bluuebunny/crossref_metadata_2025 huggingface dataset:
Keratinocyte growth factor is required for hair development but not for wound healing.
Authors: Guo L , Degenstein L , Fuchs E
Published: June 2007

Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), is synthesized by skin fibroblasts. However, its mitogenic activity is on skin keratinocytes, where it is the most potent growth factor identified thus far. To explore KGF's function in vivo, we used embryonic stem cell technology to generate mice lacking KGF. Over time, their fur developed a matted appearance, very similar to that of the rough mouse, whose recessive mutation maps at or near the KGF locus on mouse chromosome 2. In contrast to the recently reported transforming growth factor-alpha (TGF-alpha) and FGF5 knockouts, which showed defects in the follicle outer-root sheath and the hair growth cycle, respectively, the hair defect in the KGF knockout seemed to be restricted to the cells giving rise to the hair shaft. Thus, we have uncovered a third, and at least partially nonoverlapping, growth factor pathway involved in orchestrating hair follicle growth and/or differentiation. Surprisingly, the absence of KGF resulted in no abnormalities in epidermal growth or wound healing. This was true even when we engineered double knockout mice, null for both KGF and TGF-alpha, two factors that are increased dramatically in the normal wound-healing process. Whereas we found no evidence of compensatory changes at the mRNA level of wounded knockout mice, these data imply that the regulation of epidermal growth is complex and involves a number of growth stimulatory factors that go beyond what are thought to be the major paracrine and autocrine growth factors. We suggest that the redundancy in epidermal growth and wound healing is likely to stem from the vitality of these functions to the organism, a feature that is not a consideration for the hair follicle.
Rescue of Drosophila labial null mutant by the chicken ortholog Hoxb-1 demonstrates that the function of Hox genes is phylogenetically conserved.
Authors: Lutz B , Lu H C , Eichele G , Miller D , Kaufman T C
Published: June 2007

Hox complexes are important players in the establishment of the body plan of invertebrates and vertebrates. Sequence comparison demonstrates a remarkable phylogenetic conservation of key structural features of Hox genes. The correlation between the physical order of genes along the chromosomes and their domains of function along the body axis is conserved between arthropods and vertebrates. Ectopic expression experiments suggest that the functions of homeo proteins also are conserved between invertebrates and vertebrates. However, it remains an open question whether vertebrate Hox genes expressed under the control of Drosophila regulatory sequences can substitute the function of Drosophila Hox genes. We have studied this issue with the Drosophila labial (lab) gene and its chicken ortholog gHoxb-1. We fused the entire protein-coding region of gHoxb-1 with previously identified regulatory sequences of lab. This approach places gHoxb-1 into the normal embryonic spatiotemporal context in which lab acts. Ten transgenic lines carrying gHoxb-1 were established and tested for their ability to rescue lab null mutant animals. Eight lines rescued with high efficiency, embryonic lethality, and abnormal head morphogenesis, two defects observed in lab null mutant embryos. The rescue with the gHoxb-1 minigene was close to the efficiency of that obtained with the Drosophila lab minigene. This indicates that gHoxb-1 protein can regulate lab target genes and thereby restore embryonic viability. This is striking, as Lab and gHoxb-1 proteins are divergent except for their homeo domains and a short stretch of amino acids amino-terminal to the homeo domain. Our findings demonstrate a functional conservation of the lab class homeo proteins between insects and vertebrates and support the view that function of Hox genes resides in relatively few conserved motifs and largely in the homeo domain.
Transcriptional enhancers act in cis to suppress position-effect variegation.
Authors: Walters M C , Magis W , Fiering S , Eidemiller J , Scalzo D , Groudine M , Martin D I
Published: June 2007

We have examined the basis of enhancer effects on gene expression by altering the action of enhancers on expression of a stably integrated reporter gene. We used two distinct experimental approaches: recombinase-mediated deletion of an enhancer and modulation of the activity of another enhancer composed of downstream metal response elements (MREs). The flp recombinase was used to delete the 5'HS2 globin enhancer from a site downstream of beta-geo at nine separate integration sites in K562 erythroleukemia cells. In no case does deletion of 5'HS2 have a significant effect on the level of expression; however, the deletion does increase dramatically the rate at which expression of beta-geo is silenced. Zinc stimulation of a metallothionein enhancer has no effect on the level of reporter expression, but slows the rate of silencing. Silencing in both cases is highly site dependent, and resembles position-effect variegation (PEV). These results strongly support a binary mode of enhancer action, as in both cases the enhancer maintains reporter expression without a strong effect on the level of expression. Taken together, these findings suggest that transcriptional activators have a direct interaction with repressive chromatin structures, which is independent of an effect on the rate of transcription. We propose that cis-acting transcriptional control elements may act primarily through this mechanism.
Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.
Authors: Doucas V , Ishov A M , Romo A , Juguilon H , Weitzman M D , Evans R M , Maul G G
Published: June 2007

