bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete transposition requires four active monomers in the mu transposase tetramer.
Authors: Baker T A , Kremenstova E , Luo L
Published: June 2007

A tetramer of Mu transposase (MuA) cleaves and joins multiple DNA strands to promote transposition. Derivatives of MuA altered at two acidic residues that are conserved among transposases and retroviral integrases form tetramers but are defective in both cleavage and joining. These mutant proteins were used to analyze the contribution of individual monomers to the activity of the tetramer. The performance of different protein combinations demonstrates that not all monomers need to be catalytically competent for the complex to promote an individual cleavage or joining reaction. Furthermore, the results indicate that each pair of essential residues is probably donated to the active complex by a single monomer. Although stable, tetramers composed of a mixture of mutant and wild-type MuA generate products cleaved at only one end and with only one end joined to the target DNA. The abundance of these abortive products and the ratios of the two proteins in complexes stalled at different steps indicate that the complete reaction requires the activity of all four monomers. Thus, each subunit of MuA appears to use the conserved acidic amino acids to promote one DNA cleavage or one DNA joining reaction.
DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts.
Authors: Di Leonardo A , Linke S P , Clarkin K , Wahl G M
Published: June 2007

The tumor suppressor p53 is a cell cycle checkpoint protein that contributes to the preservation of genetic stability by mediating either a G1 arrest or apoptosis in response to DNA damage. Recent reports suggest that p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase (Cdk)-inhibitor Cip1. Here, we characterize the p53-dependent G1 arrest in several normal human diploid fibroblast (NDF) strains and p53-deficient cell lines treated with 0.1-6 Gy gamma radiation. DNA damage and cell cycle progression analyses showed that NDF entered a prolonged arrest state resembling senescence, even at low doses of radiation. This contrasts with the view that p53 ensures genetic stability by inducing a transient arrest to enable repair of DNA damage, as reported for some myeloid leukemia lines. Gamma radiation administered in early to mid-, but not late, G1 induced the arrest, suggesting that the p53 checkpoint is only active in G1 until cells commit to enter S phase at the G1 restriction point. A log-linear plot of the fraction of irradiated G0 cells able to enter S phase as a function of dose is consistent with single-hit kinetics. Cytogenetic analyses combined with radiation dosage data indicate that only one or a small number of unrepaired DNA breaks may be sufficient to cause arrest. The arrest also correlated with long-term elevations of p53 protein, Cip1 mRNA, and Cip1 protein. We propose that p53 helps maintain genetic stability in NDF by mediating a permanent cell cycle arrest through long-term induction of Cip1 when low amounts of unrepaired DNA damage are present in G1 before the restriction point.
Homotypic and heterotypic protein associations control Rad51 function in double-strand break repair.
Authors: Donovan J W , Milne G T , Weaver D T
Published: June 2007

Rad51 is essential for efficient repair of DNA double-strand breaks (DSBs) and recombination in Saccharomyces cerevisiae. Here, we examine Rad51 protein-protein interactions and their biological significance. GAL4 two-hybrid fusion analysis demonstrated that the amino-terminal region of Rad51 mediates both a strong Rad51:Rad51 self-association and a Rad51:Rad52 interaction. Several Rad51 variants were characterized that imparted DSB repair defects; these defects appear to result from Rad51 protein-protein interactions. First, a rad51 allele bearing a missense mutation in the consensus ATP-binding sequence disrupted DSB repair in wild-type yeast. The effect of this allele was dependent on the presence of wild-type Rad51 because MMS sensitivity of rad51 delta strains were not increased by its expression. Second, we identified a highly conserved RAD51 homolog from Kluyveromyces lactis (KlRAD51) that only partially complemented rad51 delta strains and impaired DSB repair in wild-type S. cerevisiae. Third, fusions of Gal4 domains to Rad51 disrupted DSB repair in a manner that required the presence of either Rad51 or Rad52. Because K. lactis RAD51 and RAD52 did not complement a S. cerevisiae rad51 delta rad52 delta strain, Rad51-Rad52 functions appear to be mediated through additional components. Thus, multiple types of Rad51 protein interactions, including self-association, appear to be important for DSB repair.
A functional "knockout" of human keratin 14.
Authors: Rugg E L , McLean W H , Lane E B , Pitera R , McMillan J R , Dopping-Hepenstal P J , Navsaria H A , Leigh I M , Eady R A
Published: June 2007

The importance of keratins and other intermediate filaments in the maintenance of tissue structure is emphasized by the discovery that many hereditary skin-blistering diseases are caused by mutations in keratin genes. Here, we describe a situation in which keratin 14 (K14) is missing altogether in the epidermis: A homozygous 2-nucleotide deletion in exon I of the K14 gene causes premature termination of the mRNA transcripts upstream from the start of the rod domain and results in a K14 null phenotype. In this individual no keratin intermediate filaments are visible in basal epidermal cells, although filaments are present in the upper layers of the epidermis. No compensating keratin expression is detected in vivo, and K14 mRNA is down-regulated. The individual, diagnosed as Köbner (generalized) EBS, suffers from severe widespread keratinocyte fragility and blistering at many body sites, but although the phenotype is severe, it is not lethal. This K14-/- phenotype confirms that only one K14 gene is expressed in human epidermis and provides an important model system for examining the interdependence of different keratin filament systems and their associated structures in the skin.
Role of the AGA/AGG codons, the rarest codons in global gene expression in Escherichia coli.
Authors: Chen G T , Inouye M
Published: June 2007

