bluuebunny/crossref_metadata_2025 huggingface dataset:
The WT1 gene product stabilizes p53 and inhibits p53-mediated apoptosis.
Authors: Maheswaran S , Englert C , Bennett P , Heinrich G , Haber D A
Published: June 2007

The Wilms' tumor-suppressor gene product WT1 coimmunoprecipitates with p53 from baby rat kidney (BRK) cells and Wilms' tumor specimens, and expression of WT1 in BRK cells is associated with increased levels of endogenous wild-type p53 protein. To study the effect of WT1 on p53 function, we cotransfected expression constructs into Saos-2 cells, an osteosarcoma cell line without endogenous expression of either gene. Expression of WT1 resulted in increased steady-state levels of p53, attributable to a prolongation in protein half-life, and associated with protection against papillomavirus E6-mediated degradation of p53. This effect mapped to zinc fingers 1 and 2 of WT1 and was not observed with the closely related EGR1 protein. The stabilized p53 demonstrated enhanced binding to its target DNA sequence and increased trans-activation of a promoter containing this RGC site, but reduced transcriptional repression of a TATA-containing promoter lacking this site. Expression of WT1 inhibited p53-mediated apoptosis triggered by UV irradiation or by expression of temperature-sensitive p53 in the wild-type conformation, but did not affect p53-mediated cell cycle arrest. We conclude that WT1 protein can stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis. This effect may contribute to the elevated levels of wild-type p53 protein that are observed in Wilms' tumors.
Induction of apoptosis in HeLa cells by trans-activation-deficient p53.
Authors: Haupt Y , Rowan S , Shaulian E , Vousden K H , Oren M
Published: June 2007

The p53 tumor suppressor protein is a transcriptional activator, which can mediate apoptotic cell death in a variety of cell types. To determine whether sequence-specific trans-activation is a prerequisite for the induction of apoptosis by p53, the apoptotic effects of various p53 deletion mutants were monitored in an assay based on the transient transfection of HeLa cells. A truncated protein (p53dl214), containing only the first 214 amino-terminal residues of murine p53, induced extensive apoptosis, albeit at a slower rate than trans-activation-competent wild-type p53. p53dl214 also suppressed the transformation of rat fibroblasts by several oncogene combinations and particularly by myc plus ras and HPV E7 plus ras. p53dl214 lacks a major portion of the DNA-binding domain and cannot activate p53-responsive promoters. Moreover, a human p53 protein carrying mutations in residues 22 and 23 also triggered HeLa cell apoptosis, despite failing to induce significant activation of relevant p53 target promoters. These data suggest the existence of two p53-dependent apoptotic pathways--one requiring activation of specific target genes, and the other independent of sequence-specific trans-activation. The latter pathway may actually be totally uncoupled from the binding of p53 to its consensus DNA sites. The relative contribution of trans-activation-independent apoptosis to tumor suppression by p53 may be dictated by the specific genetic lesions present in the particular tumor.
Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus.
Authors: Fiering S , Epner E , Robinson K , Zhuang Y , Telling A , Hu M , Martin D I , Enver T , Ley T J , Groudine M
Published: June 2007

The beta-globin locus control region (LCR) is a complex regulatory element that is essential for the appropriate red cell-specific expression of all cis-linked beta-globin genes. Of the five hypersensitive sites that define the LCR, only 5'HS2 has been shown to augment gene expression in vitro in both transient and stable assays, as well as in transgenic mice. Thus, 5'HS2 has been assumed to be an important element for the function of the LCR in vivo. We have utilized homologous recombination in murine embryonic stem (ES) cells and phenotypic analysis in derived mice to investigate the function of 5'HS2 in its normal chromosomal position in the murine beta-globin locus. Replacement of 5'HS2 with a selectable marker gene (delta HS2 + neo) causes a 2-5-fold reduction in expression of all of the genes in the locus, and a more pronounced effect (10-12-fold) on the most 5' embryonic globin gene, Ey, when expression of this gene is first detectable during embryogenesis. The mutation produces no alterations in the developmental timing of expression of the globin genes. When homozygous, the deletion/replacement mutation is lethal in utero, with the embryos dying during the stage of yolk sac and early fetal liver erythropoiesis. To distinguish phenotypic effects resulting from the deletion of 5'HS2 from those attributable to insertion of the selectable marker, the selectable marker was removed by expressing the FLP site-specific recombinase in ES cells harboring the homologous recombination event. Mice derived from these ES cells (delta HS2 delta neo) demonstrated nearly full expression of all the beta-like globin genes on the mutated chromosome. These results indicate that although 5'HS2 demonstrates significant regulatory activities in a variety of assays, deletion of this element from the endogenous beta-globin locus has no significant effect on the timing or extent of expression of the locus. In addition, this result emphasizes that when using homologous recombination to analyze complex regulatory elements in vivo, the inserted selectable marker must be removed to avoid influencing the phenotype of the mutation.
Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis.
Authors: Wu Z , Xie Y , Bucher N L , Farmer S R
Published: June 2007

Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.
scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development.
Authors: Roark M , Sturtevant M A , Emery J , Vaessin H , Grell E , Bier E
Published: June 2007

The Drosophila scratch (scrt) gene is expressed in most or all neuronal precursor cells and encodes a predicted zinc finger transcription factor closely related to the product of the mesoderm determination gene snail (sna). Adult flies homozygous for scrt null alleles have a reduced number of photoreceptors in the eye, and embryos lacking the function of both scrt and the pan-neural gene deadpan (dpn), which encodes a basic helix-loop-helix (bHLH) protein, exhibit a significant loss of neurons. Conversely, ectopic expression of a scrt transgene during embryonic and adult development leads to the production of supernumerary neurons. Consistent with scrt functioning as a transcription factor, various genes are more broadly expressed than normal in scrt null mutants. Reciprocally, these same genes are expressed at reduced levels in response to ectopic scrt expression. We propose that scrt promotes neuronal cell fates by suppressing expression of genes promoting non-neuronal cell fates. We discuss the similarities between the roles of the ancestrally related scrt, sna, and escargot (esc) genes in regulating cell fate choices.
Formation and resolution of double-strand break intermediates in V(D)J rearrangement.
Authors: Ramsden D A , Gellert M
Published: June 2007

A recently described pre-B cell line can be induced at high temperature to actively rearrange its immunoglobulin light-chain loci. We used this cell line to determine the fate of double-strand breaks generated by V(D)J rearrangement. After induction, 30%-40% of K loci had broken JK1 signal ends. JK1-coding ends were detectable, but 10- to 100-fold less frequent. Both covalently closed (hairpin) and open, blunt, processed coding ends were observed. Coding junctions involving JK1 accumulated with similar kinetics as JK1 signal ends, arguing that coding ends can be resolved quickly and efficiently to coding junctions, whereas signal ends remain mostly unjoined. Signal ends are then joined rapidly when cells are returned to the low temperature. These results support the model that broken signal ends and hairpin coding ends are authentic intermediates in V(D)J recombination. It appears that hairpin coding ends are rapidly opened, processed, and resolved to coding junctions, whereas joining of signal ends is clearly uncoupled from the joining of coding ends and can be much slower. Efficient formation of signal junctions may require cell cycle progression, or down-regulation of the recombination machinery.
Multiple functions for the poly(A)-binding protein in mRNA decapping and deadenylation in yeast.
Authors: Caponigro G , Parker R
Published: June 2007

The first step in the decay of many eukaryotic mRNAs is shortening of the poly(A) tail. In yeast, deadenylation leads to mRNA decapping and subsequent 5' --> 3' exonucleolytic degradation of the transcript body. We have determined that the major poly(A)-binding protein Pab1p plays at least two critical roles in this pathway. First, mRNAs in pab1 delta strains were decapped prior to deadenylation. This observation defines a new function for Pab1p as an inhibitor of mRNA decapping. Moreover, mutations that inhibit mRNA turnover suppress the inviability of a pab1 delta mutation, suggesting that premature mRNA decapping in pab1 delta strains contributes to cell death. Second, we find that Pab1p is not required for deadenylation, although in its absence poly(A) tail shortening rates are significantly reduced. In addition, in the absence of Pab1p, newly synthesized mRNAs had poly(A) tails longer than those in wild-type strains and showed an unexpected temporal delay prior to the initiation of deadenylation and degradation. These results define new and critical functions for Pab1p in the regulation of mRNA decapping and deadenylation, two important control points in the specification of mRNA half-lives. Moreover, these results suggest that Pab1p functions in additional phases of mRNA metabolism such as mRNP maturation.
U14 base-pairs with 18S rRNA: a novel snoRNA interaction required for rRNA processing.
Authors: Liang W Q , Fournier M J
Published: June 2007

U14 is a conserved small nucleolar RNA (snoRNA) required for processing of yeast 18S rRNA. The presence of two long sequences (13 and 14 nucleotides) with strong complementarity to 18S rRNA suggests that U14 base-pairs with pre-rRNA. Evidence of direct binding was developed by showing that mutations in these U14 elements mimic U14 depletion and that function can be rescued by a compensatory sequence change in 18S RNA. The U14 elements are functionally interdependent, indicating that both participate in binding. Folding models predict that binding might occur through both rRNA elements simultaneously. Potential roles of U14 in rRNA folding, maturation, and ribosome assembly are discussed. U14 is one of several snoRNAs with long complementarities to rRNA and the first snoRNA in this class shown to interact directly with rRNA.
Integrated control of seed maturation and germination programs by activator and repressor functions of Viviparous-1 of maize.
Authors: Hoecker U , Vasil I K , McCarty D R
Published: June 2007

