit of RNA polymerase II in which amanitin-resistance substitutions occur. Region 4a substitutions (residues 933-936) occur in a segment thought to contact the transcript 3' end. Region 5 substitutions (residues 1308-1356) are tightly clustered in conserved region H near the carboxyl terminus of beta'. A representative set of mutant RNA polymerases were purified and revealed unexpected variation in percent termination at six different rho-independent terminators. Based on the location and properties of these substitutions, we suggest a hypothesis for the relationship of subunits in the transcription complex.
Dynamic association of proteins with the pre-mRNA branch region.
Authors: MacMillan A M , Query C C , Allerson C R , Chen S , Verdine G L , Sharp P A
Published: June 2007

The association of proteins with the branch site region during pre-mRNA splicing was probed using a novel methodology to site-specifically modify the pre-mRNA with the photo-reagent benzophenone. Three sets of proteins were distinguished by the kinetics of their associations with pre-mRNAs, by their association with discrete splicing complexes, and by their differing factor requirements. An early U1 snRNP-dependent cross-link of the branch region to a p80 species was followed by cross-links to p14, p35, and p150 polypeptides associated with the U2 snRNP-pre-mRNA complex. Concomitant with formation of the spliceosome, a rearrangement of protein factors about the branch region occurred, in which the p35 and p150 cross-links were replaced by p220 and p70 species. These results establish that the branch region is recognized in a dynamic fashion by multiple distinct proteins during the course of spliceosomal assembly.
Location, structure, and function of the target of a transcriptional activator protein.
Authors: Tang H , Severinov K , Goldfarb A , Fenyo D , Chait B , Ebright R H
Published: June 2007

We have isolated and characterized single-amino-acid substitution mutants of RNA polymerase alpha subunit defective in CAP-dependent transcription at the lac promoter but not defective in CAP-independent transcription. Our results establish that (1) amino acids 258-265 of alpha constitute an "activation target" essential for CAP-dependent transcription at the lac promoter but not essential for CAP-independent transcription, (2) amino acid 261 is the most critical amino acid of the activation target, (3) amino acid 261 is distinct from the determinants for alpha-DNA interaction, and (4) the activation target may fold as a surface amphipathic alpha-helix. We propose a model for transcriptional activation at the lac promoter that integrates these and other recent results regarding transcriptional activation and RNA polymerase structure and function.
Igf2r and Igf2 gene expression in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos: absence of regulation by genomic imprinting.
Authors: Latham K E , Doherty A S , Scott C D , Schultz R M
Published: June 2007

Genomic imprinting in mammals is believed to result from modifications to chromosomes during gametogenesis that inactivate the paternal or maternal allele. The genes encoding the insulin-like growth factor type 2 (Igf2) and its receptor (Igf2r) are reciprocally imprinted and expressed from the paternal and maternal genomes, respectively, in the fetal and adult mouse. We find that both genes are expressed in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos. These results indicate that inactivation of imprinted genes occurs postfertilization (most likely postimplantation) and that genomic imprinting and gene inactivation are separate processes. We propose that imprinting marks the chromosome so that regulatory factors expressed in cells at later times can recognize the imprint and selectively inactivate the maternal or paternal allele. For these genes, this finding invalidates models of genomic imprinting that require them to be inactive from the time of fertilization.
The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5.
Authors: Kranz J E , Satterberg B , Elion E A
Published: June 2007

Activation of the Saccharomyces cerevisiae MAP kinase Fus3 is thought to occur via a linear pathway involving the sequential action of three proteins: Ste5, a protein of unknown function, Ste11, a MAPKK kinase homolog, and Ste7, a MAPK kinase homolog which phosphorylates and activates Fus3. In this report, we present evidence for a novel mechanism of Fus3 activation that involves a direct association with Ste5, a protein not predicted to interact with Fus3. First, overexpression of Ste5 suppresses fus3 point mutations in an allele-specific manner and increases Fus3 kinase activity in vitro. Second, Ste5 associates with Fus3 in vivo as demonstrated by the two-hybrid system and by two methods of copurification. Third, Ste5 and Fus3 associate prior to pheromone stimulation even when Fus3 is inactive, and in strains lacking Ste7 and Ste11. Fourth Ste5 is phosphorylated by Fus3 in purified complexes and copurifies with an additional protein kinase(s). These observations suggest the possibility that Ste5 promotes signal transduction by tethering Fus3 to its activating protein kinase(s).
The fission yeast mating pheromone P-factor: its molecular structure, gene structure, and ability to induce gene expression and G1 arrest in the mating partner.
Authors: Imai Y , Yamamoto M
Published: June 2007

