bluuebunny/crossref_metadata_2025 huggingface dataset:
Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice.
Authors: Suh T T , Holmbäck K , Jensen N J , Daugherty C C , Small K , Simon D I , Potter S , Degen J L
Published: June 2007

To explore the role of the key coagulation factor, fibrinogen, in development, hemostasis, wound repair, and disease pathogenesis, we disrupted the fibrinogen A alpha chain gene in mice. Homozygous, A alpha chain-deficient (A alpha-/-) mice are born normal in appearance, and there is no evidence of fetal loss of these animals based on the Mendelian pattern of transmission of the mutant A alpha chain allele. All of the component chains of fibrinogen (A alpha, B beta, and gamma) are immunologically undetectable in the circulation of both neonatal and adult A alpha-/- mice, and blood samples fail to either clot or support platelet aggregation in vitro. Overt bleeding events develop shortly after birth in approximately 30% of A alpha-/- mice, most frequently in the peritoneal cavity, skin, and soft tissues around joints. Remarkably, most newborns displaying signs of bleeding ultimately control the loss of blood, clear the affected tissues, and survive the neonatal period. Juveniles and young adult A alpha-/- mice are predisposed to spontaneous fatal abdominal hemorrhage, but long-term survival is variable and highly dependent on genetic background. The periodic rupture of ovarian follicles in breeding-age A alpha-/- females does not appear to significantly diminish life expectancy relative to males; however, pregnancy uniformly results in fatal uterine bleeding around the tenth day of gestation. Microscopic analysis of spontaneous lesions found in A alpha-/- mice suggests that fibrin(ogen) plays a fundamental role in the organization of cells at sites of injury.
Protein phosphorylation on serine, threonine, and tyrosine residues modulates membrane-protein interactions and transcriptional regulation in Salmonella typhimurium.
Authors: Ostrovsky P C , Maloy S
Published: June 2007

There exists a plethora of tyrosine kinases that play essential roles in regulation of eukaryotic proteins. Several dual specificity kinases that phosphorylate proteins on threonine, serine, and tyrosine residues also play critical roles in eukaryotic phosphorylation cascades. In contrast, very few prokaryotic proteins have been shown to be phosphorylated on tyrosine residues, and the functions of the rare examples remain obscure. Furthermore, no dual specificity kinases have been described in prokaryotes. Our results indicate that PutA protein from the bacterium Salmonella typhimurium autophosphorylates on several threonine, serine, and tyrosine residues. PutA protein both represses the proline utilization (put) operon and degrades proline to glutamate. These two opposing functions are regulated by the availability of proline and the membrane sites needed for the proline dehydrogenase activity of PutA protein. In addition, these functions are modulated by phosphorylation of PutA protein. The rate of dephosphorylation of PutA protein is determined by the availability of proline and membranes. Dephosphorylated PutA protein has a higher DNA binding affinity than the phosphorylated protein and thus may prevent toxic overexpression of PutA protein in the absence of available membrane sites.
Asymmetry in active complexes of FLP recombinase.
Authors: Qian X H , Cox M M
Published: June 2007

The FLP recombinase promotes a site-specific recombination reaction in the 2mu plasmid of yeast. The protein-DNA complex that carries out the reaction is asymmetric. Three FLP monomers bound to specific FLP-recognition sequences are required to efficiently carry out one set of reciprocal DNA cleavage and strand exchange events on a Holliday junction substrate. If a fourth monomer plays an auxiliary role in the reaction, it is bound without sequence specificity. The data suggest a modified model for cleavage of DNA in trans by the FLP recombinase that might help reconcile some seemingly conflicting resulted obtained with integrase class recombinases.
Epigenetic self-regulation of developmental excision of an internal eliminated sequence on Paramecium tetraurelia.
Authors: Duharcourt S , Butler A , Meyer E
Published: June 2007

Differentiation of the somatic macronucleus of ciliates after sexual events involves the programmed excision of thousands of single-copy internal eliminated sequences (IESs) from the germ-line genome. We have studied two cell lines of Paramecium tetraurelia that have identical germ-line genomes but differ in their macronuclear genomes. In the IES- cell line, a 222-bp IES interrupting a coding sequence is reproducibly excised during macronuclear differentiation, whereas it is not in the IES+ cell line. In a cross between the two lines, the developmental alternative in maternally inherited, suggesting that it is epigenetically controlled by the old (prezygotic) macronucleus in each cell. Transformation of the macronucleus of both lines with plasmids carrying fragments of either version of the gene shows that the presence of the IES sequence in the old macronucleus results in retention of the IES in the new macronuclear genome of sexual progeny. This could be attributable to (1) inhibition of excision, or (2) repair of a double-strand gap left in the genomic sequence after constitutive excision of the IES, by a polymerization mechanism using a homologous IES+ template from the old macronucleus. The latter possibility is ruled out by experiments showing that modified IESs can inhibit excision without being copied in the new macronuclear genome. Possible mechanisms are discussed in the light of a quantitative analysis of excision inhibition by the maternal IES sequence.
An enhancer deletion affects both H19 and Igf2 expression.
Authors: Leighton P A , Saam J R , Ingram R S , Stewart C L , Tilghman S M
Published: June 2007

