bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple HOM-C gene interactions specify cell fates in the nematode central nervous system.
Authors: Salser S J , Loer C M , Kenyon C
Published: June 2007

Intricate patterns of overlapping HOM-C gene expression along the A/P axis have been observed in many organisms; however, the significance of these patterns in establishing the ultimate fates of individual cells is not well understood. We have examined the expression of the Caenorhabditis elegans Antennapedia homolog mab-5 and its role in specifying cell fates in the posterior of the ventral nerve cord. We find that the pattern of fates specified by mab-5 not only depends on mab-5 expression but also on post-translational interactions with the neighboring HOM-C gene lin-39 and a second, inferred gene activity. Where mab-5 expression overlaps with lin-39 activity, they can interact in two different ways depending on the cell type: They can either effectively neutralize one another where they are both expressed or lin-39 can predominate over mab-5. As observed for Antennapedia in Drosophila, expression of mab-5 itself is repressed by the next most posterior HOM-C gene, egl-5. Thus, a surprising diversity in HOM-C regulatory mechanisms exists within a small set of cells even in a simple organism.
The erythroid protein cGATA-1 functions with a stage-specific factor to activate transcription of chromatin-assembled beta-globin genes.
Authors: Barton M C , Madani N , Emerson B M
Published: June 2007

The chick beta-globin gene is regulated developmentally within erythroid cells by the interaction of multiple proteins with the promoter and the 3' enhancer. These interactions are correlated with changes in chromatin structure, which are characteristic of the actively expressed gene. Using in vitro chromatin assembly and transcription with staged erythroid extracts, we have determined the critical proteins required to activate expression of nucleosome-reconstituted beta-globin genes. These genes contain a specialized TATA box at -30 (GATA) through which the erythroid-restricted protein cGATA-1 and TFIID both function to regulate different steps in beta-globin expression. We find that TBP (TATA-binding protein) alone can activate transcription of beta-globin chromatin templates from promoters mutated to a canonical TATA box but is ineffective on those containing the normal -30 GATA site. The occupancy of this site by cGATA-1 also fails to generate efficient expression of beta-globin chromatin unless combined with a stage-specific protein, NF-E4, that binds to an adjacent site. However, NF-E4 does not function with TBP to derepress nucleosome-assembled beta-globin genes. We propose that the developmental regulation of beta-globin expression is achieved, in part, by the requirement of an erythroid protein and a stage-specific factor, rather than TBP, to activate chromatin through a specialized TATA box.
Functional interaction of adenovirus E1A with holo-TFIID.
Authors: Boyer T G , Berk A J
Published: June 2007

The activation domains of several regulatory transcription factors have been shown to bind directly in vitro to the TATA box-binding protein (TBP). Yet TBP must also interact with multiple associated polypeptides, called TAFs, for these same activators to stimulate transcription. These findings raise the question of how TBP can interact with so many proteins, both activators and TAFs, simultaneously. Here, we show that the activation domain of the adenovirus large E1A protein can bind specifically and stably to isolated holo-TFIID, the multisubunit protein complex consisting of TBP plus TAFs. Consequently, the surface of TBP that interacts with E1A must be exposed in the holo-TFIID complex. To assess the functional significance of this interaction, we established an in vitro transcription system responsive to the E1A activation domain. The addition of excess E1A to this system inhibits (squelches) both E1A-dependent and E1A-independent transcription by sequestering a target factor required for E1A activation. From among the component activities that collectively reconstitute E1A-responsive transcription in this system, holo-TFIID alone is singularly capable of reversing the inhibition of transcription mediated by excess E1A, indicating that holo-TFIID is the direct functional target of the E1A activation domain.
Activation of the c-Src tyrosine kinase is required for the induction of mammary tumors in transgenic mice.
Authors: Guy C T , Muthuswamy S K , Cardiff R D , Soriano P , Muller W J
Published: June 2007

Transgenic mice expressing the polyomavirus (PyV) middle T oncogene in the mammary epithelium develop multifocal mammary tumors that metastasize with high frequency. The potent transforming activity of PyV middle T antigen can, in part, be attributed to its ability to associate with and to activate a number of c-Src family tyrosine kinases (c-Src, c-Yes, and Fyn). As a first step toward assessing the role of individual c-Src family tyrosine kinases in PyV middle T antigen-induced mammary tumorigenesis, we have crossed transgenic mice carrying the mouse mammary tumor virus (MMTV)/PyV middle T antigen fusion gene with mice bearing a disrupted c-src proto-oncogene. In contrast to the rapid tumor progression seen in the original MMTV/PyV middle T antigen strains, mice expressing the transgene in the absence of functional c-Src rarely developed mammary tumors. After long latency, these mice did eventually develop abnormal hyperplastic mammary tissue. This growth disturbance was correlated with elevated expression of the PyV middle T antigen and the activation of the PyV middle T antigen-associated c-Yes tyrosine kinase. However, transgenic mice expressing the PyV middle T antigen in the mammary epithelium of wild-type or Yes-deficient mice developed multifocal mammary tumors with comparable kinetics. Taken together, these findings suggest that c-Src tyrosine kinase activity is required for PyV middle T antigen-induced mammary tumorigenesis and also illustrate an in vivo genetic approach to the dissection of mitogenic signal transduction pathways.
unc-101, a gene required for many aspects of Caenorhabditis elegans development and behavior, encodes a clathrin-associated protein.
Authors: Lee J , Jongeward G D , Sternberg P W
Published: June 2007