Schizosaccharomyces pombe h+ cells secrete a diffusable mating pheromone called P-factor. Here we show that the map2 gene, a defect of which confers h(+)-specific sterility, encodes the precursor of P-factor. We purified P-factor from cells overexpressing map2 and determined its amino acid sequence. P-factor is a peptide of 23 residues, with the sequence Thr-Tyr-Ala-Asp-Phe-Leu-Arg-Ala-Tyr-Gln-Ser- Trp-Asn-Thr-Phe-Val-Asn-Pro-Asp-Arg-Pro-Asn-Leu. A synthetic peptide of this sequence gave the same specific activity and chromatographic profile as the purified P-factor, suggesting that P-factor is unmodified. h- cells starved for nutrition showed a morphological response to P-factor. Transcription of the sxa2 gene, which encodes a protease thought to degrade P-factor, was activated in these cells. The cry1 null mutant, which lacks adenylyl cyclase and has little intracellular cAMP, was susceptible to P-factor even in the presence of nutrients. Combination of the cyr1 and sxa2 mutations enhanced this susceptibility. P-factor induced not only responses toward mating but also arrest of the cell cycle at the G1 phase in h- cyr1 sxa2 cells. This proves that the S. pombe mating pheromone has the ability to arrest cell cycle progression, which has previously been obscured by the usual requirement for mating of nutritional starvation and subsequent growth arrest.
Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.
Authors: Ringquist S , Schneider D , Gibson T , Baron C , Böck A , Gold L
Published: June 2007

In Escherichia coli the unusual amino acid selenocysteine is incorporated cotranslationally at an in-frame UGA codon. Incorporation of selenocysteine relies, in part, on the interaction between a specialized elongation factor, the SELB protein, and a cis-acting element within the mRNA. Boundary and toeprint experiments illustrate that the SELB-GTP-Sec-tRNA(Sec) ternary complex binds to the selenoprotein encoding mRNAs fdhF and fdnG, serving to increase the concentration of SELB and Sec-tRNA(Sec) on these mRNAs in vivo. Moreover, toeprint experiments indicate that SELB recognizes the ribosome-bound message and that, upon binding, SELB may protrude out of the ribosomal-mRNA track so as to approach the large ribosomal subunit. The results place the mRNA-bound SELB-GTP-Sec-tRNA(Sec) ternary complex at the selenocysteine codon (as expected) and suggest a mechanism to explain the specificity of selenocysteine insertion. Cis-acting mRNA regulatory elements can tether protein factors to the translation complex during protein synthesis.
The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.
Authors: Luetteke N C , Phillips H K , Qiu T H , Copeland N G , Earp H S , Jenkins N A , Lee D C
Published: June 2007

Mice harboring the waved-1 (wa-1) and waved-2 (wa-2) mutations exhibit skin and eye abnormalities that are strikingly similar to those of TGF-alpha-deficient mice, and wa-1 and TGF-alpha were recently shown to be allelic. Because the wa-2 mutation was mapped previously to the vicinity of the EGF/TGF-alpha receptor (EGFR) gene on mouse chromosome 11, we hypothesized that the wa-2 phenotype might result from a defect in either the expression or activity of EGFR, or both. In the present report, we show that EGFR mRNA and protein of normal size are expressed in wa-2 liver and skin at levels that are comparable to those in the corresponding normal tissues, and that the ability of wa-2 EGFR to bind ligand is unaltered. However, ligand-dependent autophosphorylation of wa-2 EGFR is diminished 5- to 10-fold in vitro, and the ability of wa-2 EGFR to phosphorylate an exogenous substrate is reduced by > 90% compared with that of the control receptor. EGF-induced tyrosine phosphorylation, including that of EGFR itself, is also diminished in skin, particularly at lower dose of exogenous EGF. To establish the nature of the wa-2 mutation, we determined the nucleotide sequence of the coding region of normal and wa-2 murine EGFR cDNAs. A comparison of these sequences revealed a single-nucleotide transversion resulting in the substitution of a glycine for a conserved valine residue near the amino terminus of the tyrosine kinase domain. The importance of this mutation was confirmed by showing that its introduction into an otherwise normal EGFR markedly reduced the receptor's tyrosine kinase activity in transfected Chinese hamster ovary cells. Finally, in situ hybridization analysis demonstrated expression of EGFR predominantly in the outer root sheath of active hair follicles in neonatal mice. As we previously localized TGF-alpha mRNA to the inner root sheath, this pattern of EGFR expression is consistent with the effect of the wa-2 mutation on hair structure, and together with our previous characterization of TGF-alpha-deficient mice, reveals a critical role for signaling by this ligand/receptor system in skin.
Molecular analysis of reverse mutations from nonagouti (a) to black-and-tan (a(t)) and white-bellied agouti (Aw) reveals alternative forms of agouti transcripts.
Authors: Bultman S J , Klebig M L , Michaud E J , Sweet H O , Davisson M T , Woychik R P
Published: June 2007