The distal end of mouse Chromosome 7 contains four tightly linked genes whose expression is dependent on their parental inheritance. Mash-2 and H19 are expressed exclusively from the maternal chromosome, whereas Insulin-2 (Ins-2) and Insulin-like growth factor 2 (Igf2) are paternally expressed. The identical expression during development of the 3'-most genes in the cluster, Igf2 and H19, led to the proposal that their imprinting was mechanistically linked through a common set of transcriptional regulatory elements. To test this hypothesis, a targeted deletion of two endoderm-specific enhancers that lie 3' of H19 was generated by homologous recombination in embryonic stem cells. Inheritance of the enhancer deletion through the maternal lineage led to a loss of H19 gene expression in cells of endodermal origin, including cells in the liver, gut, kidney, and lung. Paternal inheritance led to a very similar loss in the expression of Igf2 RNA in the same tissues. These results establish that H19 and Igf2 utilize the same endoderm enhancers, but on different parental chromosomes. Mice inheriting the enhancer deletion from fathers were 80% of normal size, reflecting a partial loss-of-function of Igf2. The reduction was uniformly observed in a number of internal organs, indicating that insulin-like growth factor II (IGFII), the product of Igf2, acts systemically in mice to affect prenatal growth. A modest decline in Ins-2 RNA was observed in the yolk sac. In contrast Mash-2, which is expressed in spongiotrophoblast cells of the placenta, was unaffected by the enhancer deletion.
Temporally distinct patterns of p53-dependent and p53-independent apoptosis during mouse lens development.
Authors: Pan H , Griep A E
Published: June 2007

Programmed cell death, or apoptosis, is a critical event in the development of multicellular organisms, and its perturbation is implicated in many diseases including cancer. The tumor suppressor protein p53 is known to mediate apoptosis induced by the DNA tumor virus oncoproteins, adenovirus E1A (AdE1A) and SV40 T antigen (SV40 Tag). We have recently demonstrated that the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) modulate apoptosis when expressed in the lens of transgenic mice. In this study we have identified the pathways that mediate E7 induction and E6 inhibition of apoptosis during different stages in the development of the lens. E7 transgenic mice made p53-null were only partially rescued in their apoptotic phenotype, indicating that both p53-dependent and -independent pathways mediate E7-induced apoptosis in the lens. The E6 transgene and p53-null genotype acted additively to reduce levels of apoptosis induced by E7 in neonatal lenses, indicating that E6 modulates apoptosis at least in part through p53-independent mechanisms. The partial reduction in E7-induced apoptosis by the p53-null genotype correlated with an increased incidence of lens tumors in adult E7 transgenic mice. Analyses of embryonic lenses at E13.5, E15.5, and E17.5 revealed a temporally distinct activation of p53-dependent and -independent apoptosis in the E7 lens. During the early stages of lens development, apoptosis was highly p53-dependent, whereas at later stages, apoptosis occurred through both p53-independent and -dependent pathways. This later time correlates temporally with the time of normal fiber cell denucleation, which can be inhibited by E6 through a p53-independent mechanism. These data suggest a similarity between the mechanism regulating E7-induced, p53-independent apoptosis and the apoptotic-like developmental process of fiber cell denucleation, and the mechanisms through which E6 suppresses both processes.
Altering specific telomerase RNA template residues affects active site function.
Authors: Gilley D , Lee M S , Blackburn E H
Published: June 2007

The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA by copying a template sequence in the telomerase RNA. We studied the functional roles of specific residues in the Tetrahymena telomerase RNA template region. Unexpectedly, mutation of certain templating residues caused dramatic effects on specific aspects of the enzyme reaction, including loss of enzymatic fidelity and premature product dissociation. None of these fundamental changes in enzymatic action are explainable by altered base-pairing between the telomerase RNA and DNA substrate. These influences of specific template bases of the telomerase RNA on enzymatic properties of telomerase provide evidence for critical roles of these RNA residues in two active site functions--fidelity and DNA substrate/enzyme interaction.
sur-2, a novel gene, functions late in the let-60 ras-mediated signaling pathway during Caenorhabditis elegans vulval induction.
Authors: Singh N , Han M
Published: June 2007

We describe here a new gene acting downstream of let-60 ras in the vulval signaling pathway of Caenorhabditis elegans. The sur-2 (suppressor of ras) gene is defined by eight mutations identified in a genetic screen for suppressors of the Multivulva phenotype of let-60(n1046), an activated let-60 ras mutation. sur-2 mutations result in pleiotropic, incompletely penetrant phenotypes that include a Vulvaless phenotype in hermaphrodites, defects in development of the male tail, gonadal abnormalities, and larval lethality, indicating a role for the sur-2 gene product in multiple developmental events. Genetic epistasis analyses suggest that sur-2 is required late in the vulval signaling pathway, downstream of let-60 Ras, and is likely to act downstream of the Raf/MAP Kinase cascade. We cloned the sur-2 gene by DNA-mediated transformation and have shown that it encodes a novel protein. We also show that a sur-2::lacZ transgene is expressed in the vulval precursor cells at the time of vulval determination.
Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.
Authors: Lydon J P , DeMayo F J , Funk C R , Mani S K , Hughes A R , Montgomery C A , Shyamala G , Conneely O M , O'Malley B W
Published: June 2007