AGA and AGG codons for arginine are the least used codons in Escherichia coli. Previous findings have shown that these codons are used preferentially within the first 25 codons in E. coli genes. More than 100 genes having a single AGA/AGG codon within the first 25 codons were identified to be associated with various essential cellular functions. The lacZ gene, containing 5 AGG codons after the tenth codon from the initiation codon, was constructed as a model system. The production of beta-galactosidase was inhibited almost completely during the stationary phase, whereas the production of the control beta-galactosidase without AGG codons was not. The inhibitory effect by the 5 AGG codons was substantially suppressed either by coexpressing the argU gene for tRNA(ArgUCU/CCU) or by moving the 5 AGG codons by > 50 codons away from the initiation codon. In addition, the production of a number of proteins resolved by two-dimensional gel electrophoresis was enhanced significantly during the stationary phase in the cells harboring a plasmid containing argU. At least one of them was identified as the hns product encoded by an ORF having an AGA codon at the nineteenth position. On the basis of these results, it is proposed that the expression of a group of essential genes for various cellular functions that have a single AGA/AGG codon very close to the initiation codon are globally regulated by the availability of the least abundant tRNA(ArgUCU/CCU). A model for this regulation is proposed.
E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo.
Authors: Beijersbergen R L , Kerkhoven R M , Zhu L , Carlée L , Voorhoeve P M , Bernards R
Published: June 2007

The E2F family of transcription factors controls the expression of genes that are involved in cell cycle regulation. E2F DNA-binding activity is found in complex with the retinoblastoma protein, pRb, and with the pRb-related p107 and p130. To date, cDNAs for three members of the E2F gene family have been isolated. However, all three E2Fs associate in vivo exclusively with pRb. We report here the cloning and functional analysis of a fourth E2F family member. E2F-4 encodes a 413-amino-acid protein with significant homology to E2F-1. E2F-4 antibodies recognize a 60-kD protein in anti-p107 immunoprecipitates, indicating that E2F-4 associates with p107 in vivo. Like the other E2Fs, E2F-4 requires DP-1 for efficient DNA binding and transcriptional activation of E2F site-containing promoters. Increased expression of E2F-4 and DP-1 in SaoS-2 osteosarcoma cells causes a shift from G1-phase cells to S and G2/M-phase cells, suggesting a role for E2F-4 in regulation of cell-cycle progression. We show that expression of E2F-4 and DP-1 together with an activated ras oncogene in rat embryo fibroblasts, causes transformation, indicating that E2F-4 has oncogenic activity.
Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice.
Authors: van Genderen C , Okamura R M , Fariñas I , Quo R G , Parslow T G , Bruhn L , Grosschedl R
Published: June 2007

Lymphoid enhancer factor 1 (LEF-1) is a sequence-specific DNA-binding protein that is expressed in pre-B and T lymphocytes of adult mice, and in the neural crest, mesencephalon, tooth germs, whisker follicles, and other sites during embryogenesis. We have generated mice carrying a homozygous germ-line mutation in the LEF-1 gene that eliminates its protein expression and causes postnatal lethality. The mutant mice lack teeth, mammary glands, whiskers, and hair but show no obvious defects in lymphoid cell populations at birth. The LEF-1-deficient mice also lack the mesencephalic nucleus of the trigeminal nerve, the only neural crest-derived neuronal populations. Together, the pattern of these defects suggest an essential role for LEF-1 in the formation of several organs and structures that require inductive tissue interactions.
SR proteins can compensate for the loss of U1 snRNP functions in vitro.
Authors: Tarn W Y , Steitz J A
Published: June 2007

SR proteins are essential splicing factors that also influence 5' splice site choice. We show that addition of excess mixed SR proteins to a HeLa in vitro splicing system stimulates utilization of a novel 5' splice site (site 125) within the intron of the standard adenovirus pre-mRNA substrate. When U1 snRNPs are debilitated by sequestering the 5' end of U1 snRNA with a 2'-O-methyl oligoribonucleotide, excess SR proteins not only rescue splicing at the normal site and site 125 but also activate yet another 5' splice site (site 47) in the adenovirus intron. One SR protein, SC35, is sufficient to exhibit the above activities. The possibility that excess SR proteins recruit residual unblocked U1 snRNPs to participate in 5' splice site recognition has been ruled out by psoralen cross-linking studies, which demonstrate that the 2'-O-methyl oligoribonucleotide effectively blocks 5' splice site/U1 interaction. Native gel analysis reveals a nearly normal splicing complex profile in the 2'-O-methyl oligoribonucleotide pretreated, SR protein-supplemented extract. These results indicate that SR proteins can replace some functions of the U1 snRNP but underscore the contribution of U1 to the fidelity of 5' splice site selection.
Negative regulation of proneural gene activity: hairy is a direct transcriptional repressor of achaete.
Authors: Van Doren M , Bailey A M , Esnayra J , Ede K , Posakony J W
Published: June 2007