The Viviparous-1 (VP1) transcriptional activator of maize is required for abscisic acid induction of maturation-specific genes late in seed development leading to acquisition of desiccation tolerance and arrest in embryo growth. Here, we show that VP1 also inhibits induction of the germination-specific alpha-amylase genes in aleurone cells of the developing seed and thereby appears to be involved in preventing precocious hydrolyzation of storage compounds accumulating in the endosperm. In developing seeds of the somatically instable vp1-m2 mutant, hydrolase activity was derepressed specifically in endosperm sectors underlying vp1 mutant aleurone. A barley alpha-amylase promoter-GUS reporter construct (Amy-GUS) was induced in developing vp1 mutant aleurone cells but not in wild-type aleurone cells. Moreover, transient expression of recombinant VP1 and vp1 mutant aleurone cells strongly inhibited expression of Amy-GUS and thus effectively complemented this aspect of the mutant phenotype. VP1 specifically repressed induction of Amy-GUS by the hormone gibberellic acid in aleurone of germinating barley seeds. Deletion of the acidic transcriptional activation domain of VP1 did not affect the inhibitory activity, indicating that VP1 has a discrete repressor function. Hence, physically combining activator and repressor functions in one protein may provide a mechanism to integrate the control of two normally consecutive developmental programs, seed maturation and seed germination.
Cappuccino, a Drosophila maternal effect gene required for polarity of the egg and embryo, is related to the vertebrate limb deformity locus.
Authors: Emmons S , Phan H , Calley J , Chen W , James B , Manseau L
Published: June 2007

We report the molecular isolation of cappuccino (capu), a gene required for localization of molecular determinants within the developing Drosophila oocyte. The carboxy-terminal half of the capu protein is closely related to that of the vertebrate limb deformity locus, which is known to function in polarity determination in the developing vertebrate limb. In addition, capu shares both a proline-rich region and a 70-amino-acid domain with a number of other genes, two of which also function in pattern formation, the Saccharomyes cerevisiae BNI1 gene and the Aspergillus FigA gene. We also show that capu mutant oocytes have abnormal microtubule distributions and premature microtubule-based cytoplasmic streaming within the oocyte, but that neither the speed nor the timing of the cytoplasmic streaming correlates with the strength of the mutant allele. This suggests that the premature cytoplasmic streaming in capu mutant oocytes does not suffice to explain the patterning defects. By inducing cytoplasmic streaming in wild-type oocytes during mid-oogenesis, we show that premature cytoplasmic streaming can displace staufen protein from the posterior pole, but not gurken mRNA from around the oocyte nucleus.
Null mutation of Dlx-2 results in abnormal morphogenesis of proximal first and second branchial arch derivatives and abnormal differentiation in the forebrain.
Authors: Qiu M , Bulfone A , Martinez S , Meneses J J , Shimamura K , Pedersen R A , Rubenstein J L
Published: June 2007

Genetic analysis of the development and evolution of the vertebrate head is at a primitive stage. Many homeo box genes, including the Distal-less family, are potential regulators of head development. To determine the function of Dlx-2, we generated a null mutation in mice using gene targeting. In homozygous mutants, differentiation within the forebrain is abnormal and the fate of a subset of cranial neural crest cells is respecified. The latter causes abnormal morphogenesis of the skeletal elements derived from the proximal parts of the first and second branchial arches. We hypothesize that the affected skull bones from the first arch have undergone a transformation into structures similar to those found in reptiles. These results show that Dlx-2 controls development of the branchial arches and the forebrain and suggests its role in craniofacial evolution.
Abi-2, a novel SH3-containing protein interacts with the c-Abl tyrosine kinase and modulates c-Abl transforming activity.
Authors: Dai Z , Pendergast A M
Published: June 2007

A protein has been identified that interacts specifically with both the Src homologous 3 (SH3) domain and carboxy-terminal sequences of the c-Abl tyrosine kinase. The cDNA encoding the Abl interactor protein (Abi-2), was isolated from a human lymphocyte library using the yeast two-hybrid system with the Abl SH3 domain as bait. Abi-2 binds to c-Abl in vitro and in vivo. Abi-2 is a novel protein that contains an SH3 domain and proline-rich sequences critical for binding to c-Abl. A basic region in the amino terminus of Abi-2 is homologous to the DNA-binding sequence of homeo-domain proteins. We show that Abi-2 is a substrate for the c-Abl tyrosine kinase. Expression of an Abi-2 mutant protein that lacks sequences required for binding to the Abl SH3 domain but retains binding to the Abl carboxyl terminus activates the transforming capacity of c-Abl. The properties of Abi-2 are consistent with a dual role as regulator and potential effector of the c-Abl protein and suggest that Abi-2 may function as a tumor suppressor in mammalian cells.
Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.
Authors: Zhou X Y , Morreau H , Rottier R , Davis D , Bonten E , Gillemans N , Wenger D , Grosveld F G , Doherty P , Suzuki K
Published: June 2007