Although progesterone has been recognized as essential for the establishment and maintenance of pregnancy, this steroid hormone has been recently implicated to have a functional role in a number of other reproductive events. The physiological effects of progesterone are mediated by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. In most cases the PR is induced by estrogen, implying that many of the in vivo effects attributed to progesterone could also be the result of concomitantly administered estrogen. Therefore, to clearly define those physiological events that are specifically attributable to progesterone in vivo, we have generated a mouse model carrying a null mutation of the PR gene using embryonic stem cell/gene targeting techniques. Male and female embryos homozygous for the PR mutation developed normally to adulthood. However, the adult female PR mutant displayed significant defects in all reproductive tissues. These included an inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development, and an inability to exhibit sexual behavior. Collectively, these results provide direct support for progesterone's role as a pleiotropic coordinator of diverse reproductive events that together ensure species survival.
fyn tyrosine kinase is involved in keratinocyte differentiation control.
Authors: Calautti E , Missero C , Stein P L , Ezzell R M , Dotto G P
Published: June 2007

Induction of tyrosine phosphorylation is an early and specific event which is required for mouse keratinocyte differentiation to occur, in response to both calcium and TPA (12-0-tetradecanoylphorbol-13-acetate). We report here that there is an increase of tyrosine kinase activity immunoprecipitable with anti-phosphotyrosine antibodies specifically in response to calcium--and a number of other divalent cations--within 2 min of exposure. Such an activity does not correspond to any of the known tyrosine kinases that were tested. A second tyrosine kinase activity is induced in response to both calcium and TPA, and has been identified as fyn, a nonreceptor tyrosine kinase of the src family. fyn activation is induced in keratinocytes within 6 hr of calcium exposure, but already within 2 min of TPA treatment. Cortactin, a p80-85 substrate of src- and fyn-related kinases that localizes with actin at cell adhesion sites, is increasingly tyrosine phosphorylated in calcium- and TPA-induced differentiation, with a time course which parallels that of fyn activation. Keratinocytes with a specific disruption of the fyn, but not yes kinase gene show no induction of phosphorylation of p80-85 proteins, and are significantly altered in their differentiation response both in vitro and in vivo. Thus, at least two tyrosine kinase activities are induced in keratinocyte differentiation, one of which has been identified as fyn and shown to be specifically involved in this process.
Ectopic expression of the knox homeo box gene rough sheath1 alters cell fate in the maize leaf.
Authors: Schneeberger R G , Becraft P W , Hake S , Freeling M
Published: June 2007

Rough sheath1 (Rs1) is a dominant mutation that alters cell fate and causes unregulated cell division and expansion in the maize leaf. A knox (Kn1 like-homeo box gene) sequence closely linked to the Rs1-O mutation was cloned and shown by transposon mutagenesis to encode the rs1 gene. The deduced amino acid sequence of the RS1 protein is highly similar to KN1 in the homeo domain but contains a unique amino-terminal region. rs1 is expressed in the shoot apical meristem in a circular pattern preceding leaf initiation, but is not detectable in leaf primordia or mature leaves in normal plants. Rings of rs1 expression subtend leaf insertion sites in the shoot, and lateral organ primordia in inflorescence and floral meristems. The timing and position of rs1 expression in meristems suggests a possible role for rs1 in patterning the placement of lateral organs along the axis of the shoot. In contrast to wild type, rs1 is expressed in early leaf primordia of Rs1 mutant plants, suggesting that ectopic expression causes the mutant phenotype. Ectopic expression in Rs1-O plants suggests the ligular [corrected] region is more competent to respond to rs1 than blade or sheath tissues.
A novel bacterial transcription cycle involving sigma 54.
Authors: Tintut Y , Wang J T , Gralla J D
Published: June 2007

sigma 54 is the promoter recognition subunit of the form of bacterial RNA polymerase that transcribes from promoters with enhancer elements. DNase footprinting experiments show that sigma 54 is attached selectively to the template strand, which must be single-stranded for transcription initiation. sigma 54 remains bound at the promoter after core polymerase begins elongation, in contrast to the well-established sigma 70-holoenzyme transcription cycle. Permanganate footprinting experiments show that the bound sigma 54 and the elongating core RNA polymerase downstream of it are each associated with a single-strand DNA region. Template commitment assays show that the promoter-bound sigma 54 must be reconfigured before reinitiation of transcription can occur. This unexpected pathway raises interesting possibilities for transcriptional regulation, especially with regard to control at the level of reinitiation.
A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing.
Authors: Crispino J D , Sharp P A
Published: June 2007