hairy (h) acts as a negative regulator in both embryonic segmentation and adult peripheral nervous system (PNS) development in Drosophila. Here, we demonstrate that h, a basic-helix-loop-helix (bHLH) protein, is a sequence-specific DNA-binding protein and transcriptional repressor. We identify the proneural gene achaete (ac) as a direct downstream target of h regulation in vivo. Mutation of a single, evolutionarily conserved, high-affinity h binding site in the upstream region of ac results in the appearance of ectopic sensory organs in adult flies, in a pattern that strongly resembles the phenotype of h mutants. This indicates that direct repression of ac by h plays an essential role in pattern formation in the PNS. Our results demonstrate that HLH proteins negatively regulate ac transcription by at least two distinct mechanisms.
cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis.
Authors: Gold S , Duncan G , Barrett K , Kronstad J
Published: June 2007

The fungal pathogen Ustilago maydis exhibits a dimorphic switch from budding to filamentous growth in response to mating interactions and environmental conditions. We have found that disruption of the uac1 gene, encoding adenylate cyclase, results in a constitutively filamentous phenotype. Budding is restored to the uac1 mutant upon growth in the presence of cAMP or by extragenic suppression because of a mutation in the ubc1 gene. The ubc1 gene encodes a type II regulatory subunit of cAMP-dependent protein kinase (PKA); defects in this gene attenuate the filamentous growth that normally occurs in response to mating and exposure to air. Growth of wild-type cells in cAMP and mutation of the ubc1 gene also cause defects in the separation of mother and daughter cells (cytokinesis) and alter bud site selection. These results indicate a key role for cAMP and PKA in morphogenesis in U. maydis; this role may be common among dimorphic fungal pathogens.
TFIIF-TAF-RNA polymerase II connection.
Authors: Henry N L , Campbell A M , Feaver W J , Poon D , Weil P A , Kornberg R D
Published: June 2007

RNA polymerase transcription factor IIF (TFIIF) is required for initiation at most, if not all, polymerase II promoters. We report here the cloning and sequencing of genes for a yeast protein that is the homolog of mammalian TFIIF. This yeast protein, previously designated factor g, contains two subunits, Tfg1 and Tfg2, both of which are required for transcription, essential for yeast cell viability, and whose sequences exhibit significant similarity to those of the mammalian factor. The yeast protein also contains a third subunit, Tfg3, which is less tightly associated and at most stimulatory to transcription, dispensable for cell viability, and has no known counterpart in mammalian TFIIF. Remarkably, the TFG3 gene encodes yeast TAF30, and furthermore, is identical to ANC1, a gene implicated in actin cytoskeletal function in vivo (Welch and Drubin 1994). Tfg3 is also a component of the recently described mediator complex (Kim et al. 1994), whose interaction with the carboxy-terminal repeat domain of RNA polymerase II enables transcriptional activation. Deletion of TFG3 results in diminished transcription in vivo.
Conserved functional domains of the RNA polymerase III general transcription factor BRF.
Authors: Khoo B , Brophy B , Jackson S P
Published: June 2007

In Saccharomyces cerevisiae, two components of the RNA polymerase III (Pol III) general transcription factor TFIIIB are the TATA-binding protein (TBP) and the B-related factor (BRF), so called because its amino-terminal half is homologous to the Pol II transcription factor IIB (TFIIB). We have cloned BRF genes from the yeasts Kluyveromyces lactis and Candida albicans. Despite the large evolutionary distance between these species and S. cerevisiae, the BRF proteins are conserved highly. Although the homology is most pronounced in the amino-terminal half, conserved regions also exist in the carboxy-terminal half that is unique to BRF. By assaying for interactions between BRF and other Pol III transcription factors, we show that it is able to bind to the 135-kD subunit of TFIIIC and also to TBP. Surprisingly, in addition to binding the TFIIB-homologous amino-terminal portion of BRF, TBP also interacts strongly with the carboxy-terminal half. Deleting two conserved regions in the BRF carboxy-terminal region abrogates this interaction. Furthermore, TBP mutations that selectively inhibit Pol III transcription in vivo impair interactions between TBP and the BRF carboxy-terminal domain. Finally, we demonstrate that BRF but not TFIIB binds the Pol III subunit C34 and we define a region of C34 necessary for this interaction. These observations provide insights into the roles performed by BRF in Pol III transcription complex assembly.
Regulation of rDNA transcription in chloroplasts: promoter exclusion by constitutive repression.
Authors: Iratni R , Baeza L , Andreeva A , Mache R , Lerbs-Mache S
Published: June 2007