Wild-type PML and at least four other novel proteins are localized within discrete nuclear structures known as PODs. We demonstrate here that during adenovirus infection, immediate early viral proteins from the E1 and E4 transcription units associate with the POD, which in turn undergoes a dramatic morphological change. During this process, the auto-antigen Sp-100 and NDP55 but not PML, relocate from the POD to the viral inclusion bodies, the sites of adenovirus DNA replication and late RNA transcription. The E4-ORF3 11-kD protein alone will induce this reorganization and reciprocally, viruses carrying mutations in the E4-domain fail to do so. These same viral mutants are defective in viral replication as well as the accumulation of late viral mRNAs and host cell transcription shutoff. We show that interferon (INF) treatment enhances the expression of PML, reduces or blocks PODs reorganization, and inhibits BrdU incorporation into viral inclusion bodies. In addition, cell lines engineered to overexpress PML prevent PODs from viral-induced reorganization and block or severely delay adenovirus replication. These results suggest that viral replication relies on components of the POD and that the structure is a target of early viral proteins.
Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A.
Authors: Gozani O , Feld R , Reed R
Published: June 2007

A critical step in the pre-mRNA splicing reaction is the stable binding of U2 snRNP to the branchpoint sequence (BPS) to form the A complex. The multimeric U2 snRNP protein complexes SF3a and SF3b are required for A complex assembly, but their specific roles in this process are not known. Saccharomyces cerevisiae homologs of all of the SF3a, but none of the SF3b, subunits have been identified. Here we report the isolation of a cDNA encoding the mammalian SF3b subunit SAP 145 and the identification of its probable yeast homolog (29% identity). This first indication that the homology between yeast and metazoan A complex proteins can be extended to SF3b adds strong new evidence that the mechanism of A complex assembly is highly conserved. To investigate this mechanism in the mammalian system we analyzed proteins that cross-link to 32P-site-specifically labeled pre-mRNA in the A complex. This analysis revealed that SAP 145, together with four other SF3a/SF3b subunits, UV cross-links to pre-mRNA in a 20-nucleotide region upstream of the BPS. Mutation of this region, which we have designated the anchoring site, has no apparent effect on U2 snRNP binding. In contrast, when a 2'O methyl oligonucleotide complementary to the anchoring site is added to the spliceosome assembly reaction, A complex assembly and cross-linking of the SF3a/SF3b subunits are blocked. These data indicate that sequence-independent binding of the highly conserved SF3a/SF3b subunits upstream of the branch site is essential for anchoring U2 snRNP to pre-mRNA.
Assembly of the isomerized TFIIA--TFIID--TATA ternary complex is necessary and sufficient for gene activation.
Authors: Chi T , Carey M
Published: June 2007

The prevailing view of eukaryotic gene activation poses that activators stimulate transcription by recruiting limiting components of the general transcription machinery to a core promoter. In one such model case, activation by the Epstein-Barr virus ZEBRA protein correlated closely with recruitment of the general transcription factors TFIIA and TFIID (the DA complex) as measured by DNase I footprinting and gel mobility shift assays. We now report that simple recruitment is not sufficient for full-level activation. An additional concentration-independent, rate-limiting step is activator-mediated isomerization of the DA complex characterized by an extended TFIID footprint. The isomerized complex supports both binding of TFIIB in gel mobility shift assays and activated transcription in heat-treated nuclear extracts, even after removal of ZEBRA. Surprisingly, the regulatory phenomenon of synergy was manifested only when the concentration of TFIID was limiting. When the DA complex was saturating, transcription was not synergistic, as indicated by the ability of a single activator to induce isomerization effectively and turn on a gene. On the basis of these observations, we propose a new biochemical model for eukaryotic gene activation and synergy.
Mitotic chromosome condensation in the rDNA requires TRF4 and DNA topoisomerase I in Saccharomyces cerevisiae.
Authors: Castaño I B , Brzoska P M , Sadoff B U , Chen H , Christman M F
Published: June 2007

DNA topoisomerase I (topo I) is known to participate in the process of DNA replication, but is not essential in Saccharomyces cerevisiae. The TRF4 gene is also nonessential and was identified in a screen for mutations that are inviable in combination with a top1 null mutation. Here we report the surprising finding that a top1 trf4-ts double mutant is defective in the mitotic events of chromosome condensation, spindle elongation, and nuclear segregation, but not in DNA replication. Direct examination of rDNA-containing mitotic chromosomes demonstrates that a top1 trf4-ts mutant fails both to establish and to maintain chromosome condensation in the rDNA at mitosis. We show that the Trf4p associates physically with both Smclp and Smc2p, the S. cerevisiae homologs of Xenopus proteins that are required for mitotic chromosome condensation in vitro. The defect in the top1 trf4-ts mutant is sensed by the MAD1-dependent spindle assembly checkpoint but not by the RAD9-dependent DNA damage checkpoint, further supporting the notion that chromosome structure influences spindle assembly. These data indicate that TOP1 (encoding topo I) and TRF4 participate in overlapping or dependent steps in mitotic chromosome condensation and serve to define a previously unrecognized biological function of topo I.
Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.
Authors: Wang J , Takagaki Y , Manley J L
Published: June 2007