The full set of consensus sequences at the 5' splice site is recognized during splicing of pre-mRNA in extracts depleted of U1 snRNP. High concentrations of HeLa SR proteins or purified SC35 alone promote the splicing of specific RNA substrates, bypassing the requirement for U1 snRNP in formation of the U2 snRNP-pre-mRNA complex. Under these conditions, mutations in the substrate that increase the sequence complementarity between U6 snRNA and the 5' splice site region can facilitate splicing. This provides additional strong evidence that U1 snRNP is not essential for splicing. Thus, the consensus sequence at the 5' splice site is probably recognized twice during splicing of most introns; however, some pre-mRNAs could potentially be processed in the absence of interactions with U1 snRNP in regions of the nucleus containing high concentrations of SR protein.
Loss of methylation activates Xist in somatic but not in embryonic cells.
Authors: Beard C , Li E , Jaenisch R
Published: June 2007

The mouse Xist gene, which is expressed only from the inactive X chromosome, is thought to play a role in the initiation of X inactivation. The 5' end of this gene is fully methylated on the active X chromosome and completely demethylated on the inactive X chromosome, suggesting that DNA methylation may be involved in controlling allele-specific transcription of this gene. To directly investigate the importance of DNA methylation in the control of Xist expression, we have examined its methylation patterns and expression in ES cells and embryos that are deficient in DNA methyltransferase activity. We report here that demethylation of the Xist locus in male mutant embryos induces Xist expression, thus establishing a direct link between demethylation and expression of the Xist gene in the postgastrulation embryo. The transcriptional activity of Xist in undifferentiated ES cells, however, appears to be independent of its methylation status. These results suggest that methylation may only become essential for Xist repression after ES cells have differentiated or after the embryo has undergone gastrulation.
Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.
Authors: Cheng S , Schmidt-Grimminger D C , Murant T , Broker T R , Chow L T
Published: June 2007

mRNA transcription, DNA amplification, and progeny production of human papillomaviruses (HPVs) are closely linked to squamous epithelial differentiation in patient papillomas. Because suprabasal, differentiated keratinocytes have exited the cell cycle for days or weeks and because viral DNA synthesis requires the host DNA replication machinery, HPVs must have a mechanism to reactivate the essential host genes. In this study, we show via acute recombinant retrovirus infection that an intact E7 gene of either high-risk or of low-risk HPV genotypes, under the control of its respective native enhancer-promoter, induced proliferating cell nuclear antigen (PCNAs) expression in the suprabasal cells of epithelial raft cultures of primary human foreskin keratinocytes (PHK). The cellular differentiation program was unaltered by the viral oncoprotein; it was essential for high HPV promoter activity. Furthermore, extensive host chromosomal DNA replication took place in differentiated cells of HPV-18 E7-expressing raft cultures and of patient laryngeal papillomas caused by HPV-6. These results indicate that the main function of the E7 protein is to reactivate host DNA replication machinery to support viral replication in differentiated, noncycling cells.
Lethal of scute, a proneural gene, participates in the specification of muscle progenitors during Drosophila embryogenesis.
Authors: Carmena A , Bate M , Jiménez F
Published: June 2007

The mechanisms that underlie the segregation of muscle founder cells in the Drosophila embryo are undefined. We show that the proneural gene lethal of scute (l'sc) is expressed in clusters of cells in the somatic mesoderm, from which individual muscle progenitors are singled out by progressive restriction of l'sc expression. Coexpression of l'sc and S59 (a putative muscle identity gene) in a subset of muscle progenitors shows that muscle founders are produced by division of muscle progenitors. In neurogenic mutant embryos the restriction of l'sc expression fails and all cells in a cluster coexpress l'sc and S59. Loss-of-function and overexpression phenotypes indicate a role for l'sc in the segregation of muscle progenitors and the formation of the muscle pattern.
Masquerade: a novel secreted serine protease-like molecule is required for somatic muscle attachment in the Drosophila embryo.
Authors: Murugasu-Oei B , Rodrigues V , Yang X , Chia W
Published: June 2007

Diverse developmental processes, such as neuronal growth cone migration and cell shape changes, are mediated by the interactions of cells with the extracellular matrix. We describe here a secreted molecule encoded by the Drosophila masquerade (mas) gene. Total loss of mas function causes defective muscle attachment. This mutant phenotype suggests that mas normally acts to stabilize cell-matrix interaction and represents a novel functional and limiting component in the adhesion process. mas encodes a 1047-amino-acid preproprotein that is further processed by proteolytic cleavage to generate two polypeptides. The carboxy-terminal polypeptide is highly similar to serine proteases and has an extracellular localization; however, it is unlikely to possess proteolytic activity, because the catalytic site serine has been substituted by a glycine residue. During embryonic development, the mas amino- and carboxy-terminal polypeptides are differentially localized. The mas carboxy-terminal polypeptide accumulates at all somatic muscle attachment sites, which corresponds well with the morphological defect seen in the mas mutants. Our findings demonstrate the involvement of an extracellular component in somatic muscle attachment. We propose that mas acts via its modified serine protease motif, either as a novel adhesion molecule and/or as a competitive antagonist of serine proteases, to stabilize muscle attachment.
DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I.
Authors: Kuhn A , Gottlieb T M , Jackson S P , Grummt I
Published: June 2007