Spinach chloroplasts contain two types of RNA polymerases. One is multimeric and Escherichia coli-like. The other one is not E. coli-like and might represent a monomeric enzyme of 110 kD. The quantitative relation of the two polymerases changes during plant development. This raises the question, how are plastid genes transcribed that contain E. coli-like and non-E. coli-like promoter elements during developmental phases when both enzymes are present? Transcription of the spinach plastid rrn operon promoter is initiated at three sites: P1, PC, and P2. P1 and P2 are preceded by E. coli-like promoter elements that are recognized by E. coli RNA polymerase in vitro. However, in vivo, transcription starts exclusively at PC. We analyzed different promoter constructions using in vitro transcription and gel mobility-shift studies to understand why P1 and P2 are not used in vivo. Our results suggest that the sequence-specific DNA-binding factor CDF2 functions as a repressor for transcription initiation of the E. coli-like enzyme at P1 and P2. We propose a mechanism of constitutive repression to keep the rrn operon in all developmental phases under the transcriptional control of the non-E. coli-like RNA polymerase.
Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function.
Authors: Guan K L , Jenkins C W , Li Y , Nichols M A , Wu X , O'Keefe C L , Matera A G , Xiong Y
Published: June 2007

The D-type cyclin-dependent kinases CDK4 and CDK6 are complexed with many small cellular proteins (p14, p15, p16, p18, and p20). We have isolated cDNA sequences corresponding to the MTS2 genomic fragment that encodes the CDK4- and CDK6-associated p14 protein. By use of a yeast interaction screen to search for CDK6-interacting proteins, we have also identified an 18-kD human protein, p18, that is a homolog of the cyclin D-CDK4 inhibitors p16 (INK4A/MTS1) and p14 (MTS2/INK4B). Both in vivo and in vitro, p18 interacts strongly with CDK6, weakly with CDK4, and exhibits no detectable interaction with the other known CDKs. Recombinant p18 inhibits the kinase activity of cyclin D-CDK6. Distinct from the p21/p27 family of CDK inhibitors that form ternary complexes with cyclin-CDKs, only binary complexes of p14, p16, and p18 were found in association with CDK4 and/or CDK6. Ectopic expression of p18 or p16 suppresses cell growth with a correlated dependence on endogenous wild-type pRb.
Colorectal hyperplasia and inflammation in keratin 8-deficient FVB/N mice.
Authors: Baribault H , Penner J , Iozzo R V , Wilson-Heiner M
Published: June 2007

We report that keratin 8 (mK8) gene disruption causes colorectal hyperplasia in FVB/N mice. The intestinal lesions affect uniformly the cecum, colon, and rectum but not the small intestine. The elongation of the crypts is accompanied by an inflammation of the lamina propria and submucosa. Hepatic, renal, and pancreatic functions tested in clinical assays are within nonpathological range, suggesting that the major defect lies in colonic epithelial cells. Still, small but consistent elevation in the hepatic enzymes alanine (AST) and asparate (ALT) aminotransferase are observed, along with a 70% increase in spleen weight. No homozygous mouse line has been established, because of a markedly reduced fertility of the mK8-/- females. Previously, we reported that the mK8- targeted mutation causes embryonic lethality in (C57B1/6x129Sv) mice. This strong effect of the genetic background on the mK8- mutant phenotype emphasizes the importance of using several inbred mouse strains to reveal the polygenic contribution to mutant phenotypes. Our results demonstrate that genetic modifiers of K8/K18 filament functions, with profound effects on embryogenesis and gut functional integrity, are differentially active in the FVB/N and C57B1/6 genetic backgrounds. More importantly, the increase in mK8-/- gut epithelial cell number, rather than cell disruption, contrasts with the known function of epidermal keratins in providing mechanical strength.
Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth.
Authors: Roberts R L , Fink G R
Published: June 2007

Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from a colonial form of growth to a filamentous pseudohyphal form. This dimorphism requires a polar budding pattern and elements of the MAP kinase signal transduction pathway essential for mating pheromone response in haploids. We report here that haploid strains exhibit an invasive growth behavior with many similarities to pseudohyphal development, including filament formation and agar penetration. Haploid filament formation depends on a switch from an axial to a bipolar mode of bud site selection. Filament formation is distinct from agar penetration in both haploids and diploids. We find that the same components of the MAP kinase cascade necessary for diploid pseudohyphal development (STE20, STE11, STE7, and STE12) are also required for both filament formation and agar penetration in haploids. Thus, haploid yeast cells can enter either of two developmental pathways: mating or invasive growth, both of which depend on elements of a single MAP kinase cascade. Our results provide a novel developmental model to study the dynamics of signal transduction, with implications for higher eukaryotes.
Ribonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA.
Authors: Alifano P , Rivellini F , Piscitelli C , Arraiano C M , Bruni C B , Carlomagno M S
Published: June 2007