Our genetic analysis indicates that the unc-101 gene of Caenorhabditis elegans is required for many aspects of development and behavior, including negative regulation of vulval differentiation. We have cloned unc-101 and found that it encodes a homolog of the mammalian medium chains of clathrin-associated protein complexes located at the trans-Golgi and the plasma membrane, AP47 and AP50, respectively. Therefore, clathrin-mediated events might contribute to the negative regulation of vulval differentiation. Comparison of sequences, including a full-length sequence of a C. elegans AP50 homolog, reveals that UNC-101 is most closely related to AP47. Mouse AP47 and nematode UNC-101 proteins are functionally equivalent as assayed in transgenic nematodes. We have sequenced the mutant alleles of unc-101 identified in various genetic screens and shown that all but one are deletions or nonsense mutations, suggesting that these alleles severely reduce unc-101 function.
p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest.
Authors: Polyak K , Kato J Y , Solomon M J , Sherr C J , Massague J , Roberts J M , Koff A
Published: June 2007

Cell-cell contact and TGF-beta can arrest the cell cycle in G1. Mv1Lu mink epithelial cells arrested by either mechanism are incapable of assembling active complexes containing the G1 cyclin, cyclin E, and its catalytic subunit, Cdk2. These growth inhibitory signals block Cdk2 activation by raising the threshold level of cyclin E necessary to activate Cdk2. In arrested cells the threshold is set higher than physiological cyclin E levels and is determined by an inhibitor that binds to cyclin E-Cdk2 complexes. A 27-kD protein that binds to and prevents the activation of cyclin E-Cdk2 complexes can be purified from arrested cells but not from proliferating cells, using cyclin E-Cdk2 affinity chromatography. p27 is present in proliferating cells, but it is sequestered and unavailable to interact with cyclin E-Cdk2 complexes. Cyclin D2-Cdk4 complexes bind competitively to and down-regulate the activity of p27 and may thereby act in a pathway that reverses Cdk2 inhibition and enables G1 progression.
Novel insights into erythroid development revealed through in vitro differentiation of GATA-1 embryonic stem cells.
Authors: Weiss M J , Keller G , Orkin S H
Published: June 2007

Mouse embryonic stem (ES) cells lacking the transcription factor GATA-1 do not produce mature red blood cells either in vivo or in vitro. To define the consequences of GATA-1 loss more precisely, we used an in vitro ES cell differentiation assay that permits enumeration of primitive (EryP) and definitive (EryD) erythroid precursors and recovery of pure erythroid colonies. In contrast to normal ES cells, GATA-1- ES cells fail to generate EryP precursors. EryD precursors, however, are normal in number but undergo developmental arrest and death at the proerythroblast stage. Contrary to initial expectations, arrested GATA-1(-)-definitive proerythroblasts express GATA target genes at normal levels. Transcripts of the related factor GATA-2 are remarkably elevated in GATA-1- proerythroblasts. These findings imply substantial interchangeability of GATA factors in vivo and suggest that GATA-1 normally serves to repress GATA-2 during erythropoiesis. The approach used here is a paradigm for the phenotypic analysis of targeted mutations affecting hematopoietic development.
N-ras oncogene causes AP-2 transcriptional self-interference, which leads to transformation.
Authors: Kannan P , Buettner R , Chiao P J , Yim S O , Sarkiss M , Tainsky M A
Published: June 2007

Genetic alterations in elements of normal signal transduction mechanisms are known to be oncogenic events often resulting in aberrant activation of programs of gene transcription. We have investigated the effect of N-ras oncogene-induced tumorigenic transformation on the transcription factor AP-2. N-ras oncogene-induced transformation of human teratocarcinoma cells PA-1 results in sixfold elevated AP-2 mRNA levels. However, the level of AP-2-mediated trans-activation is dramatically inhibited in these cells. We show here that the high-level expression of AP-2 ultimately results in transcriptional "self-interference". The activation domain of AP-2, when fused to the DNA-binding domain of GAL4, is sufficient for self-interference. Non-N-ras PA-1 cells constitutively expressing AP-2 or GAL4-AP-2 fusion protein from an SV40 promoter exhibit reduced AP-2-mediated transcriptional activation, inhibition of differentiation, and promotion of anchorage-independent growth, properties that are similar to N-ras-transformed PA-1 cells. Thus, AP-2 is placed in the N-ras signal transduction pathway, and many of the biological effects of N-ras can be accomplished by overexpression of AP-2. This is the first evidence that inhibition of the activity of a transcription factor by self-interference contributes to a physiological process.
The helix-loop-helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein.
Authors: Iavarone A , Garg P , Lasorella A , Hsu J , Israel M A
Published: June 2007