The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of beta-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein.
Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3.
Authors: Yuan H , Corbi N , Basilico C , Dailey L
Published: June 2007

Fibroblast growth factor 4 (FGF-4) has been shown to be a signaling molecule whose expression is essential for postimplantation mouse development and, at later embryonic stages, for limb patterning and growth. The FGF-4 gene is expressed in the blastocyst inner cell mass and later in distinct embryonic tissues but is transcriptionally silent in the adult. In tissue culture FGF-4 expression is restricted to undifferentiated embryonic stem (ES) cells and embryonal carcinoma (EC) cell lines. Previously, we determined that EC cell-specific transcriptional activation of the FGF-4 gene depends on a synergistic interaction between octamer-binding proteins and an EC-specific factor, Fx, that bind adjacent sites on the FGF-4 enhancer. Through the cloning and characterization of an F9 cell cDNA we now show that the latter activity is Sox2, a member of the Sry-related Sox factors family. Sox2 can form a ternary complex with either the ubiquitous Oct-1 or the embryonic-specific Oct-3 protein on FGF-4 enhancer DNA sequences. However, only the Sox2/Oct-3 complex is able to promote transcriptional activation. These findings identify FGF-4 as the first known embryonic target gene for Oct-3 and for any of the Sox factors, and offer insights into the mechanisms of selective gene activation by Sox and octamer-binding proteins during embryogenesis.
Mouse Otx2 functions in the formation and patterning of rostral head.
Authors: Matsuo I , Kuratani S , Kimura C , Takeda N , Aizawa S
Published: June 2007

The anterior part of the vertebrate head expresses a group of homeo box genes in segmentally restricted patterns during embryogenesis. Among these, Otx2 expression covers the entire fore- and midbrains and takes place earliest. To examine its role in development of the rostral head, a mutation was introduced into this locus. The homozygous mutants did not develop structures anterior to rhombomere 3, indicating an essential role of Otx2 in the formation of the rostral head. In contrast, heterozygous mutants displayed craniofacial malformations designated as otocephaly; affected structures appeared to correspond to the most posterior and most anterior domains of Otx expression where Otx1 is not expressed. The homo- and heterozygous mutant phenotypes suggest Otx2 functions as a gap-like gene in the rostral head where Hox code is not present. The evolutionary significance of Otx2 mutant phenotypes was discussed for the innovation of the neurocranium and the jaw.
A role for nucleosome assembly in both silencing and activation of the Xenopus TR beta A gene by the thyroid hormone receptor.
Authors: Wong J , Shi Y B , Wolffe A P
Published: June 2007

We have assembled the thyroid hormone-inducible promoter of the Xenopus thyroid hormone receptor (TR)beta A gene into chromatin using replication-coupled and -independent assembly pathways in vivo. We establish that heterodimers of TR and 9-cis retinoic acid receptors (RXR) can bind to their recognition sites within chromatin both in vivo and in vitro and alternately repress or activate transcription dependent on the absence or presence of thyroid hormone. Maximal transcriptional repression requires the presence of unliganded TR/RXR heterodimers during replication-coupled chromatin assembly. We demonstrate an increase in transcription directed by the TR beta A promoter of over two orders of magnitude in vivo, following the addition of thyroid hormone. This increase in transcription involves the relief of the repressed state that is established by the unliganded TR/RXR heterodimer during replication-coupled chromatin assembly. The association of thyroid hormone with the chromatin-bound TR/RXR heterodimer leads to the disruption of local chromatin structure in a transcription-independent process. Thus, chromatin structure has multiple roles in the regulation of TR beta A gene expression in vivo: The TR/RXR heterodimer recognizes the response element within chromatin, TR/RXR makes use of the chromatin assembly process to silence transcription more efficiently, and TR/RXR directs the disruption of local chromatin structure in response to thyroid hormone.
Transgenic mice with targeted inactivation of the Col2 alpha 1 gene for collagen II develop a skeleton with membranous and periosteal bone but no endochondral bone.
Authors: Li S W , Prockop D J , Helminen H , Fässler R , Lapveteläinen T , Kiraly K , Peltarri A , Arokoski J , Lui H , Arita M
Published: June 2007