The agouti gene regulates the differential production of eumelanin (black or brown) and phaeomelanin (yellow) pigment granules by melanocytes in the hair follicles of mice. The original nonagouti (a) allele, which confers a predominantly black coat color, has been shown to revert to two other more dominant agouti alleles, black-and-tan (a(t)) and white-bellied agouti (Aw), with an exceptionally high frequency. The a(t) and Aw alleles confer phenotypes in which the pigmentation is not uniformly distributed over the dorsal and ventral surfaces of the animal; in both cases the ventral surface of the animal is markedly lighter than the dorsal surface due to an increase in phaeomelanin production. To understand the unusually high reversion rate of a to a(t) or Aw, and to decipher the molecular events associated with the different pigmentation patterns associated with these three agouti alleles, we have characterized a, a(t) and Aw at the molecular level. Here, we report that insertions of 11, 6, and 0.6 kb are present at precisely the same position in the first intron of the agouti gene in a, a(t), and Aw, respectively. The a insertion consists of a 5.5-kb VL30 element that has incorporated 5.5 kb of additional sequence internally; this internal sequence is flanked by 526-bp direct repeats. The a(t) allele contains only the VL30 element and a single, internal 526-bp repeat. The Aw allele has only a solo VL30 LTR. Based on the comparison of the structure of the a(t) and Aw insertions, we propose that reverse mutations occur by excision of inserted sequences in a through homologous recombination, utilizing either the 526-bp direct repeats to generate a(t) or the VL30 LTRs to generate Aw. Moreover, the analysis of these three alleles has allowed us to identify additional exons of the agouti gene that give rise to alternatively processed forms of agouti mRNA. We demonstrate that the distinct insertions in a, a(t) and Aw cause pigmentation differences by selectively inactivating the expression of different forms of agouti transcripts.
Raf acts downstream of the EGF receptor to determine dorsoventral polarity during Drosophila oogenesis.
Authors: Brand A H , Perrimon N
Published: June 2007

In Drosophila, as in mammalian cells, the Raf serine/threonine kinase appears to act as a common transducer of signals from several different receptor tyrosine kinases. We describe a new role for Raf in Drosophila development, showing that Raf acts in the somatic follicle cells to specify the dorsoventral polarity of the egg. Targeted expression of activated Raf (Rafgof) within follicle cells is sufficient to dorsalize both the eggshell and the embryo, whereas reduced Raf activity ventralizes the eggshell. We show that Raf functions downstream of the EGF receptor to instruct the dorsal follicle cell fate. In this assay, human and Drosophila Rafgof are functionally similar, in that either can induce ventral follicle cells to assume a dorsal fate.
The Aspergillus nidulans fluG gene is required for production of an extracellular developmental signal and is related to prokaryotic glutamine synthetase I.
Authors: Lee B N , Adams T H
Published: June 2007

Mutations in the Aspergillus nidulans fluG gene disrupt the programmed induction of asexual sporulation and result in formation of fluffy colonies that are characterized by undifferentiated cotton-like masses of vegetative cells. We show that the fluG mutant phenotype is suppressed when fluG mutant colonies are grown next to wild-type colonies even if the two strains are separated by dialysis membrane with a 6000- to 8000-dalton pore size. fluG encodes a cytoplasmically localized approximately 96,000-dalton polypeptide that is present at relatively constant levels during vegetative growth and following developmental induction. Sequence analysis of fluG demonstrated that the carboxy-terminal 436 amino acids predicted by the 864-codon FluG open reading frame shares approximately 28% identity with GSI-type prokaryotic glutamine synthetases. We consider it unlikely that FluG functions in synthesis of glutamine but instead propose that FluG functions as a GSI-related enzyme in synthesizing an extracellular signal directing asexual sporulation and perhaps other aspects of colony growth. The relationships between fluG and other genes identified by fluffy mutants are discussed.
Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.
Authors: White A E , Livanos E M , Tlsty T D
Published: June 2007