Alternative splicing factor/splicing factor 2 (ASF/SF2) is the prototype of a family of nuclear proteins highly conserved throughout metazoa, the SR (serine/arginine) proteins. Based largely on in vitro studies, SR proteins have been suggested to play important roles in constitutive and alternative splicing of pre-mRNAs. Here we describe the development of a genetic system employing the chicken B-cell line DT40 to study the function of ASF/SF2 in vivo. The high level of homologous recombination and rapid growth rate of these cells allowed us to show first that ASF/SF2 is an essential gene, and then to perform targeted disruption of both ASF/SF2 alleles, by creating a cell line in which the only source of ASF/SF2 is a human cDNA controlled by a tetracycline (tet)-repressible promoter. We show that addition of tet to these cells results in rapid depletion of ASF/SF2, concomitant accumulation of incompletely processed pre-mRNA, and subsequent cell death. The tet-induced lethality could be rescued by plasmids expressing wild-type ASF/SF2, but not several mutant derivatives, or other SR proteins. Heterozygous cell lines overexpressing human ASF/SF2 displayed significant reductions of endogenous ASF/SF2 mRNA, suggesting that ASF/SF2 mRNA levels are controlled by an autoregulatory loop. This system provides a novel method for genetic analysis of factors that function in basic processes in vertebrate cells.
smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo.
Authors: Smibert C A , Wilson J E , Kerr K , Macdonald P M
Published: June 2007

nanos mRNA, which encodes the localized component of the Drosophila posterior body patterning determinant, is normally translated only at the posterior pole of the embryo, where the mRNA is concentrated. Here we identify two similar cis-acting sequences in the nanos mRNA 3' untranslated region that mediate translational repression. These sequences bind an embryonic protein of 135 kD, smaug, and we refer to them as smaug recognition elements (SREs). Analysis of point mutations in the SREs reveals a strong correlation between smaug binding and translational repression; mutants unable to bind smaug in vitro are not repressed translationally in vivo, whereas mutants that do bind smaug remain repressed translationally. These results strongly suggest that smaug acts in translational repression of unlocalized nanos mRNA. Translational repression is essential, as embryos expressing a nanos mRNA with mutated SREs develop with anterior body patterning defects and die, despite correct localization of the RNA.
The ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase.
Authors: Grallert B , Nurse P
Published: June 2007

In a screen for new cell-cycle genes in Schizosaccharomyces pombe we have isolated cdc30, which is identical to orp1, a putative homolog of the Saccharomyces cerevisiae ORC1 gene. Analysis of the temperature-sensitive orp1-4 and the orp1(delta) mutants indicates that orp1 is required at the onset of S phase for an early step of DNA replication. Orp1p is found in the nucleus and is present at a constant level throughout the cell cycle. Genetic interactions occur between orp1 and cdc18 and cdc21 (an MCM homolog). Orp1p forms protein complexes with both cdc18p and cdc21p in vivo, suggesting that interactions between these proteins and ORC are important for controlling the initiation of DNA replication at the onset of S phase. The orp1 gene is also required for the control that prevents entry into mitosis in the absence of DNA replication, suggesting a role for ORC in this checkpoint pathway.
KSR modulates signal propagation within the MAPK cascade.
Authors: Therrien M , Michaud N R , Rubin G M , Morrison D K
Published: June 2007

Kinase suppressor of Ras (KSR) is a recently identified component of Ras-dependent signaling pathways. In this report, we show that murine KSR1 (mKSR1) cooperates with activated Ras to promote Xenopus oocyte maturation and cellular transformation and provide evidence that this cooperation occurs by accelerating mitogen and extracellular regulated kinase (MEK) and mitogen-activated protein kinase (MAPK) activation. We also find that mKSR1 associates with Raf-1 at the plasma membrane in a Ras-dependent manner, indicating the presence of a membrane-bound kinase signaling complex. Although mKSR1 is related structurally to Raf-1, our findings reveal striking functional differences between these proteins. In marked contrast to the isolated amino- and carboxy-terminal domains of Raf-1, the KSR amino terminus also cooperates with Ras, whereas the carboxy-terminal kinase domain blocks Ras signaling as well as MEK and MAPK activation. The isolated KSR kinase domain suppressed Xenopus oocyte maturation, cellular transformation, and Drosophila eye development, suggesting that separation of the amino- and carboxy-terminal domains has uncoupled the normal regulation of KSR as a positive effector of Ras signaling. Together, our findings indicate that mKSR1 is an integral component of the MAPK module functioning via a novel mechanism to modulate signal propagation between Raf-1, MEK1, and MAPK.
v-Myb of E26 leukemia virus up-regulates bcl-2 and suppresses apoptosis in myeloid cells.
Authors: Frampton J , Ramqvist T , Graf T
Published: June 2007