The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized rne-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.
fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation.
Authors: Yamaguchi T P , Harpal K , Henkemeyer M , Rossant J
Published: June 2007

Experiments in amphibians have implicated fibroblast growth factors (FGFs) in the generation and patterning of mesoderm during embryogenesis. We have mutated the gene for fibroblast growth factor receptor 1 (fgfr-1) in the mouse to genetically dissect the role of FGF signaling during development. In the absence of fgfr-1 signaling, embryos displayed early growth defects; however, they remained capable of gastrulating and generating mesoderm. The nascent mesoderm of fgfr-1 homozygous mutant embryos differentiated into diverse mesodermal subtypes, but mesodermal patterning was aberrant. Somites were never generated and axial mesoderm was greatly expanded at the expense of paraxial mesoderm. These results suggest that FGFR-1 transduces signals that specify mesodermal cell fates and regional patterning of the mesoderm during gastrulation.
Retinoid-dependent in vitro transcription mediated by the RXR/RAR heterodimer.
Authors: Valcárcel R , Holz H , Jiménez C G , Barettino D , Stunnenberg H G
Published: June 2007

The effects of retinoids on gene regulation are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Here, we provide the first biochemical evidence that, in vitro, ligand governs the transcriptional activity of RXR alpha/RAR alpha by inducing conformational changes in the ligand-binding domains. Using limited proteolytic digestion we show that binding of the cognate ligand causes a conformational change in the carboxy-terminal part of the receptor. We also show that recombinant RXR alpha/RAR alpha is partially active in the absence of exogenously added ligand. Trans-activation depends critically on the ligand-dependent transcriptional activation function AF-2 of RAR alpha. Full activation by recombinant RXR alpha/RAR alpha, however, requires the addition of either all-trans RA, 9-cis RA, or other RAR-specific agonists, whereas an RAR alpha-specific antagonist abolishes trans-activation. Intriguingly, the ligand-dependent AF-2 of RXR does not contribute to the level of transcription from the RAR beta 2 promoter in vitro even when the cognate ligand (9-cis RA) is bound. Thus, the major role of RXR in trans-activation of the RAR beta 2 promoter is to serve as an auxiliary factor required for the binding of RAR which, in turn, is directly responsible for transcriptional activity.
Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene.
Authors: Karaplis A C , Luz A , Glowacki J , Bronson R T , Tybulewicz V L , Kronenberg H M , Mulligan R C
Published: June 2007

The parathyroid hormone-related peptide (PTHrP) gene was disrupted in murine embryonic stem cells by homologous recombination, and the null allele was introduced into the mouse germ line. Mice homozygous for the PTHrP null mutation died postnatally, probably from asphyxia, and exhibited widespread abnormalities of endochondral bone development. Histological examination revealed a diminution of chondrocyte proliferation, associated with premature maturation of chondrocytes and accelerated bone formation. Analysis of earlier developmental stages revealed that disturbance in cartilage growth preceded abnormal endochondral bone formation. There were no morphological abnormalities apparent in other tissues. These results provide direct evidence implicating PTHrP in normal skeletal development and serve to emphasize its potential involvement in human osteochondrodysplasias.
Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila.
Authors: Binari R , Perrimon N
Published: June 2007

We describe the characterization of the Drosophila gene, hopscotch (hop), which is required maternally for the establishment of the normal array of embryonic segments. In hop embryos, although expression of the gap genes appears normal, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu, as well as the segment-polarity genes engrailed and wingless. We demonstrate that the effect of hop on the expression of these genes is stripe-specific. The hop gene encodes a putative nonreceptor tyrosine kinase of the Janus kinase family, based on an internal duplication of the catalytic domain. We present a model in which the Hop tyrosine kinase is involved in the control of pair-rule gene transcription in a stripe-specific manner. Our results provide the first evidence for stripe-specific regulation of pair-rule genes by a tyrosine kinase.
Functional overlap in the src gene family: inactivation of hck and fgr impairs natural immunity.
Authors: Lowell C A , Soriano P , Varmus H E
Published: June 2007

We have generated mice with targeted disruptions of the src-like genes hck and fgr to assess the role of these kinases in myeloid cell development and function. Hematopoiesis appears to proceed normally in both hck-l- and fgr-l- animals, and in hck-l(-)-fgr-l- double homozygotes, but phagocytosis is impaired in hck-l- macrophages. Macrophages cultured from doubly homozygous, hck-l(-)-fgr-l- animals retain many other normal functional properties, suggesting that the deficiency of these kinases is complemented by other src family members. The specific activity of the Lyn protein kinase is increased in hck-l- macrophages, implying that Lyn may compensate for a deficiency in Hck. Doubly mutant animals, however, have a novel immunodeficiency characterized by an increased susceptibility to infection with Listeria monocytogenes, indicating that either hck or fgr is required to maintain a normal natural immune response. These data provide the first direct example of genetic interactions between src gene family members.
Activin/inhibin beta B subunit gene disruption leads to defects in eyelid development and female reproduction.
Authors: Vassalli A , Matzuk M M , Gardner H A , Lee K F , Jaenisch R
Published: June 2007