Homologous recombination in embryonic stem cells was used to prepare transgenic mice with an inactivated Col2a1 gene for collagen II, the major protein component of the extracellular matrix of cartilage. Heterozygous mice had a minimal phenotype. Homozygous mice developed into fetuses that were delivered vaginally but died either just before or shortly after birth. The cartilage in the mice consisted of highly disorganized chondrocytes with a complete lack of extracellular fibrils discernible by electron microscopy. There was no endochondrial bone or epiphyseal growth plate in long bones. However, many skeletal structures such as the cranium and ribs were normally developed and mineralized. The results demonstrate that a well-organized cartilage matrix is required as a primary tissue for development of some components of the vertebrate skeleton, but it is not essential for others.
E2F-1 accumulation bypasses a G1 arrest resulting from the inhibition of G1 cyclin-dependent kinase activity.
Authors: DeGregori J , Leone G , Ohtani K , Miron A , Nevins J R
Published: June 2007

Numerous experiments have defined a critical role for the G1 cyclins and associated kinases in allowing a normal progression of cells from a quiescent state, through G1, and into S phase. We now demonstrate that G1 cyclin-dependent kinase activity is critical for the accumulation of E2F activity late in G1. Moreover, E2F-1 overexpression can overcome a G1 arrest caused by the inhibition of G1 cyclin-dependent kinase activity, consistent with E2F activation being an important consequence of the action of G1 cyclins. E2F-1 also overcomes a G1 block caused by gamma irradiation and leads to an apparent complete replication of the cellular genome and entry into mitosis. This E2F-1-mediated induction of S phase and mitosis is not accompanied by the rise in either cyclin D-associated kinase activity or cdk2 activity that is normally observed during the G1 phase of the cell cycle. We conclude that one key function for G1 cyclin-dependent kinase activity is the activation of E2F-1, that the accumulation of E2F activity may be sufficient to allow initiation and completion of S phase, but that additional events, including G1 cyclin kinase activity, are likely necessary for a normal proliferative event.
Combinatorial signaling by Sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite.
Authors: Münsterberg A E , Kitajewski J , Bumcrot D A , McMahon A P , Lassar A B
Published: June 2007

We have demonstrated previously that a combination of signals from the neural tube and the floor plate/notochord complex synergistically induce the expression of myogenic bHLH genes and myogenic differentiation markers in unspecified somites. In this study we demonstrate that Sonic hedgehog (Shh), which is expressed in the floor plate/notochord, and a subset of Wnt family members (Wnt-1, Wnt-3, and Wnt-4), which are expressed in dorsal regions of the neural tube, mimic the muscle inducing activity of these tissues. In combination, Shh and either Wnt-1 or Wnt-3 are sufficient to induce myogenesis in somitic tissue in vitro. Therefore, we propose that myotome formation in vivo may be directed by the combinatorial activity of Shh secreted by ventral midline tissues (floor plate and notochord) and Wnt ligands secreted by the dorsal neural tube.
The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections.
Authors: Hu S , Fambrough D , Atashi J R , Goodman C S , Crews S T
Published: June 2007

Motor axons make synaptic connections with specific muscles, and this specificity unfolds during development as motoneuron growth cones make specific pathway choices and ultimately recognize and synapse on their specific muscle targets. The Drosophila clueless mutation was identified previously in a genetic screen for mutations that disrupt motoneuron guidance and connectivity. We show here that clueless is allelic to abrupt. The abrupt gene is required for the embryonic formation of specific synaptic connections between a subset of motoneurons and a subset of muscles. Mutations in abrupt also reveal its role in establishing and maintaining muscle attachments, adult sensory cell formation, and morphogenesis of adult appendages. The abrupt gene encodes a zinc finger protein with a conserved BTB domain. Abrupt is expressed in muscle nuclei but not motoneurons, suggesting that abrupt controls the muscle expression of molecules required for correct motoneuron targeting, as well as molecules required for correct muscle attachments.
Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae.
Authors: Stevenson B J , Ferguson B , De Virgilio C , Bi E , Pringle J R , Ammerer G , Sprague G F
Published: June 2007

We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype. One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit. Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p.
A tactile sensory system of Myxococcus xanthus involves an extracellular NAD(P)(+)-containing protein.
Authors: Lee B U , Lee K , Mendez J , Shimkets L J
Published: June 2007