Cell growth and differentiation are usually antagonistic. Proteins of the basic helix-loop-helix (bHLH) family bind DNA and play important roles in the differentiation of specific cell types. Id proteins heterodimerize with bHLH transcription factors, blocking their activation of lineage-specific gene expression and thereby inhibiting cellular differentiation. To examine the effect of Id-2 on cell proliferation, we overexpressed Id-2 in the human osteosarcoma cell line U2OS. Id-2 expression in U2OS reduced the serum requirement for growth and stimulated cellular proliferation by shortening the doubling time and increasing the percentage of cells in S phase. We demonstrated that Id-2 expression was able to reverse the inhibition of cellular proliferation and the block in cell cycle progression mediated by the product of the retinoblastoma tumor suppressor gene pRB. This effect was not associated with changes in the state of pRb phosphorylation in transfected cells. In vitro, unphosphorylated pRb from cell lysates specifically bound Id-2 but was not able to bind a mutated form of Id-2 lacking the HLH domain that also did not antagonize the growth arrest by pRb. In vitro-synthesized pRb containing mutations within the E1A/large T-binding pocket did not bind Id-2. However, wild-type pRb was able to bind to a region of Id-2 corresponding to only the HLH domain. In vivo, a physical association between Id-2 and pRb was seen in cross-linked extracts from SAOS-2 cells transfected with Id-2 and pRb. Our data identify a role for Id-2 in the regulation of cellular proliferation and suggest that the interaction between Id-2 and pRB is a molecular pathway over which synchronous changes in growth and differentiation are mediated in vivo.
Altered cell cycle regulation in the lens of HPV-16 E6 or E7 transgenic mice: implications for tumor suppressor gene function in development.
Authors: Pan H , Griep A E
Published: June 2007

Tumor suppressor proteins are believed to play a role in regulating cell cycle control during mammalian development. The E6 and E7 oncoproteins from human papillomavirus type 16 are known to affect cell growth control, at least in part, through their inactivation of cellular tumor suppressor gene products, p53 and Rb, respectively. Therefore, these viral proteins can serve as trans-dominant repressors of tumor suppressor gene function. To study the potential role of p53 and Rb in murine lens morphogenesis, we generated transgenic mice in which the expression of E6 or E7 was directed to the developing lens. Transgenic mice expressing E7 exhibited microphthalmia and cataracts, whereas transgenic mice expressing E6 exhibited cataracts without noticeable microphthalmia. Microscopic analysis of the lenses from neonatal and adult E7 transgenic mice revealed inhibition of lens fiber cell differentiation, induction of cell proliferation in spatially inappropriate regions of the lens, and apoptosis. Transgenic mice expressing a mutant E7 that is defective in Rb/p107 binding exhibited normal eyes, suggesting that the activity of Rb and/or Rb-like proteins is required for the perturbation of lens development and induction of apoptosis in E7 mice. Microscopic analysis of lenses from E6 neonatal and adult transgenic mice indicated the presence of nuclei in elongated fiber cells, suggesting that E6 inhibits lens fiber cell denucleation. Furthermore, expression of E6 inhibited the apoptotic-like DNA degradation observed in the lenses of nontransgenic 15.5-day embryos. In lenses from neonatal E6 x E7 double transgenic mice, the level of apoptosis was reduced compared with that seen in lenses from neonatal E7 mice. In adults E6 x E7 double transgenic mice, lens tumors developed, whereas in E6 or E7 only transgenic mice, tumors did not. Taken together, these results point to specific roles in lens morphogenesis for Rb and p53 and to the necessity of these tumor suppressor gene products in regulating exit from the normal cell division cycle in differentiating lens fiber cells.
Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair.
Authors: Harrington J J , Lieber M R
Published: June 2007

Structure-specific nucleases catalyze critical reactions in DNA replication, recombination, and repair. Recently, a structure-specific endonuclease, FEN-1, has been purified and shown to cleave DNA flap structures. Here, we describe the cloning of the murine FEN-1 gene. The nucleotide sequence of FEN-1 is highly homologous to the Saccharomyces cerevisiae genes YKL510 and RAD2. We show that YKL510 and a truncated RAD2 protein are also structure-specific endonucleases. The substrate specificity of the truncated RAD2 protein implicates branched DNA structures as important intermediates in nucleotide excision repair. The polarity of these branched DNA structures allows us to predict the placement of DNA scissions by RAD2 and RAD1/RAD10 in this reaction.
The yeast homolog of H < beta > 58, a mouse gene essential for embryogenesis, performs a role in the delivery of proteins to the vacuole.
Authors: Bachhawat A K , Suhan J , Jones E W
Published: June 2007

The highly conserved and ubiquitously expressed mouse gene H < beta > 58, identified through insertional mutagenesis, has been shown to be essential for early postimplantation development in mouse, but the mechanism by which it acts is unknown (Radice et al. 1991; Lee et al. 1992). We report here the identification of a yeast gene related to the H < beta > 58 gene and provide biochemical and genetic evidence for its function within the cell. The gene, PEP8, plays a role in the delivery of proteins to the vacuole. Disruption of the gene did not affect cell viability. However, the disruptants were shown to have a defect in the processing of the soluble vacuolar proteases but not the membrane vacuolar hydrolases. The processing defect appeared to be a consequence of the inability of the soluble vacuolar hydrolase to reach the vacuole. Although a small amount of the vacuolar precursors was mis-sorted to the extracellular medium, mis-sorting did not appear to be the primary defect in these cells. Pep8p was identified by epitope tagging of the protein. Biochemical fractionation indicated that the protein was peripherally bound to membranes. Immuno-gold electron microscopy indicated that the Pep8p localized to vacuolar membranes. Complementation experiments with the mouse H < beta > 58 cDNA revealed that a Pep8p-H < beta > 58 fusion protein in which the carboxy-terminal 85 amino acids of Pep8p were replaced by the carboxy-terminal 115 amino acids of H < beta > 58 was functional.(ABSTRACT TRUNCATED AT 250 WORDS)
Net, a new ets transcription factor that is activated by Ras.
Authors: Giovane A , Pintzas A , Maira S M , Sobieszczuk P , Wasylyk B
Published: June 2007