Many oncogenes have been shown to be deregulated transcription factors, yet direct target genes mediating cell transformation remain elusive. Here we describe such a target for v-Myb by exploiting a temperature-sensitive mutant of the E26 avian leukemia virus encoding Myb-Ets. Myeloblasts transformed by the mutant differentiate into macrophages or die by apoptosis when shifted to the nonpermissive temperature as a result of inactivation of v-Myb. During this process mRNA of the antiapoptotic oncoprotein Bcl-2 is down-regulated with kinetics similar to those of Mim-1, a differentiation-related protein whose expression is directly regulated by Myb. Forced expression of bcl-2 rescues the cells from apoptosis, without preventing either their withdrawal from the cell cycle or their differentiation. v-Myb appears to act directly on the bcl-2 gene, because a bcl-2 promoter-driven reporter is activated by Myb-Ets and v-Myb-VP16 and requires intact Myb binding sites within the promoter. Surprisingly, inactivation of v-Myb in multipotent progenitors transformed by E26 virus does not induce apoptosis, indicating that bcl-2 regulation by the oncoprotein is required for the transformation of some cell types but not others.
A JNK signal transduction pathway that mediates morphogenesis and an immune response in Drosophila.
Authors: Sluss H K , Han Z , Barrett T , Davis R J , Ip Y T
Published: June 2007

The Drosophila MAP kinase DJNK is a homolog of the mammalian c-Jun amino-terminal kinase (JNK). Mutations in the DJNK gene correspond to the complementation group basket. DJNK is phosphorylated and activated by the Drosophila MAP kinase kinase HEP. Substrates of DJNK include the transcription factor DJun. DJNK participates in multiple physiological processes. Exposure to endotoxic lipopolysaccharide initiates an insect immune response and leads to DJNK activation. In addition, embryos lacking DJNK are defective in dorsal closure, a process in which the lateral epithelial cells migrate over the embryo and join at the dorsal midline. These data demonstrate that the DJNK signal transduction pathway mediates an immune response and morphogenesis in vivo.
Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro.
Authors: Paull T T , Carey M , Johnson R C
Published: June 2007

Nonhistone proteins 6A and 6B (NHP6A/B) are nonsequence-specific DNA-binding proteins from Saccharomyces cerevisiae that are related structurally and functionally to the mammalian high mobility group proteins 1 and 2. These DNA architectural proteins distort DNA structure severely and have been shown to promote assembly of specialized recombination complexes. Here we show that the yeast NHP6A/B proteins are required for the induction of a subset of genes transcribed by RNA polymerase II (pol II). Activation of the CUP1, CYC1, GAL1, and DDR2 genes was decreased or abolished completely in the delta nhp6A/B strain. No significant change in basal expression was observed for any of the 10 genes examined. Analysis of chimeric gene constructs localized the regions dependent on NHP6A/B to be primarily at the core promoters, although the GAL1 UAS also requires NHP6A/B for activity. In vitro, NHP6A stimulated transcription by pol II at the GAL1 promoter three- to fivefold above the level of activation by GAL4-VP16 alone. Gel mobility shift assays showed that NHP6A promotes the formation of a complex with TBP and TFIIA at the TATA box that has enhanced affinity for TFIIB.
Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.
Authors: Hoppler S , Brown J D , Moon R T
Published: June 2007

During gastrulation of Xenopus embryos the prospective mesoderm is induced initially with domains of dorsal and ventral fate, then further patterned to generate somitic mesoderm by signals from the gastrula organizer. Although Xwnt-8 is expressed in future ventrolateral mesoderm and induces prospective epidermis to differentiate in vitro as ventral mesoderm, no loss-of-function studies have demonstrated a requirement for Wnt signaling for the normal expression of mesodermal genes in the gastrula. We report development of a dominant-negative Wnt (dnXwnt-8) that inhibits embryonic responses to Wnt signaling in a cell-nonautonomous fashion. By expressing dnXwnt-8 in Xenopus embryos, we uncover a requirement of Wnt signaling for localized expression in prospective mesoderm of XMyoDa and Xenopus-posterior (Xpo). Because ectopic expression of functional Xwnt-8 in the dorsal marginal zone of the gastrula induces ectopic XMyoDa and Xpo, both gain-of-function and loss-of-function experiments support a model in which endogenous Xwnt-8 functions to induce expression of genes involved in specification of ventral and somitic mesoderm.
Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae.
Authors: Cook J G , Bardwell L , Kron S J , Thorner J
Published: June 2007