Inhibins and activins are dimeric growth factors of the transforming growth factor-beta superfamily, a class of peptides that can regulate the growth and differentiation of a variety of cell types. Recently, activins have been implicated in early vertebrate development through their ability to evoke, in Xenopus embryo explants, both morphological and molecular changes characteristic of mesoderm induction. To understand these processes further, we have used homologous recombination in embryonic stem cells to create mouse strains carrying mutations in the gene encoding the activin/inhibin beta B subunit. These mice are expected to be deficient in activin B (beta B:beta B), activin AB (beta A:beta B), and inhibin B (alpha:beta B). Viable mutant animals were generated, indicating that the beta B subunit is not essential for mesoderm formation in the mouse. Mutant animals suffered, however, from distinct developmental and reproductive defects. An apparent failure of eyelid fusion during late embryonic development led to eye lesions in mutant animals. Whereas beta B-deficient males bred normally, mutant females manifested a profoundly impaired reproductive ability, characterized by perinatal lethality of their offspring. The phenotype of mutant mice suggests that activin beta B (1) plays a role in late fetal development and (2) is critical for female fecundity. In addition, we have found that expression of the related beta A subunit of activin is highly upregulated in ovaries of mutant females. Altered regulation of beta A activin in beta B-deficient mice may contribute to the mutant phenotype.
Elucidation of the role of breathless, a Drosophila FGF receptor homolog, in tracheal cell migration.
Authors: Reichman-Fried M , Dickson B , Hafen E , Shilo B Z
Published: June 2007

DFGF-R1 (breathless), a Drosophila FGF receptor homolog, is required for the migration of tracheal cells and the posterior midline glial cells during embryonic development. To define the role of this receptor in cell migration, we have monitored the biological effects of a deregulated receptor containing the extracellular and transmembrane regions of the torso dominant allele and the cytoplasmic domain of DFGF-R1. Ubiquitous expression of the chimeric receptor at the time of tracheal cell migration did not disrupt migration in wild-type embryos. However, induction of the chimeric receptor corrected the tracheal defects of breathless (btl) mutant embryos, allowing the tracheal cells to migrate along their normal tracts. This result indicates that the normal activity of DFGF-R1 in promoting cell migration does not require spatially restricted cues. Late inductions of the chimeric construct, after the normal initiation of tracheal migration, allowed the definition of a broad time window during which the external signals guiding migration persist and the tracheal cells retain the capacity to respond to these cues. Rescue of tracheal migration in btl mutant embryos by the chimeric construct provides a sensitive biological assay for the activity of other Drosophila receptor tyrosine kinases (RTKs). Deregulated receptors containing the cytoplasmic domains of DFGF-R2, DER, torso, and sevenless were all able to partially rescue the migration defects. Consistent with the notion that these RTKs share a common signaling pathway, constructs containing the activated downstream elements Dras1 and Draf were also able to rescue tracheal migration, demonstrating that these two proteins are key players in the DFGF-R1 signaling pathway.
CHOP (GADD153) and its oncogenic variant, TLS-CHOP, have opposing effects on the induction of G1/S arrest.
Authors: Barone M V , Crozat A , Tabaee A , Philipson L , Ron D
Published: June 2007

The growth arrest and DNA damage-inducible gene CHOP (GADD153) encodes a small nuclear protein from the C/EBP family, originally isolated from adipocytes in culture. Although inactive in cells under normal conditions, the CHOP gene is markedly induced by a variety of cellular stresses, including nutrient deprivation and metabolic perturbations. These lead to accumulation of CHOP protein in the nucleus. Because cellular stress normally leads to growth arrest, we examined the implication of CHOP in this process. Microinjection of CHOP expression plasmids into NIH-3T3 cells blocked the cells from progressing through the cell cycle, measured by an attenuation in the fraction of cells incorporating BrdU, an S-phase marker. The precise point in the cell cycle at which CHOP acts was mapped by microinjection of bacterially expressed CHOP protein into synchronized cells--this blocked the cells from progressing from G1 to S phase. This effect of CHOP was observed only when the protein was introduced early after serum stimulation suggesting that CHOP works at or around the so-called G1/S checkpoint. CHOP dimerizes with other C/EBP proteins and the CHOP-C/EBP dimers are directed away from "classical" C/EBP sites recognizing instead unique "nonclassical" sites. Mutant forms of the CHOP protein that lack the leucine zipper dimerization domain or the unusually structured basic region, potentially involved in DNA binding, fail to induce growth arrest. A tumor-specific form of CHOP, TLS-CHOP, that has been found so far exclusively in the human adipose tissue tumor myxoid liposarcoma, fails to cause growth arrest and furthermore interferes with the ability of normal CHOP to induce growth arrest. CHOP has been shown recently to be markedly inducible by nutritional deprivation of cells. This suggests that CHOP may play a role in an inducible growth arrest pathway that is triggered by metabolic cues and is of particular importance in adipose tissue--an organ that undergoes marked changes in its metabolic activity. Blocking of this pathway by TLS-CHOP may play a mechanistic role in the establishment of myxoid liposarcoma.
Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal.
Authors: Lutz C S , Alwine J C
Published: June 2007