Ras signaling appears to be mediated in part by transcription factors that belong to the ets gene family. To identify downstream targets for the Ras signal transduction pathway, we have used Ras-transformed mouse fibroblasts to isolate a new member of the ets gene family, net. Net has sequence similarity in three regions with the ets factors Elk1 and SAP1, which have been implicated in the serum response of the fos promoter. Net shares various properties with these proteins, including the ability to bind to ets DNA motifs through the Ets domain of the protein and form ternary complexes with the serum response factor SRF on the fos serum response element, SRE. However, Net differs from Elk1 and SAP1 in a number of ways. The pattern of net RNA expression in adult mouse tissues is different. Net has negative effects on transcription in a number of assays, unlike Elk1. Strikingly, Ras, Src, and Mos expression switch Net activity to positive. The study of Net should help in understanding the interplay between Net and other members of the Elk subfamily and their contribution to signal transduction through Ras to the nucleus.
Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.
Authors: Coetzee T , Herschlag D , Belfort M
Published: June 2007

To address the effect of host proteins on the self-splicing properties of the group I introns of bacteriophage T4, we have purified an activity from Escherichia coli extracts that facilitates both trans- and cis-splicing of the T4 introns in vitro. The activity is attributable to a number of proteins, several of which are ribosomal proteins. Although these proteins have variable abilities to stimulate splicing, ribosomal protein S12 is the most effective. The activity mitigates the negative effects on splicing of the large internal open reading frames (ORFs) common to the T4 introns. In contrast to proteins shown previously to facilitate group I splicing, S12 does not bind strongly or specifically to the intron. Rather, S12 binds RNA with broad specificity and can also facilitate the action of a hammerhead ribozyme. Addition of S12 to unreactive trans-splicing precursors promoted splicing, suggesting that S12 can resolve misfolded RNAs. Furthermore, incubation with S12 followed by its proteolytic removal prior to the initiation of the splicing reaction still resulted in splicing enhancement. These results suggest that this protein facilitates splicing by acting as an RNA chaperone, promoting the assembly of the catalytically active tertiary structure of ribozymes.
A splicing enhancer exhibits both constitutive and regulated activities.
Authors: Tian M , Maniatis T
Published: June 2007

The Drosophila proteins Transformer (Tra) and Transformer2 (Tra2) regulate the sex-specific alternative splicing of Drosophila doublesex (dsx) pre-mRNA by specifically binding to a splicing enhancer (dsx repeat element; dsxRE) located 300 nucleotides (nt) downstream from a female-specific 3' splice site. In this paper we show that the dsxRE can function as a Tra and Tra2-independent splicing enhancer in vitro when located within 150 nucleotides of the 3' splice site. Based on the relative levels of SR proteins that bind stably to the dsxRE in the presence or absence of Tra and Tra2, we propose that the constitutive splicing activity of the dsxRE is mediated by its weak interactions with SR proteins and possibly other general splicing factors. In contrast, Tra and Tra2 allow the dsxRE to function at a distance from the intron by stabilizing the interactions between these proteins and the dsxRE.
Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion.
Authors: Luo L , Liao Y J , Jan L Y , Jan Y N
Published: June 2007

The small GTPases of the Rac/Rho/Cdc42 subfamily are implicated in actin cytoskeleton-membrane interaction in mammalian cells and budding yeast. The in vivo functions of these GTPases in multicellular organisms are not known. We have cloned Drosophila homologs of rac and CDC42, Drac1, and Dcdc42. They share 70% amino acid sequence identity with each other, and both are highly expressed in the nervous system and mesoderm during neuronal and muscle differentiation, respectively. We expressed putative constitutively active and dominant-negative Drac1 proteins in these tissues. When expressed in neurons, Drac1 mutant proteins cause axon outgrowth defects in peripheral neurons without affecting dendrites. When expressed in muscle precursors, they cause complete failure of, or abnormality in, myoblast fusion. Expressions of analogous mutant Dcdc42 proteins cause qualitatively distinct morphological defects, suggesting that similar GTPases in the same subfamily have unique roles in morphogenesis.
A sigma 54 transcriptional activator also functions as a pole-specific repressor in Caulobacter.
Authors: Wingrove J A , Gober J W
Published: June 2007