Haploid cells of budding yeast Saccharomyces cerevisiae respond to mating pheromones by inducing genes required for conjugation, arresting cell cycle progression, and undergoing morphological changes. The same cells respond to nutrient deprivation by altering budding pattern and inducing genes required for invasive growth. Both developmental alternatives to vegetative proliferation require the MAP kinase Kss1 and the transcriptional transactivator Ste12. Using a two-hybrid screen for gene products that interact with Kss1, two homologous and previously uncharacterized loci (DIG1 and DIG2, for down-regulator of invasive growth) were identified. DIG2 is pheromone-inducible, whereas DIG1 is constitutively expressed. Dig1 colocalizes with Kssl in the nucleus, coimmunoprecipitates with Kss1 from cell extracts in a pheromone-independent manner, and is phosphorylated by Kss1 in immune complexes in a pheromone-stimulated manner. Kss1 binds specifically to a GST-Dig1 fusion in the absence of any other yeast protein. Using the two-hybrid method, both Dig1 and Dig2 also interact with the other MAP kinase of the pheromone response pathway, Fus3. However, neither dig1 or dig2 single mutants, nor a dig1 dig2 double mutant, have a discernible effect on mating. In contrast, dig1 dig2 cells constitutively invade agar medium, whereas a dig1 dig2 ste12 triple mutant does not, indicating that Dig1 and Dig2 share a role in negatively regulating the invasive growth pathway. High-level expression of Dig1 suppresses invasive growth and also causes cells to appear more resistant to pheromone-imposed cell cycle arrest. Ste12 also binds specifically to GST-Dig1 in the absence of any other yeast protein. Collectively, these findings indicate that Dig1, and most likely Dig2, are physiological substrates of Kssl and suggest that they regulate Ste12 function by direct protein-protein interaction.
BID: a novel BH3 domain-only death agonist.
Authors: Wang K , Yin X M , Chao D T , Milliman C L , Korsmeyer S J
Published: June 2007

The BCL-2 family of proteins consists of both antagonists (e.g., BCL-2) and agonists (e.g., BAX) that regulate apoptosis and compete through dimerization. The BH1 and BH2 domains of BCL-2 are required to heterodimerize with BAX and to repress cell death; conversely, the BH3 domain of BAX is required to heterodimerize with BCL-2 and to promote cell death. To extend this pathway, we used interactive cloning to identify Bid, which encodes a novel death agonist that heterodimerizes with either agonists (BAX) or antagonists (BCL-2). BID possesses only the BH3 domain, lacks a carboxy-terminal signal-anchor segment, and is found in both cytosolic and membrane locations. BID counters the protective effect of BCL-2. Moreover, expression of BID, without another death stimulus, induces ICE-like proteases and apoptosis. Mutagenesis revealed that an intact BH3 domain of BID was required to bind the BH1 domain of either BCL-2 or BAX. A BH3 mutant of BID that still heterodimerized with BCL-2 failed to promote apoptosis, dissociating these activities. In contrast, the only BID BH3 mutant that retained death promoting activity interacted with BAX, but not BCL-2. This BH3-only molecule supports BH3 as a death domain and favors a model in which BID represents a death ligand for the membrane-bound receptor BAX.
The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.
Authors: Rusconi C P , Cech T R
Published: June 2007

The import of nuclear-encoded RNAs into mitochondria is required for proper mitochondrial function in most organisms. However, the mechanisms used to achieve RNA import are largely unknown. In particular, the RNA elements that direct import have not been identified in any organism. In Tetrahymena, only one of three nuclear-encoded glutamine accepting tRNAs is imported into mitochondria. We transform Tetrahymena with marked glutamine tRNAs and quantitate their level of accumulation in mitochondria. Of several isostructural nucleotide substitutions tested, alteration of the anticodon sequence uniquely abolishes import. Furthermore, substitution of a single anticodon nucleotide (UUA-->UUG) confers import on a normally nonimported glutamine tRNA. Thus, the anticodon functions as a mitochondrial localization signal and is both necessary and sufficient for tRNA import. Given the prior evidence that neither the cytoplasmic nor the mitochondrial glutaminyl-tRNA synthetase distinguishes between the imported and nonimported glutamine tRNAs with respect to aminoacylation, we propose that some mitochondrial import factor distinct from a synthetase recognizes the anticodon of the imported glutamine tRNA.
A protein of the SR family of splicing factors binds extensively to exonic Balbiani ring pre-mRNA and accompanies the RNA from the gene to the nuclear pore.
Authors: Alzhanova-Ericsson A T , Sun X , Visa N , Kiseleva E , Wurtz T , Daneholt B
Published: June 2007

We report on the molecular cloning and intracellular localization of a heterogeneous nuclear ribonucleoprotein (hnRNP), Ct-hrp45, one of the major components of pre-mRNP particles in Chironomus tentans. It is shown that hrp45 belongs to the SR family of splicing factors and exhibits high sequence similarity to Drosophila SRp55/B52 and human SF2/ASF. The distribution of hrp45 within the C. tentans salivary gland cells is studied by immunocytology. The hrp45 protein is found to be abundant in the nucleus, whereas it is undetectable in the cytoplasm. The fate of hrp45 in specific pre-mRNP particles, the Balbiani ring (BR) granules, is revealed by immunoelectron microscopy. It is observed that hrp45 is associated with the growing BR pre-mRNP particles and is being added continuously concomitant with the growth of the transcript, indicating that hrp45 is bound extensively to exon 4, which comprises 80-90% of the primary transcript. Furthermore, hrp45 remains bound to the BR RNP particles in the nucleoplasm and is not released until the particles translocate through the nuclear pore. Thus, hrp45 behaves as an hnRNP protein linked to exon RNA (and perhaps also to the introns) rather than as a spliceosome component connected to the assembly and disassembly of spliceosomes. It seems that hrp45, and possibly also other SR family proteins, is playing an important role in the structural organization of pre-mRNP particles and is perhaps participating not only in splicing but also in other intranuclear events.
SREBP transcriptional activity is mediated through an interaction with the CREB-binding protein.
Authors: Oliner J D , Andresen J M , Hansen S K , Zhou S , Tjian R
Published: June 2007