An integral component of the splicing machinery, the U1 snRNP, is here implicated in the efficient polyadenylation of SV40 late mRNAs. This occurs as a result of an interaction between U1 snRNP-A protein and the upstream efficiency element (USE) of the polyadenylation signal. UV cross-linking and immunoprecipitation demonstrate that this interaction can occur while U1 snRNP-A protein is simultaneously bound to U1 RNA as part of the snRNP. The target RNA of the first RRM (RRM1) has been shown previously to be the second stem-loop of U1 RNA. We have found that a target for the second RRM (RRM2) is within the AUUUGURA motifs of the USE of the SV40 late polyadenylation signal. RNA substrates containing the wild-type USE efficiently bind to U1 snRNP-A protein, whereas substrates fail to bind when motifs of the USE were replaced by linker sequences. The addition of an oligoribonucleotide containing a USE motif to an in vitro polyadenylation reaction inhibits polyadenylation of a substrate representing the SV40 late polyadenylation signal, whereas a mutant oligoribonucleotide, a nonspecific oligoribonucleotide, and an oligoribonucleotide containing the U1 RNA-binding site had much reduced or no inhibitory effects. In addition, antibodies to bacterially produced, purified U1 snRNP-A protein specifically inhibit in vitro polyadenylation of the SV40 late substrate. These data suggest that the U1 snRNP-A protein performs an important role in polyadenylation through interaction with the USE. Because this interaction can occur when U1 snRNP-A protein is part of the U1 snRNP, our data provide evidence to support a link between the processes of splicing and polyadenylation, as suggested by the exon definition model.
The Drosophila orb RNA-binding protein is required for the formation of the egg chamber and establishment of polarity.
Authors: Lantz V , Chang J S , Horabin J I , Bopp D , Schedl P
Published: June 2007

The orb gene of Drosophila encodes sex-specific germ-line proteins that contain two RRM-type RNA-binding domains. Here we report the distribution of Orb protein in wild-type, tumorous, and orb mutant ovaries. The wild-type distribution of Orb protein during oogenesis resembles that of its RNA, preferentially accumulating in the cytoplasm of the developing oocyte shortly after the formation of the 16-cell cyst. As anticipated from its germ-line expression, mutations in orb lead to female sterility. Analysis of the effect of orb mutants on the distribution of RNAs known to be required for oocyte differentiation and polarity suggests that orb functions in RNA localization at multiple points during oogenesis. In addition, phenotypic characterization of the orb mutants indicates that the gene is required early in oogenesis for formation of the 16-cell cyst. It then functions in the differentiation of the oocyte and is required for the three-dimensional reorganization of the germ cells in the cyst as well as for the establishment of normal germ-line-soma interactions in the egg chamber.
Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair.
Authors: Weinert T A , Kiser G L , Hartwell L H
Published: June 2007

In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage. Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The G2-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MEC1 and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13; cdc13 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdc13 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest in different stages of the cell cycle at the restrictive temperature and by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/or DNA damage and cause arret of cells before mitosis.
The v-abl tyrosine kinase negatively regulates NF-kappa B/Rel factors and blocks kappa gene transcription in pre-B lymphocytes.
Authors: Klug C A , Gerety S J , Shah P C , Chen Y Y , Rice N R , Rosenberg N , Singh H
Published: June 2007

Transformation of B-lineage precursors by the Abelson murine leukemia virus appears to arrest development at the pre-B stage. Abelson-transformed pre-B cell lines generally retain transcriptionally inactive, unrearranged immunoglobulin kappa alleles. We demonstrate that nontransformed pre-B cells expanded from the mouse bone marrow efficiently transcribe unrearranged kappa alleles. In addition, they contain activated complexes of the NF-kappa B/Rel transcription factor family, in contrast with their Abelson-transformed counterparts. Using conditionally transformed pre-B cell lines, we show that the v-abl viral transforming protein, a tyrosine kinase, blocks germ-line kappa gene transcription and negatively regulates NF-kappa B/Rel activity. An active v-abl kinase specifically inhibits the NF-kappa B/Rel-dependent kappa intron enhancer, which is implicated in promoting both transcription and rearrangement of the kappa locus. v-abl inhibits the activated state of NF-kappa B/Rel complexes in a pre-B cell via a post-translational mechanism that results in increased stability of the inhibitory subunit I kappa B alpha. This analysis suggests a molecular pathway by which v-abl inhibits kappa locus transcription and rearrangement.
Sex-specific regulation of the male-specific lethal-1 dosage compensation gene in Drosophila.
Authors: Palmer M J , Richman R , Richter L , Kuroda M I
Published: June 2007