DNA-dependent protein kinase (DNA-PK) comprises a catalytic subunit of approximately 350 kD (p350) and a DNA-binding component termed Ku. Although DNA-PK can phosphorylate many transcription factors, no function for this enzyme in transcription has been reported thus far. Here, we show that DNA-PK strongly represses transcription by RNA polymerase I (Pol I). Transcriptional repression by DNA-PK requires ATP hydrolysis, and DNA-PK must be colocalized on the same DNA molecule as the Pol I transcription machinery. Consistent with DNA-PK requiring DNA ends for activity, transcriptional inhibition only occurs effectively on linearized templates. Mechanistic studies including single-round transcriptions, abortive initiation assays, and factor-independent transcription on a tailed template demonstrate that DNA-PK inhibits initiation (i.e., the formation of the first phosphodiester bonds) but does not affect transcription elongation. Repression of transcription involves phosphorylation of the transcription initiation complex, and rescue experiments reveal that the inactivated factor remains bound to the promoter and thus prevents initiation complex formation. We discuss the possible relevance of these findings in regard to the control of rRNA synthesis in vivo.
Transient DNA breaks associated with programmed genomic deletion events in conjugating cells of Tetrahymena thermophila.
Authors: Saveliev S V , Cox M M
Published: June 2007

Thousands of programmed genomic deletion events occur during macronuclear development in Tetrahymena thermophila. Two of the deleted segments, called M and R, have been particularly well-characterized. Using ligation-mediated PCR, we have detected DNA strand breaks that correlate temporally and structurally with the deletion events in the M and R regions. The ends appear at positions that correspond precisely to boundaries of deleted sequences, as defined by observed chromosomal junctions found after deletion is complete. They occur exclusively during the known DNA rearrangement period in macronuclear development. The breaks are staggered by 4 bp in the complementary strands. Several alternative breaks were found at the end of one deleted region, consistent with multiple alternative chromosomal junctions detected previously. The free 5' ends generated at the breaks are phosphorylated. A purine residue always occurs at the free 3' ends, with an adenosine appearing in 11 of 12 cases. Patterns found in the detected break sites suggest rules that define the ends of the deleted segments within a transposon-like deletion mechanism.
Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases.
Authors: Gillespie D E , Berg C A
Published: June 2007

The homeless (hls) gene of Drosophila is required for anteroposterior and dorsoventral axis formation during oogenesis. At a low frequency, females homozygous for mutations in hls generate early egg chambers in which the oocyte is positioned incorrectly within the cyst. At a high frequency, late-stage egg chambers exhibit a ventralized chorion. Sequence analysis of the hls cDNA predicts a protein with amino-terminal homology to members of the DE-H family of RNA-dependent ATPases and putative helicases. Similarity of 51% in the amino-terminal third of the protein was found to two yeast splicing factors, PRP2 and PRP16, and to Drosophila Maleless, which is required for dosage compensation. To analyze Hls function, RNA localization patterns were determined for seven different transcripts in hls mutant ovaries. Previtellogenic transport to the oocyte was unaffected for all transcripts examined. Transport and localization of bicoid and oskar messages during vitellogenic stages were strongly disrupted, and the distribution and/or quantity of gurken, orb, and fs(1)K10 mRNAs were also affected, but to a lesser degree. In contrast, hu-li tai shao and Bicaudal-D transcripts were transported and localized normally in hls mutants. In addition, Kinesin heavy chain:beta-Galactosidase fusion protein failed to localize correctly to the posterior of the oocyte in vitellogenic egg chambers. Examination of the microtubule structure with anti-alpha-Tubulin antibodies revealed aberrant microtubule organizing center movement and an abnormally dense cytoplasmic microtubule meshwork. We discuss potential roles for Hls in organizing a cytoskeletal framework essential for localizing specific RNAs.
Similarities between trunk and spätzle, putative extracellular ligands specifying body pattern in Drosophila.
Authors: Casanova J , Furriols M , McCormick C A , Struhl G
Published: June 2007

The basic body plan of Drosophila is specified by four determinant systems that organize pattern along the anteroposterior and dorsoventral axes. Two of these systems (anterior and posterior) depend on localized mRNAs. In contrast, the other two (ventral and terminal) require locally generated extracellular ligands that are transduced, respectively, by the transmembrane receptors Toll and torso (tor). The ligand for the Toll receptor is thought to be spätzle (spz), a secreted protein that is activated by proteolytic cleavage. Here we report that trunk (trk), a gene required for activity of the tor receptor, encodes a protein that resembles spz in several respects. In particular, the sequence suggests that trk is a secreted protein and that it contains an internal site for proteolytic cleavage. Furthermore, the carboxy-terminal domain of trk has a similar arrangement of cysteines to that of spz. We propose that trk encodes an extracellular ligand involved in specifying terminal body pattern and suggest by analogy with spz that a cleaved form of trk constitutes the ligand for the tor receptor.
Enhancer-independent variants of phage Mu transposase: enhancer-specific stimulation of catalytic activity by a partner transposase.
Authors: Yang J Y , Jayaram M , Harshey R M
Published: June 2007