The sterol regulatory element binding proteins (SREBP-1 and -2) activate transcription of genes whose products are involved in the cellular uptake and synthesis of cholesterol. Although considerable effort has been exerted to define the events regulating the levels of active SREBP, little is known about the transcriptional cofactors mediating SREBP function. In an unbiased search for potential coactivators of SREBP, we isolated a protein of 265 kD from HeLa cells that directly bound SREBP-1 and SREBP-2. Peptide sequencing and Western blot analysis established that the 265-kD protein was CBP (CREB-binding protein), a recently identified transcriptional coactivator. The putative activation domain of SREBP was shown to bind specifically to amino-terminal domains of recombinant CBP and p300 (a CBP-related protein). Moreover, transfection studies demonstrated that CBP enhances the ability of SREBP to activate transcription of reporter genes in HeLa cells. Together, these data suggest that CBP mediates SREBP transcriptional activity, thus revealing a new step in the biochemical pathway regulating cholesterol metabolism.
araucan and caupolican provide a link between compartment subdivisions and patterning of sensory organs and veins in the Drosophila wing.
Authors: Gómez-Skarmeta J L , Modolell J
Published: June 2007

The homeo box prepattern genes araucan (ara) and caupolican (caup) are coexpressed near the anterior-posterior (AP) compartment border of the developing Drosophila wing in two symmetrical patches located one at each side of the dorsoventral (DV) compartment border. ara-caup expression at these patches is necessary for the specification of the prospective vein L3 and associated sensory organs through the transcriptional activation, in smaller overlapping domains, of rhomboid/veinlet and the proneural genes achaete and scute. We show that ara-caup expression at those patches is mediated by the Hedgehog signal through its induction of high levels of Cubitus interruptus (Ci) protein in anterior cells near to the AP compartment border. The high levels of Ci activate decapentaplegic (dpp) expression, and, together, Ci and Dpp positively control ara-caup. The posterior border of the patches is apparently defined by repression by engrailed. Wingless accumulation at the DV border sets, also by repression, the gap between the two patches. Thus, ara and caup integrate the inputs of genes effecting the primary subdivisions of the wing disc into compartments to define two smaller territories. These in turn help create the even smaller domains of rhomboid/veinlet and achaete-scute expression.
Wild-type p53 negatively regulates the expression of a microtubule-associated protein.
Authors: Murphy M , Hinman A , Levine A J
Published: June 2007

The product of the p53 tumor suppressor gene has a well-documented activity as a transcriptional activator, and several studies indicate that this function is at least in part essential for the ability of p53 to suppress cellular proliferation. However, there is growing evidence that some activities of wild-type p53 may be independent of its trans-activation function; in fact, recent investigations have indicated that the transcriptional repression function of p53, rather than its trans-activation function, may be influential in p53-mediated apoptosis. The focus of this study has been on the identification of genes that exhibit decreased expression during p53-dependent apoptosis, and therefore represent potential p53-repressed genes influential in programmed cell death. This report identifies the gene encoding the microtubule-associated protein MAP4 as one whose mRNA and protein expression decrease in cells following induction of wild-type p53. Importantly, decreased MAP4 expression following p53 induction can be inhibited by molecules that prevent p53-mediated transcriptional repression and apoptosis, such as the adenovirus E1B-19K protein and the Wilms tumor gene product WT1. Additionally, overexpression of MAP4 in cells induced to undergo p53-dependent apoptosis significantly delays this process, indicating that the negative regulation of this gene by p53 may be influential in the rapid progression of apoptosis.
Heartless, a Drosophila FGF receptor homolog, is essential for cell migration and establishment of several mesodermal lineages.
Authors: Beiman M , Shilo B Z , Volk T
Published: June 2007

A Drosophila FGF receptor homolog (DFGF-R2/DFRI) termed Heartless (Htl) is expressed in the embryonic mesoderm. The phenotypes of null mutant embryos demonstrated that Htl is a central player that is required for the development of several mesodermal lineages. No abnormalities in the primary specification of the mesoderm were observed. The first defects were seen as irregular migration and spreading of the mesoderm over the ectoderm. Subsequently, cell fates were not induced in several lineages including the visceral mesoderm, heart, and the dorsal somatic muscles. The defects in the induction of cell fates are likely to result from failure of the mesoderm to spread over the ectoderm and receive patterning signals. The defective spreading could be circumvented in htl mutant embryos by providing an ectopic Dpp patterning signal, leading to the formation of heart and dorsal muscle cells. Htl appears to be required also subsequently during the migration and morphogenesis of the different lineages. Expression of a dominant-negative htl construct after the initial induction of cell fates gave rise to aberrant migration and organization of the visceral mesoderm, heart, and somatic muscles. Thus, a common role for Htl in cell migration and tissue organization may account for the pleiotropic defects of the htl mutation.
The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.
Authors: Wang Z F , Whitfield M L , Ingledue T C , Dominski Z , Marzluff W F
Published: June 2007

Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop. Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA. The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events. We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system. The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein. The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts. These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.
A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation.
Authors: Ha I , Wightman B , Ruvkun G
Published: June 2007

The Caenorhabditis elegans heterochronic gene lin-14 generates a temporal gradient of the LIN-14 proteins to control stage-specific patterns of cell lineage during development. Down-regulation of LIN-14 is mediated by the lin-14 3' untranslated region (UTR), which bears seven sites that are complementary to the regulatory lin-4 RNA. Here we report molecular and genetic evidence that RNA duplexes between the lin-4 and lin-14 RNAs form in vivo and are necessary for LIN-14 temporal gradient generation. lin-4 RNA binds in vitro to a lin-14 mRNA bearing the seven lin-4 complementary sites but not to a lin-14 mRNA bearing point mutations in these sites. In vivo, the lin-4 complementary regions are necessary for lin-14 3' UTR-mediated temporal gradient formation. Based on lin-14 3' UTR sequence comparisons between C. elegans and C. briggsae, four of the seven lin-4/lin-14 RNA duplexes are predicted to bulge a lin-4 C residue, and three sites are predicted to form nonbulged RNA duplexes. Reporter genes bearing multimerized bulged C lin-4 binding sites show almost wild-type temporal gradient formation, whereas those bearing multimerized nonbulged lin-4 binding sites do not form a temporal gradient. Paradoxically, lin-4 RNA binds in vitro to nonbulged lin-14 RNA more avidly than to the bulged lin-14 RNA. This suggests that a specific secondary structure of lin-4/lin-14 RNA duplex that may be recognized by an accessory protein, rather than an RNA duplex per se, is required in vivo for the generation of the LIN-14 temporal gradient.
pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis.
Authors: Zacksenhaus E , Jiang Z , Chung D , Marth J D , Phillips R A , Gallie B L
Published: June 2007

Mice deficient for the RB gene (RB-/-), prior to death at embryonic day 14.5, show increased cell death in all tissues that normally express RB1: the nervous system, liver, lens, and skeletal muscle precursor cells. We have generated transgenic mice (RBlox) that express low levels of pRb, driven by an RB1 minigene. RBlox/RB-/- mutant fetuses die at birth with specific skeletal muscle defects, including increased cell death prior to myoblast fusion, shorter myotubes with fewer myofibrils, reduced muscle fibers, accumulation of elongated nuclei that actively synthesized DNA within the myotubes, and reduction in expression of the late muscle-specific genes MCK and MRF4. Thus, insufficient pRb results in failure of myogenesis in vivo, manifest in two ways. First, the massive apoptosis of myoblasts implicates a role of pRb in cell survival. Second, surviving myotubes failed to develop normally and accumulated large polyploid nuclei, implicating pRb in permanent withdrawal from the cell cycle. These results demonstrate a role for pRb during terminal differentiation of skeletal muscles in vivo and place pRb at a nodal point that controls cell proliferation, differentiation, and death.
The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.
Authors: Missero C , Di Cunto F , Kiyokawa H , Koff A , Dotto G P
Published: June 2007

p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in differentiation-associated growth arrest, as its expression is augmented in many terminally differentiating cells. A general involvement of p21 in growth/differentiation control and tumor suppression has been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in any of their organs, and exhibit no increase in spontaneous tumor development. However, a significant imbalance between growth and differentiation could be unmasked under conditions where normal homeostatic mechanisms are impaired. We report here that primary keratinocytes derived from p21 knockout mice, transformed with a ras oncogene, and injected subcutaneously into nude mice exhibit a very aggressive tumorigenic behavior, which is not observed with wild-type control keratinocytes nor with keratinocytes with a disruption of the closely related p27 gene. p21 knockout keratinocytes tested under well-defined in vitro conditions show a significantly increased proliferative potential, which is also observed but to a lesser extent with p27 knockout cells. More profound differences were found in the differentiation behavior of p21 versus p27 knockout keratinocytes, with p21 (but not p27) deficiency causing a drastic down-modulation of differentiation markers linked with the late stages of the keratinocyte terminal differentiation program. Thus, our results reveal a so far undetected role of p21 in tumor suppression, demonstrate that this function is specific as it cannot be attributed to the closely related p27 molecule, and point to an essential involvement of p21 in terminal differentiation control, which may account for its role in tumor suppression.
Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p.
Authors: Cohen-Fix O , Peters J M , Kirschner M W , Koshland D
Published: June 2007