Dosage compensation in Drosophila occurs by a twofold increase in transcription per copy of X-linked genes in males (XY) compared with females (XX). msl-1 is one of four genes that are essential for dosage compensation in males, and MSL-1 protein is associated specifically with the male X chromosome. To explore the basis for the sex specificity of dosage compensation, we examined MSL-1 expression in males, females, and dosage compensation mutants. MSL-1 protein levels are negatively regulated by Sxl in females, resulting in male-specific expression of MSL-1. In addition, msl-2 is required for translation and/or stability of MSL-1 in males. Furthermore, the wild-type pattern of MSL-1 localization to the X chromosome is dependent on mle and msl-3 function, although a subset of sites are stained with MSL-1 antibodies in these mutants. Collectively, these data provide the first evidence for an order of msl gene function and suggest that male-specific expression of MSL-1 plays a key role in the sex specificity of dosage compensation.
Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites.
Authors: Ren R , Ye Z S , Baltimore D
Published: June 2007

To understand the normal and oncogenic functions of the protein-tyrosine kinase Abl, the yeast two-hybrid system has been used for identifying proteins that interact with it. One interacting protein is Crk-I, an SH3/SH2-containing adapter protein that was originally identified as the oncogenic element in the avian sarcoma virus CT10. Direct interaction between the Crk-I SH3 and Abl at novel, approximately 10 amino acid sites just carboxy-terminal to the Abl kinase domain occurs in vitro and in mammalian cells. There is a nearby site specific for binding another adapter, Nck, and these sites also bind Grb-2. When bound to Abl, Crk-I was phosphorylated on tyrosine. Thus, the SH3-binding sites on Abl serve as substrate recognition sites for the relatively nonspecific kinase of Abl. In Crk-I-transformed cells, Crk-I associates with endogenous c-Abl and is phosphorylated on tyrosine. The association of Crk and Abl suggests that Abl could play a role in v-Crk and Crk-I transformation and that normal Abl function may be partly mediated through bound adapter molecules.
Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites.
Authors: Bailly V , Lamb J , Sung P , Prakash S , Prakash L
Published: June 2007

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for postreplication repair of UV-damaged DNA, DNA damage induced mutagenesis, sporulation, and amino-end rule protein degradation. RAD6 interacts physically with the UBR1 gene product in carrying out the multiubiquitination of amino-end rule proteolytic substrates. In mediating postreplication repair, it has remained unclear whether RAD6 acts in a pleiotropic manner distal from the site of DNA damage or is targeted to the damage site via interaction with another repair component. Here, we show that RAD6 forms a specific complex with the product of the DNA repair gene RAD18. The biological significance of this interaction is attested by the observation that overproduction of the rad6 Ala-88 mutant protein, which lacks ubiquitin-conjugating activity but retains the ability to interact with RAD18 protein, confers a high level of UV sensitivity on wild-type RAD+ cells that can be corrected by the concomitant overexpression of RAD18. We demonstrate that whereas RAD6 has no affinity for DNA, RAD18 binds single-stranded DNA. Thus, association of RAD6 with RAD18 could provide a means for targeting RAD6 to damage-containing DNA regions, where the RAD6 ubiquitin-conjugating function could modulate the activity of a stalled DNA replication machinery. We also show that RAD6 forms separate complexes with RAD18 and with UBR1, and the extremely conserved amino terminus of RAD6 that is required for complex formation with UBR1 is dispensable for complex formation with RAD18.
pct1+, which encodes a new DNA-binding partner of p85cdc10, is required for meiosis in the fission yeast Schizosaccharomyces pombe.
Authors: Zhu Y , Takeda T , Nasmyth K , Jones N
Published: June 2007

The transcriptional activation of genes at late G1 is an important regulatory step in the commitment to a new cell division cycle. In Schizosaccharomyces pombe, this regulation is mediated by MCB elements that serve as binding sites for the MBF/DSC-1 complex. The cdc10(+)-encoded protein is a component of this complex. We report the cloning of a new gene, pct1+, encoding a 73-kD protein that interacts with p85cdc10 to form an MCB-binding heteromer. Pct1+ is related to, but distinct from, the res1+/sct1+ gene that also encodes a p85cdc10 partner. p73pct1 has centrally located ankyrin repeats and a putative amino-terminal DNA-binding domain that has extensive sequence similarity to the DNA-binding domains of the Saccharomyces cerevisiae SWI4 and MBP1 proteins. The p73pct1/p85cdc10 complex binds both in vitro and in vivo to MCB but not SCB or E2F sites. Overexpression of pct1+ is sufficient to rescue the growth of the cdc10-129 temperature-sensitive mutant at the restrictive temperature, although it is unable to rescue a cdc10 null mutation. A deletion of pct1+ is not lethal but does result in a severe meiotic defect. Our results indicate that there are two cdc10-containing heteromeric complexes that bind to MCB elements and play differential roles in mitotic division and meiosis.