Assembly of the functional tetrameric form of phage Mu transposase (A protein) requires specific interactions between the Mu A monomer and its cognate sequences at the ends of the Mu genome (attL and attR) as well as those internal to it (the enhancer element). We describe here deletion variants of Mu A that show enhancer-independence in the assembly of the strand cleavage complex. These deletions remove the amino-terminal region of Mu A required for its interactions with the enhancer elements. The basal enhancer-independent activity of the variant proteins can be stimulated by a partner variant harboring an intact enhancer-binding domain. By exploiting the identical att-binding, and nonidentical enhancer-binding specificities of Mu A and D108 A (transposase of the Mu related phage D108), we show that the stimulation of activity is enhancer-specific. Taken together, these results suggest that the domain of Mu A that includes the enhancer-interacting region may exert negative as well as positive modulatory effects on the strand cleavage reaction. We discuss the implications of these results in the framework of a recent model for the assembly of shared active sites within the Mu A tetramer.
Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity.
Authors: Shi Y , Alin K , Goff S P
Published: June 2007

A novel cellular protein, Abl-interactor-1 (Abi-1), which specifically interacts with the carboxy-terminal region of Abl oncoproteins, has been identified in a mouse leukemia cell line. The protein exhibits sequence similarity to homeotic genes, contains several polyproline stretches, and includes a src homology 3 (SH3) domain at its very carboxyl terminus that is required for binding to Abl proteins. The abi-1 gene has been mapped to mouse chromosome 2 and is genetically closely linked to the c-abl locus. The gene is widely expressed in the mouse, with highest levels of mRNA found in the bone marrow, spleen, brain, and testes. The Abi-1 protein coimmunoprecipitates with v-Abl and serves as a substrate for kinase activity. When overexpressed in NIH-3T3 cells, abi-1 potently suppresses the transforming activity of Abelson leukemia virus expressing the full-length p160v-abl kinase but does not affect the transforming activity of viruses expressing a truncated p90v-abl or v-src kinases. We suggest that the Abi-1 protein may serve as a regulator of Abl function in transformation or in signal transduction.
The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling.
Authors: Lecourtois M , Schweisguth F
Published: June 2007

The Notch protein (N) acts as a transmembrane receptor for intercellular signals controlling cell fate choices in vertebrates and invertebrates. The signal of N activation may be transduced directly from the cell surface into the nucleus by an evolutionarily conserved transcription factor, Suppressor of Hairless [Su(H)], by its regulated nuclear import. Su(H) is shown here to play a direct role in the immediate response of the genome to N signaling in Drosophila. First, Su(H) mutant embryos derived from mutant germ-line clones exhibited a "neurogenic" phenotype of neural hypertrophy similar to the N phenotype. Second, the lack of N lateral signaling in these Su(H) mutant embryos was associated with a failure to express the m5 and m8 genes from the Enhancer of split Complex [E(spl)-C]. Finally, the Su(H) protein bound to the regulatory sequences of the E(spl)-C m5 and m8 genes, and these binding sites were required for the activation of the m5 and m8 promoters in the ventral neuroectoderm. The expression of the E(spl)-C m8 gene was found to be similarly regulated by Su(H) during wing imaginal disc development. Thus, the transcriptional activation of these E(spl)-C genes by Su(H) appears to be a direct and relatively general response to the activation of N. However, we also present evidence indicating that N signals in an Su(H)-independent manner during mesectoderm formation.
The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation.
Authors: Murthy K G , Manley J L
Published: June 2007

Cleavage-polyadenylation specificity factor (CPSF) is a multisubunit protein that plays a central role in 3' processing of mammalian pre-mRNAs. CPSF recognizes the AAUAAA signal in the pre-mRNA and interacts with other proteins to facilitate both RNA cleavage and poly(A) synthesis. Here we describe the isolation of cDNAs encoding the largest subunit of CPSF (160K) as well as characterization of the protein product. Antibodies raised against the recombinant protein inhibit polyadenylation in vitro, which can be restored by purified CPSF. Extending previous studies, which suggested that 160K contacts the pre-mRNA, we show that purified recombinant 160K can, by itself, bind preferentially to AAUAAA-containing RNAs. While the sequence of 160K reveals similarities to the RNP1 and RNP2 motifs found in many RNA-binding proteins, no clear match to a known RNA-binding domain was found, and RNA recognition is therefore likely mediated by a highly diverged or novel structure. We also show that 160K binds specifically to both the 77K (suppressor of forked) subunit of the cleavage factor CstF and to poly(A) polymerase (PAP). These results provide explanations for previously observed cooperative interactions between CPSF and CstF, which are responsible for poly(A) site specification, and between CPSF and PAP, which are necessary for synthesis of the poly(A) tail. Also supporting a direct role for 160K in these interactions is the fact that 160K by itself retains partial ability to cooperate with CstF in binding pre-mRNA and, unexpectedly, inhibits PAP activity in in vitro assays. We discuss the significance of these multiple functions and also a possible evolutionary link between yeast and mammalian polyadenylation suggested by the properties and sequence of 160K.
Roles for two RecA homologs in promoting meiotic chromosome synapsis.
Authors: Rockmill B , Sym M , Scherthan H , Roeder G S
Published: June 2007