CsgA is a cell surface protein that plays an essential role in tactile responses during Myxococcus xanthus fruiting body formation by producing the morphogenic C-signal. The primary amino acid sequence of CsgA exhibits homology with members of the short-chain alcohol dehydrogenase (SCAD) family and several lines of evidence suggest that NAD(P)+ binding is essential for biological activity. First, the predicted CsgA secondary structure based on the 3 alpha/20 beta-hydroxysteroid dehydrogenase crystal structure suggests that the amino-terminal portion of the protein contains an NAD(P)+ binding pocket. Second, strains with csgA alleles encoding amino acid substitutions T6A and R10A in the NAD(P)+ binding pocket failed to develop. Third, exogenous MalE-CsgA rescues csgA development, whereas MalE-CsgA with the amino acid substitution CsgA T6A does not. Finally, csgA spore yield increased approximately 20% when containing 100 nM of MalE-CsgA was supplemented with 10 microM of NAD+ or NADP+. Conversely, 10 microM of NADH or NADPH delayed development for approximately 24 hr and depressed spore levels approximately 10%. Together, these results argue that NAD(P)+ binding is critical for C-signaling. S135 and K155 are conserved amino acids in the catalytic domain of SCAD members. Strains with csgA alleles encoding the amino acid substitutions S135T or K155R failed to develop. Furthermore, a MalE-CsgA protein containing CsgA S135T was not able to restore development to csgA cells. In conclusion, amino acids conserved in the coenzyme binding pocket and catalytic site are essential for C-signaling.
Molecular mechanisms of Spemann's organizer formation: conserved growth factor synergy between Xenopus and mouse.
Authors: Watabe T , Kim S , Candia A , Rothbächer U , Hashimoto C , Inoue K , Cho K W
Published: June 2007

Mesoderm induction assays in Xenopus have implicated growth factors such as activin, Vg1, Xwnt-8, and noggin as important in directing the formation of dorsal mesoderm (Spemann's organizer). Because these growth factors are structurally very different, they presumably act through distinct cell surface receptors that initiate different intracellular signaling cascades. A consequence of all of these signaling pathways, however, seems to be the induction of goosecoid (gsc) gene expression. To understand how integration of these different signaling pathways results in formation of Spemann's organizer, we sought to identify growth factor-responsive elements within the gsc promoter. Through microinjection of reporter genes we have identified two cis-acting elements, a distal element (DE) and a proximal element (PE), that are required for activin/BVg1 and Wnt induction, respectively. We have shown that the DE mediates activin induction in the absence of protein synthesis and therefore constitutes the first activin response element identified to interpret transforming growth factor-beta (TGF-beta) superfamily member signaling directly. Using a reporter gene construct containing a multimerized DE, we find that an activin/BVg1-type signaling cascade is active throughout the vegetal hemisphere and marginal zone but not in the animal hemisphere. We demonstrate further that both the distal and proximal elements are essential for high-level transcription of the gsc gene, specifically in dorsal mesoderm, strongly suggesting that establishment of Spemann's organizer requires synergistic input from activin/BVg1-like and Wnt signaling pathways. Finally, mechanisms of establishing the organizer are likely to be conserved throughout vertebrate evolution.
A proline-rich TGF-beta-responsive transcriptional activator interacts with histone H3.
Authors: Alevizopoulos A , Dusserre Y , Tsai-Pflugfelder M , von der Weid T , Wahli W , Mermod N
Published: June 2007

The molecular mechanisms involved in the regulation of gene expression by transforming growth factor-beta (TGF-beta) have been analyzed. We show that TGF-beta specifically induces the activity of the proline-rich trans-activation domain of CTF-1, a member of the CTF/NF-I family of transcription factors. A TGF-beta-responsive domain (TRD) in the proline-rich transcriptional activation sequence of CTF-1 was shown to mediate TGF-beta induction in NIH-3T3 cells. Mutagenesis studies indicated that this domain is not the primary target of regulatory phosphorylations, suggesting that the growth factor may regulate a CTF-1-interacting protein. A two-hybrid screening assay identified a nucleosome component, histone H3, as a specific CTF-1-interacting protein in yeast. Furthermore, the CTF-1 trans-activation domain was shown to interact with histone H3 in both transiently and stably transfected mammalian cells. This interaction requires the TRD, and it appears to be upregulated by TGF-beta in vivo. Moreover, point mutations in the TRD that inhibit TGF-beta induction also reduce interaction with histone H3. In vitro, the trans-activation domain of CTF-1 specifically contacts histone H3 and oligomers of histones H3 and H4, and full-length CTF-1 was shown to alter the interaction of reconstituted nucleosomal cores with DNA. Thus, the growth factor-regulated trans-activation domain of CTF-1 can interact with chromatin components through histone H3. These findings suggest that such interactions may regulate chromatin dynamics in response to growth factor signaling.
Soma-specific expression and cloning of PSI, a negative regulator of P element pre-mRNA splicing.
Authors: Siebel C W , Admon A , Rio D C
Published: June 2007