The differential localization of proteins in the Caulobacter predivisional cell leads to the formation of two distinct progeny cells: a motile swarmer cell and a sessile stalked cell. Pole-specific transcription in the predivisional cell is one mechanism responsible for protein localization. Here we show that the sigma 54 transcriptional activator FlbD, which activates swarmer pole-specific transcription of a subset of late flagellar genes, is also capable of functioning as a pole-specific repressor of the early flagellar fliF operon. DNase I footprinting and methylation interference assays indicate that FlbD binds to regions of the fliF promoter at regions that would be likely to interfere with the binding of RNA polymerase. A mutation that abolishes FlbD binding results in up to a fourfold increase in fliF promoter expression. This mutation alters both the spatial and temporal pattern of fliF expression resulting in the inappropriate expression of the fliF operon in the swarmer pole of the predivisional cell. These results demonstrate that FlbD represses early flagellar gene expression in the swarmer pole of the Caulobacter predivisional cell. This is the first instance in which a protein specifically involved in pole-specific repression has been identified in Caulobacter. The restriction of FlbD activity to the swarmer pole accomplishes two regulatory missions by simultaneously activating late flagellar gene expression and repressing early flagellar genes.
Targeted mutations in the Caenorhabditis elegans POU homeo box gene ceh-18 cause defects in oocyte cell cycle arrest, gonad migration, and epidermal differentiation.
Authors: Greenstein D , Hird S , Plasterk R H , Andachi Y , Kohara Y , Wang B , Finney M , Ruvkun G
Published: June 2007

We used targeted gene inactivation to analyze the function of a Caenorhabditis elegans POU gene, ceh-18, and to dissect its functional domains in vivo. In ceh-18 mutants, oocytes exhibit an incompletely penetrant failure to arrest in diakinesis of meiotic prophase I and instead undergo multiple rounds of DNA replication without cytokinesis. ceh-18 is expressed in the gonadal sheath cells that signal the oocyte, but not in the oocyte. This suggests that ceh-18 affects, directly or indirectly, a sheath cell signal that causes oocytes to maintain diakinesis arrest. ceh-18 also participates in directing gonad migration and in specifying the differentiated phenotypes of epidermal cells during postembryonic development. Analysis of targeted deletions that disrupt half of the POU domain selectively by deleting either the POUhd or the POUsp alone, indicates that each CEH-18 POU subdomain is sufficient for partial activity in vivo.
Telomerase RNAs of different ciliates have a common secondary structure and a permuted template.
Authors: Lingner J , Hendrick L L , Cech T R
Published: June 2007

Telomerase is composed of protein and RNA. The RNA serves as a template for telomere DNA synthesis and may also be important for enzyme structure or catalysis. We have used the presence of conserved sequence elements in the promoter and template regions to amplify by PCR the telomerase RNA genes from six different hypotrichous ciliates: Oxytricha nova, Oxytricha trifallax, Stylonychia mytilis, Stylonychia lemnae, Euplotes aediculatus, and Euplotes eurystomus. RNaseH cleavage of the O. nova RNA in extracts by use of a complementary oligonucleotide leads to loss of telomerase activity, supporting the identification. Primary sequence and biochemical experiments suggest that the templates of Oxytricha and Stylonychia are circularly permuted relative to that of E. aediculatus. On the basis of the pause sites, the former two add G4T4 during a single primer elongation cycle, whereas E. aediculatus adds G3T4G. The only primary sequence element outside the template that is conserved between these phylogenetically distant telomerase RNAs is the sequence 5'-(C)UGUCA-3', which precedes the template regions by exactly two bases. We propose a common secondary structure model that is based on nucleotide covariations, a model which resembles that proposed previously for tetrahymenine telomerase RNAs.
Dual roles of the retinoblastoma protein in cell cycle regulation and neuron differentiation.
Authors: Lee E Y , Hu N , Yuan S S , Cox L A , Bradley A , Lee W H , Herrup K
Published: June 2007

To assess the functions of the retinoblastoma protein (RB) during normal development, we have analyzed mouse embryos that lack a functional copy of the retinoblastoma gene (genotype: Rb-1 delta 20/Rb-1 delta 20). Our findings demonstrate that RB plays an important role in the regulation of the neuronal cell cycle. In mutant embryos, dividing cells are found well outside of the normal neurogenic regions in both the central and peripheral nervous systems. In addition to abnormal cell cycle regulation, however, the mutant embryos show two less expected phenotypes. First, many of the ectopically dividing cells die by apoptosis shortly after their entrance into S phase. In sensory ganglia, most nerve cells die by this process, beginning at about the same time as normal target-related neuronal death. Second, although the expression of certain differentiation markers such as N-CAM and Brn-3.0 appears to be near normal, nerve cells, especially in sensory ganglia, do not mature properly. Their morphology is stunted and expression of neuronal beta II tubulin is greatly reduced. Preferential reduction in the expression of TrkA, TrkB, and the low-affinity neurotrophin receptor p75LNGFR may be relevant to neuronal cell death and lack of neuronal differentiation seen in the mutant embryos. Primary cultures of dorsal root and trigeminal ganglion cells from later stage mutant embryos reveal a decrease in neuronal cell survival and in neurite outgrowth even in the presence of the appropriate neurotrophins. Taken together, these results suggest that the p110RB protein not only regulates progression through the cell cycle but is also important for cell survival and differentiation.
Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling.
Authors: Singson A , Leviten M W , Bang A G , Hua X H , Posakony J W
Published: June 2007