Anaphase initiation has been postulated to be controlled through the ubiquitin-dependent proteolysis of an unknown inhibitor. This process involves the anaphase promoting complex (APC), a specific ubiquitin ligase that has been shown to be involved in mitotic cyclin degradation. Previous studies demonstrated that in Saccharomyces cerevisiae, Pds1 protein is an anaphase inhibitor and suggested that it may be an APC target. Here we show that in yeast cells and in mitotic Xenopus extracts Pds1p is degraded in an APC-dependent manner. In addition, Pds1p is directly ubiquitinated by the Xenopus APC. In budding yeast Pds1p is degraded at the time of anaphase initiation and nondegradable derivatives of Pds1p inhibit the onset of anaphase. We conclude that Pds1p is an anaphase inhibitor whose APC-dependent degradation is required for the initiation of anaphase.
Est1 has the properties of a single-stranded telomere end-binding protein.
Authors: Virta-Pearlman V , Morris D K , Lundblad V
Published: June 2007

In Saccharomyces cerevisiae, deletion of the EST1 gene results in phenotypes identical to those displayed by a deletion of a known component of telomerase (the yeast telomerase RNA), arguing that EST1 is also critical for telomerase function. In this study, we show that the Estl protein binds to yeast G-rich telomeric oligonucleotides in vitro. Binding is specific for single-stranded substrates and requires a free 3' terminus, consistent with the properties expected for a protein bound to the 3' single-stranded G-rich extension present at the telomere. Assessment of the in vivo function of this single-stranded DNA-binding protein has shown that EST1 acts in the same pathway of telomere replication as the TLC1 telomerase RNA, by several different genetic criteria: est1 tlc1 double mutant strains show no enhancement of phenotype relative to either single mutant strain, and EST1 dominant mutations have an effect on telomeric silencing similar to that displayed by TLC1 previously. We propose that Est1 is a telomere end-binding protein that is required to mediate recognition of the end of the chromosome by telomerase.
Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development.
Authors: Allende M L , Amsterdam A , Becker T , Kawakami K , Gaiano N , Hopkins N
Published: June 2007

Recently our laboratory described an efficient method for generating retroviral provirus insertions in the zebrafish germ line, and we showed that provirus insertions induce embryonic mutations at a frequency of roughly one mutant per 70 insertions. To date we have isolated four insertional mutants and, using the proviruses as a molecular tag, have cloned the genes disrupted in three of them. The proviruses in all three mutants lie within or just 5' of the first coding exon, point in the opposite transcriptional orientation from the gene, and disrupt transcription. Here we present a molecular characterization of two genes identified by this method and describe the associated mutant phenotypes. The pescadillo (pes) gene is predicted to encode a protein of 582 amino acids with no recognizable functional motifs, which is highly conserved from yeast to humans. pes mRNA is expressed widely and dynamically during the first 3 days of embryogenesis. Prominent sites of expression are the eyes and optic tectum on day 1, the fin buds, liver primordium, and gut on day 2, and the branchial arches on day 3. Beginning at day 3 of embryogenesis, pes mutant embryos exhibit small eyes, a reduced brain and visceral skeleton, shortened fins, and a lack of expansion of the liver and gut, and then die on the sixth day of development. The dead eye (dye) gene encodes a protein of 820 amino acids that is homologous to genes of unknown function in human, mouse, and Xenopus, and that has weak homology with the yeast NIC96 (nucleoporin-interacting component) gene. dye mutants can be recognized on day 2 of embryogenesis by the presence of necrotic cells in the tectum and eyes. dye mutants die on day 5 of development. These results demonstrate the power of insertional mutagenesis in zebrafish for rapidly finding and characterizing novel genes essential for embryonic development. Using our current methodology, we estimate that our laboratory could screen approximately 25,000 insertions in 2-3 years, identifying perhaps 250-350 embryonic lethal genes. Assuming that all genes are accessible to proviral insertion, the wider application of this approach could lead to the rapid identification of the majority of genes that are required for embryonic development of this vertebrate.
Characterization of human lysosomal neuraminidase defines the molecular basis of the metabolic storage disorder sialidosis.
Authors: Bonten E , van der Spoel A , Fornerod M , Grosveld G , d'Azzo A
Published: June 2007

Neuraminidases (sialidases) have an essential role in the removal of terminal sialic acid residues from sialoglycoconjugates and are distributed widely in nature. The human lysosomal enzyme occurs in complex with beta-galactosidase and protective protein/cathepsin A (PPCA), and is deficient in two genetic disorders: sialidosis, caused by a structural defect in the neuraminidase gene, and galactosialidosis, in which the loss of neuraminidase activity is secondary to a deficiency of PPCA. We identified a full-length cDNA clone in the dbEST data base, of which the predicted amino acid sequence has extensive homology to other mammalian and bacterial neuraminidases, including the F(Y)RIP domain and "Asp-boxes." In situ hybridization localized the human neuraminidase gene to chromosome band 6p21, a region known to contain the HLA locus. Transient expression of the cDNA in deficient human fibroblasts showed that the enzyme is compartmentalized in lysosomes and restored neuraminidase activity in a PPCA-dependent manner. The authenticity of the cDNA was verified by the identification of three independent mutations in the open reading frame of the mRNA from clinically distinct sialidosis patients. Coexpression of the mutant cDNAs with PPCA failed to generate neuraminidase activity, confirming the inactivating effect of the mutations. These results establish the molecular basis of sialidosis in these patients, and clearly identify the cDNA-encoded protein as lysosomal neuraminidase.