PSI is an RNA-binding protein involved in repressing splicing of the P element third intron in Drosophila somatic cell extracts. PSI produced in bacteria restores splicing inhibition to an extract relieved of inhibitory activity, indicating that PSI plays a direct role in somatic inhibition. Sequence analysis of cDNAs encoding PSI reveals three KH RNA-binding domains, a conserved motif also found in the yeast splicing regulator MER1. Notably, PSI is expressed highly in somatic embryonic nuclei but is undetectable in germ-line cells. In contrast, hrp48, another protein implicated in somatic inhibition, is found in the nucleus and cytoplasm of both tissues. The splicing inhibitory properties and soma-specific expression of PSI may be sufficient to explain the germ-line-specific transposition of P elements.
Targeting expression of a dominant-negative retinoic acid receptor mutant in the epidermis of transgenic mice results in loss of barrier function.
Authors: Imakado S , Bickenbach J R , Bundman D S , Rothnagel J A , Attar P S , Wang X J , Walczak V R , Wisniewski S , Pote J , Gordon J S
Published: June 2007

To study the effects of retinoic acid on the skin in vivo, we have subverted the activity of endogenous receptors by targeting expression of a dominant negative mutant of retinoic acid receptor alpha (RAR alpha) to the epidermis of transgenic mice. At birth, mice expressing the mutant RAR alpha transgene exhibited a marked phenotype of a red, shiny skin that was somewhat sticky to touch. Severely affected neonates died within 24 hr. Histological changes in the epidermis were subtle with the phenotypic stratum corneum appearing slightly thinner and more loosely packed than in controls. Electron microscopic studies revealed that lipid multilamellar structures were not present between cells in the stratum corneum of phenotypic mice. When assayed for transepidermal water loss, phenotypic skin lost water at a rate three times faster than controls, suggesting that neonatal lethality resulted from loss of epidermal barrier function. The absence of a functional lipid barrier in transgenic mice first became evident at E17 when lipids were extruded initially into the intercellular space. We have identified a potential pathway linking inhibition of retinoid signaling with disruption of the lipid barrier that involves peroxisome proliferator-activated receptors. This study documents the role of the retinoid signaling pathway in formation and maintenance of a functional epidermis and provides the first evidence that this is mediated in part by modulation of lipid metabolism.
Action of a RAP1 carboxy-terminal silencing domain reveals an underlying competition between HMR and telomeres in yeast.
Authors: Buck S W , Shore D
Published: June 2007

RAP1 is a sequence-specific DNA-binding protein in yeast that can either repress or activate transcription. Previous studies have demonstrated a direct role for RAP1 in silencing at HM mating-type loci and telomeres. Here, we show that a small carboxy-terminal domain of RAP1 is sufficient to establish repression when fused to the GAL4 DNA-binding domain (GBD) and targeted to mutated HMR silencers containing GAL4 DNA-binding sites. Silencing by GBD/RAP1 hybrids, like normal silencing at HMR, requires the trans-acting factors SIR2, SIR3, and SIR4. However, GBD/RAP1-mediated silencing is independent of SIR1, whose product is normally required for the establishment of repression at HMR. Targeted silencing also displays an unusual response to silencing-defective rap1s mutations. The incorporation of a rap1s missense mutation into GBD/RAP1 hybrids can improve targeted silencing, yet wild-type GBD/RAP1 hybrids fail to establish repression in strains in which the endogenous RAP1 locus carries a rap1s mutation. In addition, we find that telomeric silencing is increased in rap1s strains. We propose that the rap1s mutation creates an HMR-specific silencing defect by shifting a balance between silencing at HMR and telomeres in favor of telomeric silencing. This balance is regulated by telomere length and by interactions between the RAP1 carboxyl terminus and both RIF1 and SIR4 proteins. In support of this model, we show that abnormally long telomeres antagonize silencing at HMR and a rap1s hybrid protein displays a strengthened interaction with SIR4 in a two-hybrid assay.
Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis.
Authors: Solomon J M , Magnuson R , Srivastava A , Grossman A D
Published: June 2007

Development of genetic competence in Bacillus subtilis is regulated by extracellular signaling molecules, including the ComX pheromone, a modified 9- or 10-amino-acid peptide. Here, we present characterization of a second extracellular competence stimulating factor (CSF). CSF appears to be, at least in part, a small peptide of between 520 and 720 daltons. Production of CSF requires several genes that are needed both for initiation of sporulation and development of competence (spo0H, spo0A, spo0B, and spo0F). Although both peptide factors regulate competence, two different sensing pathways mediate the response to the ComX pheromone and CSF. Analysis of double mutants indicated that ComX pheromone is on the same genetic pathway as the membrane-bound histidine protein kinase encoded by comP and that CSF is on the same genetic pathway as the oligopeptide permease encoded by spo0K. Furthermore, the cellular response to partly purified ComX pheromone requires the ComP histidine protein kinase, whereas the response to partly purified CSF requires the Spo0K oligopeptide permease. These two sensing pathways converge to activate competence genes. Both factors and their convergent sensing pathways are required for normal development of competence and might function to integrate different physiological signals.
canoe encodes a novel protein containing a GLGF/DHR motif and functions with Notch and scabrous in common developmental pathways in Drosophila.
Authors: Miyamoto H , Nihonmatsu I , Kondo S , Ueda R , Togashi S , Hirata K , Ikegami Y , Yamamoto D
Published: June 2007