In Drosophila imaginal discs, the spatially restricted activities of the achaete (ac) and scute (sc) proteins, which are transcriptional activators of the basic-helix-loop-helix class, define proneural clusters (PNCs) of potential sensory organ precursor (SOP) cells. Here, we report the identification of several genes that are direct downstream targets of ac-sc activation, as judged by the following criteria. The genes are expressed in the PNCs of the wing imaginal disc in an ac-sc-dependent manner; the proximal promoter regions of all of these genes contain one or two high-affinity ac-sc binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY; where tested, these binding sites are required in vivo for PNC expression of promoter-reporter fusion genes. Interestingly, these ac-sc target genes, including Bearded, Enhancer of split m7, Enhancer of split m8, and scabrous, are all known or believed to function in the selection of a single SOP from each PNC, a process mediated by inhibitory cell-cell interactions. Thus, one of the earliest steps in adult peripheral neurogenesis is the direct activation by proneural proteins of genes involved in restricting the expression of the SOP cell fate.
The Sex-lethal amino terminus mediates cooperative interactions in RNA binding and is essential for splicing regulation.
Authors: Wang J , Bell L R
Published: June 2007

Sex-lethal (Sxl) acts as a binary switch that regulates Drosophila sexual differentiation and dosage compensation and also maintains a stable female state through autoregulation. As part of a cascade of genes that are regulated by sex-specific splicing, Sxl controls the sex-specific splicing of transformer (tra) RNA and also its own RNA. Sxl contains two RNP-CS (RNA-binding) domains and is known to bind tra pre-mRNA near the alternative 3' splice site, thus blocking use of that site to give the female-specific splicing pattern. Here, we test how Sxl protein interacts with Sxl RNA during autoregulation. We show that Sxl not only binds Sxl pre-mRNA near the alternative 3' splice site but also at distant, multiple sites surrounding the Sxl alternative exon. Moreover, Sxl binds cooperatively at these multiple sites. The Sxl amino terminus is essential for the cooperative interaction and is also required for regulatory activity in vivo. It appears that this region of Sxl protein, which resembles regions in some other RNA-binding proteins, is a domain that mediates protein-protein interactions during RNA binding and plays an important role in splicing regulation.
Germ cell-less encodes a cell type-specific nuclear pore-associated protein and functions early in the germ-cell specification pathway of Drosophila.
Authors: Jongens T A , Ackerman L D , Swedlow J R , Jan L Y , Jan Y N
Published: June 2007

The maternally supplied plasm at the posterior pole of a Drosophila embryo contains determinants that specify both the germ-cell precursors (pole cells) and the posterior axis. One pole plasma component, the product of the germ cell-less gene, has been found to be required for specification of pole cells, but not posterior somatic cells. Mothers with reduced levels of gcl give rise to progeny that lack pole cells, but are otherwise normal. Mothers overexpressing gcl, on the other hand, produce progeny exhibiting a transient increase of pole cells. Ectopic localization of gcl to the anterior pole of the embryo causes nuclei at that location to adopt characteristics of pole cell nuclei, with concurrent loss of somatic cells. We also present evidence indicating that the gcl protein associates specifically with the nuclear pores of the pole cell nuclei. This localization suggests a novel mechanism in the specification of cell fate for the germ line.
SMK1, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cerevisiae.
Authors: Krisak L , Strich R , Winters R S , Hall J P , Mallory M J , Kreitzer D , Tuan R S , Winter E
Published: June 2007

Mitogen-activated protein (MAP) kinases comprise a family of conserved, eukaryotic enzymes that mediate responses to a wide variety of extracellular stimuli. In yeast, different signal transduction pathways utilize distinct MAP kinase family members. We have identified a new yeast MAP kinase gene (named SMK1) that is required for the completion of sporulation. Molecular and cytologic markers indicate that meiotic development proceeds normally in homozygous smk1-delta 1 diploids through meiosis II. However, light and electron microscopy show that smk1 asci are defective in organizing spore wall assembly. Consistent with a defect in spore wall assembly, smk1-delta 1 mutant asci display enhanced sensitivities to enzymatic digestion, heat shock, and exposure to ether. SMK1 mRNA, which is not detectable in vegetative cells, is derepressed at least 200-fold just prior to prospore enclosure. We propose that the SMK1 MAP kinase participates in a developmentally regulated signal transduction pathway that coordinates cytodifferentiation events with the transcriptional program.
Phytochrome signal transduction pathways are regulated by reciprocal control mechanisms.
Authors: Bowler C , Yamagata H , Neuhaus G , Chua N H
Published: June 2007

Three signal transduction pathways, dependent on cGMP and/or calcium, are utilized by phytochrome to control the expression of genes required for chloroplast development and anthocyanin biosynthesis in plant cells. For example, chs is controlled by a cGMP-dependent pathway, cab is controlled by a calcium-dependent pathway, and fnr is regulated by a pathway that requires both cGMP and calcium. Using a soybean photomixotrophic cell culture and microinjection into the cells of a phytochrome-deficient tomato mutant, we have studied the regulatory mechanisms acting within and between these three signaling pathways. We provide evidence that changes in cGMP levels mediate the observed induction and desensitization of chs gene expression in response to light and demonstrate that high cGMP concentrations cause negative regulation of both the calcium- and the calcium/cGMP-dependent pathways. Conversely, high activity of the calcium-dependent pathway can negatively regulate the cGMP-dependent pathway. We have termed these opposing regulatory mechanisms reciprocal control. In all cases, the molecules that are involved appear to be downstream components of the signal transduction pathways, rather than calcium and cGMP themselves. Furthermore, we have found that the calcium/cGMP-dependent pathway has a lower requirement for cGMP than does the cGMP-dependent pathway. The role of these phenomena in the regulation of plant photoresponses is discussed.
Long-term genome stability and minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-, immortalized human uroepithelial cells.
Authors: Reznikoff C A , Belair C , Savelieva E , Zhai Y , Pfeifer K , Yeager T , Thompson K J , DeVries S , Bindley C , Newton M A
Published: June 2007