Previous studies have shown that the RAD51 and DMC1 genes of Saccharomyces cerevisiae encode homologs of the Escherichia coli RecA strand exchange enzyme. Results presented here demonstrate that the dmc1 and rad51 mutants undergo nearly complete chromosome synapsis, but synaptonemal complex formation is delayed substantially compared with wild type. In the zip1 mutant, chromosomes are paired homologously, but not synapsed, and the protein backbones (axial elements) of each pair of chromosomes are connected intimately to each other at a few sites referred to herein as axial associations. dmc1 zip1 and rad51 zip1 double mutants assemble axial elements that are not obviously associated, demonstrating that the Dmc1 and Rad51 proteins are required to establish or stabilize axial associations. We propose that axial associations serve to promote meiotic chromosome synapsis and that the absence of these associations accounts for the delayed and inefficient synapsis observed in dmc1 and rad51 strains. During meiosis in haploid yeast, chromosome synapsis takes place between nonhomologous chromosome segments. In a zip1 haploid, axial associations are not apparent, suggesting that these associations depend on interactions between homologous sequences.
Constitutive NF-kappa B activation, enhanced granulopoiesis, and neonatal lethality in I kappa B alpha-deficient mice.
Authors: Beg A A , Sha W C , Bronson R T , Baltimore D
Published: June 2007

Transcription factors belonging to the NF-kappa B family are controlled by inhibitory I kappa B proteins, mainly I kappa B alpha and I kappa B beta. Apparently normal at birth, I kappa B alpha-/- mice exhibit severe runting, skin defects, and extensive granulopoiesis postnatally, typically dying by 8 days. Hematopoietic tissues from these mice display elevated levels of both nuclear NF-kappa B and mRNAs of some, but not all, genes thought to be regulated by NF-kappa B. NF-kappa B elevation results in these phenotypic abnormalities because mice lacking both I kappa B alpha and the p50 subunit of NF-kappa B show a dramatically delayed onset of abnormalities. In contrast to hematopoietic cells, I kappa B alpha-/- embryonic fibroblasts show minimal constitutive NF-kappa B, as well as normal signal-dependent NF-kappa B activation that is concomitant with I kappa B beta degradation. Our results indicate that I kappa b beta, but not I kappa B alpha, is required for the signal-dependent activation of NF-kappa B in fibroblasts. However, I kappa B alpha is required for the postinduction repression of NF-kappa B in fibroblasts. These results define distinct roles for the two forms of I kappa B and demonstrate the necessity for stringent control of NF-kappa B.
Human TAFII250 interacts with RAP74: implications for RNA polymerase II initiation.
Authors: Ruppert S , Tjian R
Published: June 2007

Accurate and regulated transcription by RNA polymerase II requires the assembly of an initiation complex involving multiple protein-DNA and protein-protein interactions. A key event is binding of TFIID, a complex consisting of TBP and associated factors (TAFs) to the template DNA. The TAF subunits of TFIID carry out diverse functions critical for transcription, including specific contact with enhancer proteins and binding to core promoter DNA. However, the role of TAFs in RNA polymerase II-mediated transcription initiation and cross talk with other basal factors remains poorly characterized. Here, we report the specific interaction of TAFII250 with RAP74, an essential subunit of the basal transcription factor IIF. Using various in vitro binding assays we have mapped recognition interfaces between TAFII250 and RAP74. In vivo complementation of a temperature-sensitive TAFII250 cell line reveals that the RAP74 interaction is critical for cell viability. Because TFIIF is thought to be responsible for binding and recruiting RNA polymerase II, the ability of TAFII250 to interact selectively with RAP74 is likely to contribute a critical contact for the assembly of an active transcription complex.
A protein-RNA interaction network facilitates the template-independent cooperative assembly on RNA polymerase of a stable antitermination complex containing the lambda N protein.
Authors: Mogridge J , Mah T F , Greenblatt J
Published: June 2007

The stable association of the N gene transcriptional antiterminator protein of bacteriophage lambda with transcribing RNA polymerase requires a nut site (boxA+boxB) in the nascent transcript and the Escherichia coli factors NusA, NusB, NusG, and ribosomal protein S10. We have used electrophoretic mobility shift assays to analyze the assembly of N protein, the E. coli factors, and RNA polymerase onto the nut site RNA in the absence of a DNA template. We show that N binds boxB RNA and that subsequent association of NusA with the N-nut site complex is facilitated by both boxA and boxB. In the presence of N, NusA, and RNA polymerase the nut site assembles ribonucleoprotein complexes containing NusB, NusG, and S10. The effects on assembly of mutations in boxA, boxB, NusA, and RNA polymerase define multiple weak protein-protein and protein-RNA interactions (e.g., NusB with NusG; NusA with boxB; NusA, NusB, and NusG with boxA) that contribute to the overall stability of the complex. Interaction of each component of the complex with two or more other components can explain the many observed cooperative binding associations in the DNA-independent assembly of a stable antitermination complex on RNA polymerase.
The tetratricopeptide repeats of Ssn6 interact with the homeo domain of alpha 2.
Authors: Smith R L , Redd M J , Johnson A D
Published: June 2007