The canoemisty1 (cnomis1) mutation was isolated by virtue of its severe rough eye phenotype from approximately 500 fly lines, each harboring a single autosomal insertion of a P element (Bm delta w). Excision of the P element generated a lethal, null allele, cnomis10, together with many revertants with normal eye morphology. Ommatidia homozygous for cnomis10, produced in an otherwise wild-type eye by somatic recombination, typically contain a reduced number of outer photoreceptors. Some cnomis1 homozygous adults bear extra macrochaetes on the head, notum, humerus and/or scutellum. cnomis1 hemizygotes often show conspicuous wing phenotypes such as a notched blade and the loss of a cross vein. The sequence of cno cDNA clones isolated from an embryonic cDNA library revealed a long open reading frame that potentially encodes a 1893-amino-acid protein with the GLGF/DHR motif, a conserved sequence in Discs large, Dishevelled, and some other proteins associated with cellular junctions. Flies doubly mutant for cnomis1 and scabrous1 (sca1) and those for cnomis1 and the split (spl) allele of Notch (N) always have rumpled wings curved downward. The spl; cnomis1 double mutant flies also exhibit a "giant socket" phenotype. These phenotypes are rarely observed flies singly mutant for either cnomis1, sca1 or spl. The wing vein gaps caused by Abruptex1, a N allele producing an activated form of N protein, are dominantly suppressed by cnomis1. Heterozygosity for shaggy and myospheroid promotes formation of extra wing veins in cnomis1 homozygotes. The genetic interactions suggest that cno participates with members of the N pathway in regulating adhesive cell-cell interactions for the determination of cell fate.
Suppression of a cold-sensitive mutation in 16S rRNA by overexpression of a novel ribosome-binding factor, RbfA.
Authors: Dammel C S , Noller H F
Published: June 2007

A novel 15-kDa protein, RbfA, has been identified by virtue of its ability to act as a high copy suppressor of a previously characterized dominant cold-sensitive mutation (C23U) in 16S rRNA. RbfA is found associated with free 30S ribosomal subunits, but not with 70S ribosomes or polysomes, and is essential for maximal cell growth, particularly at low temperatures. Cells lacking RbfA in a wild-type rRNA background exhibit a cold-sensitive phenotype that is strikingly similar to that of the cold-sensitive C23U rRNA mutant. The observed patterns of allele specificity of suppression and synthetic lethality in cells containing an RbfA knockout in combination with various 16S rRNA mutations suggests that RbfA interacts with the 5'-terminal helix region of 16S rRNA, possibly during a late step of 30S maturation.
Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution.
Authors: Lee M H , Reynisdóttir I , Massagué J
Published: June 2007

Progression through the cell cycle is catalyzed by cyclin-dependent kinases (CDKs) and is negatively controlled by CDK inhibitors (CDIs). We have isolated a new member of the p21CIP1/p27KIP1 CDI family and named it p57KIP2 to denote its apparent molecular mass and higher similarity to p27KIP1. Three distinct p57 cDNAs were cloned that differ at the start of their open reading frames and correspond to messages generated by the use of distinct splice acceptor sites. p57 is distinguished from p21 and p27 by its unique domain structure. Four distinct domains follow the heterogeneous amino-terminal region and include, in order, a p21/p27-related CDK inhibitory domain, a proline-rich (28% proline) domain, an acidic (36% glutamic or aspartic acid) domain, and a carboxy-terminal nuclear targeting domain that contains a putative CDK phosphorylation site and has sequence similarity to p27 but not to p21. Most of the acidic domain consists of a novel, tandemly repeated 4-amino acid motif. p57 is a potent inhibitor of G1- and S-phase CDKs (cyclin E-cdk2, cyclin D2-cdk4, and cyclin A-cdk2) and, to lesser extent, of the mitotic cyclin B-Cdc2. In mammalian cells, p57 localizes to the nucleus, associates with G1 CDK components, and its overexpression causes a complete cell cycle arrest in G1 phase. In contrast to the widespread expression of p21 and p27 in human tissues, p57 is expressed in a tissue-specific manner, as a 1.5-kb species in placenta and at lower levels in various other tissues and a 7-kb mRNA species observed in skeletal muscle and heart. The expression pattern and unique domain structure of p57 suggest that this CDI may play a specialized role in cell cycle control.