Parameters of genome instability and morphological alterations associated with cell transformation were studied in an isogeneic set of clonal human uroepithelial cell (HUC) lines immortalized by the human papilloma virus 16 (HPV16) E6 and/or E7 gene(s). HPV16 E6 binds p53, leading to rapid degradation of p53, whereas E7 binds and alters pRb and other proteins. We report that two independent E7-immortalized HUC lines showed minimal phenotypic or genotypic alterations, except that both lines contained amplification of 20q DNA sequences and a greater polyploidization at an early passage. The E7-immortalized HUC line resembled normal HUC lines, except that they failed to senesce. In contrast, the E6-immortalized HUC lines were morphologically altered, contained numerous random chromosome aberrations, and showed unstable evolving karyotypes with passage in culture. No amplified DNA sequences were detected in E6-immortalized HUC lines. Instead, clonal losses of chromosome regions (i.e., -3p, -6q, -9p), putatively containing tumor suppressor or senescence genes, accompanied the E6-HUC immortalization event. E6-immortalized HUC lines showed transformed phenotypes similar to E6/E7-HUC lines. The difference in genome stability between E6- and E7-immortalized HUC was highly significant statistically (p-value < 10(-6). Thus, the HPV16 E7 gene led to HUC immortalization by a pathway that blocked cellular senescence, but did not disrupt genome stability. These results implicate p53 loss, but not pRb alteration, in genome destabilization.
Sequence and structural elements critical for U8 snRNP function in Xenopus oocytes are evolutionarily conserved.
Authors: Peculis B A , Steitz J A
Published: June 2007

We have generated mutants in Xenopus U8 RNA, a nucleolar snRNA required for the maturation of 5.8S and 28S rRNAs, to identify sequences and structural domains essential for RNA stability, particle assembly, and function of the U8 RNP. Activity of the mutants was assayed by microinjection of in vitro-synthesized U8 RNAs into the cytoplasm of Xenopus oocytes. Most of the mutant RNAs were stable, bound fibrillarin, a protein common to several of the nucleolar-specific snRNPs, and became hypermethylated. Although hypermethylation of the 5' cap of U8 RNA and fibrillarin binding can occur in either the cytoplasmic or nuclear compartment of Xenopus oocytes, neither is required for nuclear import. We find that the trimethylguanosine cap, although present on the endogenous U8 RNA, is not essential for stability, particle assembly, or functioning of U8 in the coordinate processing of pre-rRNA at sites 3' of 28S and 5' of 5.8S RNA. Several conserved single- and double-stranded sequences within the 5' domain of U8 RNA are essential for function.
Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1.
Authors: Moretti P , Freeman K , Coodly L , Shore D
Published: June 2007

The maintenance of transcriptional silencing at HM mating-type loci and telomeres in yeast requires the SIR2, SIR3, and SIR4 proteins, none of which appear to be DNA-binding proteins. Here we show that SIR3 and SIR4 interact with a carboxy-terminal domain of the silencer, telomere, and UAS-binding protein RAP1. We identified SIR3 and SIR4 in a two-hybrid screen for RAP1-interacting factors and showed that SIR3 interacts both with itself and with SIR4. The interaction between RAP1 and SIR3 can be observed in vitro in the absence of other yeast proteins. Consistent with the notion that native SIR proteins interact with the RAP1 carboxyl terminus, we show that mutation of the endogenous SIR3 and SIR4 genes increases transcriptional activation by LexA/RAP1 hybrids. To test the importance of the RAP1-SIR3 interaction for silencing, we identified mutations in the RAP1 carboxyl terminus that either diminish or abolish this interaction. When introduced into the native RAP1 protein, these mutations cause corresponding defects in silencing at both HMR and telomeres. We propose that RAP1 acts in the initiation of transcriptional silencing by recruiting a complex of SIR proteins to the chromosome via protein-protein interactions. These data are consistent with a model in which SIR3 and SIR4 play a structural role in the maintenance of silent chromatin and indicate that their action is initiated at the silencer itself.
RRN6 and RRN7 encode subunits of a multiprotein complex essential for the initiation of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae.
Authors: Keys D A , Vu L , Steffan J S , Dodd J A , Yamamoto R T , Nogi Y , Nomura M
Published: June 2007