Genomic integrity is maintained by a network of cellular activities that assess the status of the genome at a given point in time, provide signals to proceed with or halt cell cycle progression, and provide for repair of damaged DNA. Mutations in any part of these pathways can have the ultimate effect of disturbing chromosomal integrity. Recent work suggests that p53 performs this integrator function in mammalian cells. Our present study demonstrates that in mortal cells, the expression of E6 and E7 viral oncoproteins of type 16 human papillomavirus each disrupts the integration of these signals by diverged pathways. Cells expressing E6 protein, which binds and degrades the p53 protein, exhibited alterations in cell cycle control when placed in drug and displayed the ability to amplify the CAD gene. The expression of E7, which binds different cellular proteins important for transformation, including Rb, led to a p53-independent alteration in cell cycle control, a widespread cytocidal response, and polyploidy as a mechanism of drug resistance. These results demonstrate that diverse perturbations of molecular pathways can have different effects on chromosomal integrity.
An active v-abl protein tyrosine kinase blocks immunoglobulin light-chain gene rearrangement.
Authors: Chen Y Y , Wang L C , Huang M S , Rosenberg N
Published: June 2007

Lymphoid cells transformed by Abelson murine leukemia virus have provided one of the classic models for study of early B-cell development and immunoglobulin rearrangement. Most of these cells have rearranged their heavy-chain locus but not their light chain genes, suggesting that an active v-abl protein interferes with this differentiation step. To test this hypothesis, light-chain gene structure was examined in pre-B cells transformed by temperature-sensitive mutants of the Abelson virus and in derivatives that survive at the nonpermissive temperature because they express a human BCL-2 gene. Our studies reveal that inactivation of the v-abl protein tyrosine kinase triggers high-frequency rearrangement of kappa and lambda light-chain genes. These events are accompanied by marked increases in the expression of RAG-1 and RAG-2 RNAs. These increases occur in the absence of protein synthesis but are dependent on inactivation of the v-abl protein tyrosine kinase. As documented in the accompanying paper (Klug et al., this issue), an active v-abl protein also suppresses the activity of NF-kappa B/rel and expression controlled by the kappa intron enhancer. Together these data demonstrate that the v-abl protein specifically interferes with light-chain gene rearrangement by suppressing at least two pathways essential for this stage of B-cell differentiation and suggest that tyrosine phosphorylation is important in regulating RAG gene expression.
Posterior transformation, neurological abnormalities, and severe hematopoietic defects in mice with a targeted deletion of the bmi-1 proto-oncogene.
Authors: van der Lugt N M , Domen J , Linders K , van Roon M , Robanus-Maandag E , te Riele H , van der Valk M , Deschamps J , Sofroniew M , van Lohuizen M
Published: June 2007

The bmi-1 proto-oncogene has been implicated in B-cell lymphomagenesis in E mu-myc transgenic mice. Distinct domains of the Bmi-1 protein are highly conserved within the Drosophila protein Posterior Sex Combs, a member of the Polycomb group involved in maintaining stable repression of homeotic genes during development. We have inactivated the bmi-1 gene in the germ line of mice by homologous recombination in ES cells. Null mutant mice display three phenotypic alterations: (1) a progressive decrease in the number of hematopoietic cells and an impaired proliferative response of these cells to mitogens; (2) neurological abnormalities manifested by an ataxic gait and sporadic seizures; and (3) posterior transformation, in most cases along the complete anteroposterior axis of the skeleton. The observations indicate that Mbi-1 plays an important role in morphogenesis during embryonic development and in hematopoiesis throughout pre- and postnatal life. Furthermore, these data provide the first evidence of functional conservation of a mammalian Polycomb group homolog.
Dominant interfering alleles define a role for c-Myb in T-cell development.
Authors: Badiani P , Corbella P , Kioussis D , Marvel J , Weston K
Published: June 2007

The transcription activator c-Myb is expressed at high levels in immature thymocytes and during T-cell activation and may be a regulator of T-cell differentiation. To investigate the role of c-Myb in T-cell development, we generated transgenic mice in which two dominant interfering Myb alleles, one a competitive inhibitor of DNA binding, and the other, an active repressor comprising the Myb DNA-binding domain linked to the Drosophila Engrailed transcription repressor domain, were expressed from early times onward in T cells. Both alleles partially blocked thymopoiesis and inhibited proliferation of mature T cells. The Myb-En chimera was the more efficient repressor and might serve as an archetype for the manufacture of other dominant interfering transcription factor alleles.