The tetratricopeptide repeat (TPR) is a 34-amino-acid degenerate sequence motif that is found in a large variety of proteins, both prokaryotic and eukaryotic. TPRs are usually found in tandem arrays of up to 16 copies. In this paper we identify a direct interaction between the TPRs of Ssn6, a general transcriptional repressor, and alpha 2, a cell-type regulator in Saccharomyces cerevisiae. Five of the Ssn6 TPRs were tested individually, and all were found to interact specifically with alpha 2. These results suggest a model for TPR-protein interactions and for the role that a tandem array of TPRs may have in mediating transcriptional repression.
Genetic strategy for analyzing specificity of dimer formation: Escherichia coli cyclic AMP receptor protein mutant altered in its dimerization specificity.
Authors: Joung J K , Chung E H , King G , Yu C , Hirsh A S , Hochschild A
Published: June 2007

Many transcriptional regulators function in homo- or heterodimeric combinations. The same protein can carry out distinct regulatory functions depending on the partner with which it associates. Here, we describe a mutant of the Escherichia coli cAMP receptor protein (CRP) that has an altered dimerization specificity; that is, mutant/mutant homodimers form preferentially over wild-type/mutant heterodimers. CRP dimerization involves the formation of a parallel coiled-coil structure, and our CRP mutant bears an amino acid substitution affecting the first "d" position residue within the alpha-helix that mediates CRP dimerization. The genetic strategy we used to isolate this CRP altered dimerization specificity (ADS) mutant is generalizable and could be utilized to isolate ADS mutants of other dimeric transcriptional regulators.
Poly(A) site selection in the HIV-1 provirus: inhibition of promoter-proximal polyadenylation by the downstream major splice donor site.
Authors: Ashe M P , Griffin P , James W , Proudfoot N J
Published: June 2007

In common with all retroviruses, the human immunodeficiency virus type 1 (HIV-1) contains duplicated long terminal repeat (LTR) sequences flanking the proviral genome. These LTRs contain identical poly(A) signals, which are both transcribed into RNA. Therefore, to allow efficient viral expression, a mechanism must exist to either restrict promoter-proximal poly(A) site use or enhance the activity of the promoter-distal poly(A) site. We have examined the use of both poly(A) sites using proviral clones. Mutation of the previously defined upstream activatory sequences of the 3' LTR poly(A) site decreases the efficiency of polyadenylation when placed in competition with an efficient downstream processing signal. However, in the absence of competition, these mutations have no effect on HIV-1 polyadenylation. In addition, the 5' LTR poly(A) site is inactive, whereas a heterologous poly(A) site positioned in its place is utilized efficiently. Furthermore, transcription initiating from the 3' LTR promoter utilizes the 3' LTR poly(A) signal efficiently. Therefore, the main determinant of the differential poly(A) site use appears to be neither proximity to a promoter element in the 5' LTR nor the presence of upstream activating sequences at the 3' LTR. Instead, we show that the major splice donor site that is immediately downstream of the 5' LTR inhibits cleavage and polyadenylation at the promoter-proximal site. The fact that this poly(A) site is active in a proviral clone when the major splice donor site is mutated suggests that the selective use of poly(A) signals in HIV-1 is mediated by a direct inhibition of the HIV-1 poly(A) site by downstream splicing events or factors involved in splicing.
Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis.
Authors: Mishina Y , Suzuki A , Ueno N , Behringer R R
Published: June 2007

Bone morphogenetic proteins (BMPs) are secreted proteins that interact with cell-surface receptors and are believed to play a variety of important roles during vertebrate embryogenesis. Bmpr, also known as ALK-3 and Brk-1, encodes a type I transforming growth factor-beta (TGF-beta) family receptor for BMP-2 and BMP-4. Bmpr is expressed ubiquitously during early mouse embryogenesis and in most adult mouse tissues. To study the function of Bmpr during mammalian development, we generated Bmpr-mutant mice. After embryonic day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects were first detected at E7.0 and were smaller than normal. Morphological and molecular examination demonstrated that no mesoderm had formed in the mutant embryos. The growth characteristics of homozygous mutant blastocysts cultured in vitro were indistinguishable from those of controls; however, embryonic ectoderm (epiblast) cell proliferation was reduced in all homozygous mutants at E6.5 before morphological abnormalities had become prominent. Teratomas arising from E7.0 mutant embryos contained derivatives from all three germ layers but were smaller and gave rise to fewer mesodermal cell types, such as muscle and cartilage, than controls. These results suggest that signaling through this type I BMP-2/4 receptor is not necessary for preimplantation or for initial postimplantation development but may be essential for the inductive events that lead to the formation of mesoderm during gastrulation and later for the differentiation of a subset of mesodermal cell types.