Previously, we have isolated mutants of Saccharomyces cerevisiae primarily defective in the transcription of 35S rRNA genes by RNA polymerase I and have identified a number of genes (RRN genes) involved in this process. We have now cloned the RRN6 and RRN7 genes, determined their nucleotide sequences, and found that they encode proteins of calculated molecular weights of 102,000 and 60,300, respectively. Extracts prepared from rrn6 and rrn7 mutants were defective in in vitro transcription of rDNA templates. We used extracts from strains containing epitope-tagged wild-type Rrn6 or Rrn7 proteins to purify protein components that could complement these mutant extracts. By use of immunoaffinity purification combined with biochemical fractionation, we obtained a highly purified preparation (Rrn6/7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparent molecular weight of 66,000, but which did not contain the TATA-binding protein (TBP). This complex complemented both rrn6 and rrn7 mutant extracts. Template commitment experiments carried out with this purified Rrn6/7 complex and with rrn6 mutant extracts have demonstrated that the Rrn6/7 complex does not bind stably to the rDNA template by itself, but its binding is dependent on the initial binding of some other factor(s) and that the Rrn6/7 complex is required for the formation of a transcription-competent preinitiation complex. These observations are discussed in comparison to in vitro rDNA transcription systems from higher eukaryotes.
Evidence that the cis preference of the Tn5 transposase is caused by nonproductive multimerization.
Authors: Weinreich M D , Gasch A , Reznikoff W S
Published: June 2007

The transposase (Tnp) of the bacterial transposon Tn5 acts 50- to 100-fold more efficiently on elements located cis to the site of its synthesis compared with those located in trans. In an effort to understand the basis for this cis preference, we have screened for Tnp mutants that exhibit increased transposition activity in a trans assay. Two mutations in the carboxyl terminus were isolated repeatedly. The EK345 mutation characterized previously increases Tnp activity eightfold both in cis and in trans. The novel LP372 mutation, however, increases Tnp activity 10-fold specifically in trans. Combining both mutations increases Tnp activity 80-fold. Interestingly, the LP372 mutation maps to a region shown previously to be critical for interaction with Inh, an inhibitor of Tn5 transposition, and results in reduced inhibition activity by both Tnp and Inh. Tnp also inhibits Tn5 transposition in trans, and this has been suggested to occur by the formation of inactive Tnp multimers. Because Inh and (presumably) Tnp inhibit Tn5 transposition by forming defective multimers with Tnp, the inhibition defect of the trans-active LP372 mutant suggests that the cis preference of Tnp may also be attributable to nonproductive Tnp-Tnp multimerization. In addition, we show that increasing the synthesis of EK345/LP372 Tnp, but not wild-type Tnp, leads to very high levels of transposition, presumably because this altered Tnp is defective in the inhibitory activity of the wild type protein.
Region-specific activators of meiotic recombination in Schizosaccharomyces pombe.
Authors: DeVeaux L C , Smith G R
Published: June 2007

Schizosaccharomyces pombe rec mutants were previously isolated on the basis of their deficiency in meiotic recombination at the ade6 locus. We surveyed their meiotic recombination deficiencies at and between other loci. In rec10 mutants recombinant frequencies in the approximately 2-Mb region surrounding the ade6 locus were reduced 10- to 100-fold, but recombinant frequencies at or between nine other unlinked loci were reduced < 3-fold. The rec10 mutations are recessive and are on chromosome I; the ade6 region is on chromosome III. These results indicate that the rec10 gene product is required for activation of meiotic recombination in the approximately 2-Mb region surrounding ade6 but not in the other regions surveyed. Similar ade6 regional specificities were observed for rec8 and rec11. We infer that there are multiple activators of meiotic recombination, each specific for a limited set of loci, and we discuss how these regional activators may work.
Regulation of splicing at an intermediate step in the formation of the spliceosome.
Authors: Vilardell J , Warner J R
Published: June 2007

In vivo experiments have demonstrated that the ribosomal protein L32 of Saccharomyces cerevisiae brings about the inhibition of splicing of the transcript of its own gene through an RNA structure comprised largely of the first exon. We now show that L32, itself, binds specifically to this RNA. Splicing of the RPL32 transcript in vitro is blocked by the presence of L32. Furthermore, addition of the 75-nucleotide RNA representing the 5' end of the RPL32 transcript stimulates specifically the splicing of the RPL32 substrate, presumably by competing for L32 present in the extract. Use of RNAs carrying mutations shown to abolish the regulation of splicing, either as substrates or as competitors, confirmed that the in vitro reaction is a faithful representation of the situation in vivo. We conclude that the regulation of splicing occurs through the specific binding of L32 to an RNA structure within the first 75 nucleotides of the RPL32 transcript. The RPL32 substrate, bound to L32, forms a complex with U1 snRNP, the first step in spliceosome assembly. The presence of L32 prevents the ATP-dependent association of the U2 snRNP necessary to form a complete spliceosome.
Caenorhabditis elegans unc-51 gene required for axonal elongation encodes a novel serine/threonine kinase.
Authors: Ogura K , Wicky C , Magnenat L , Tobler H , Mori I , Müller F , Ohshima Y
Published: June 2007

Mutations in the unc-51 gene of the nematode Caenorhabditis elegans result in various abnormalities in axonal elongation and axonal structures. We cloned the unc-51 gene by tagging with the transposon Tc1. The wild-type unc-51 gene, which rescued the mutant phenotypes, encodes a novel serine/threonine kinase of 856 amino acids. Mutation sites were identified in the unc-51 gene of six mutants. A Lys-->Met mutation created in vitro in the kinase domain led to the loss of rescuing activity and was dominant negative, indicating that the kinase domain of Unc-51 is essential for the function. Expression of an unc-51/lacZ fusion gene was observed in many neurons at all stages. We propose that protein phosphorylation by the unc-51 product is important for axonal elongation and possibly for axonal guidance.