bluuebunny/crossref_metadata_2025 huggingface dataset:
Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.
Authors: Cosgrove D , Meehan D T , Grunkemeyer J A , Kornak J M , Sayers R , Hunter W J , Samuelson G C
Published: June 2007

A mouse model for the autosomal form of Alport syndrome was produced. These mice develop a progressive glomerulonephritis with microhematuria and proteinuria, consistent with the human disease. End-stage renal disease develops at approximately 14 weeks of age. TEM analysis of the glomerular basement membranes (GBM) during development of renal pathology revealed focal multilaminated thickening and thinning beginning in the external capillary loops at 4 weeks and spreading throughout the GBM by 8 weeks. By 14 weeks, half of the glomeruli were fibrotic with collapsed capillaries. Immunofluorescence analysis of the GBM showed the absence of type IV collagen alpha-3, alpha-4, and alpha-5 chains and a persistence of alpha-1 and alpha-2 chains (these chains normally localize to the mesangial matrix). Northern blot analysis using probes specific for the collagen chains illustrate the absence of COL4A3 in the knockout, whereas mRNAs for the remaining chains are unchanged. An accumulation of fibronectin, heparan sulfate proteoglycan, laminin-1, and entactin was observed in the GBM of the affected animals. The temporal and spatial pattern of accumulation was consistent with that for thickening of the GBM as observed by TEM. Thus, expression of these basement membrane-associated proteins may be involved in the progression of Alport renal disease pathogenesis. The levels of mRNAs encoding the basement membrane-associated proteins at 7 weeks were unchanged.
heartless encodes a fibroblast growth factor receptor (DFR1/DFGF-R2) involved in the directional migration of early mesodermal cells in the Drosophila embryo.
Authors: Gisselbrecht S , Skeath J B , Doe C Q , Michelson A M
Published: June 2007

After invagination of the mesodermal primordium in the gastrulating Drosophila embryo, the internalized cells migrate in a dorsolateral direction along the overlying ectoderm. This movement generates a stereotyped arrangement of mesodermal cells that is essential for their correct patterning by later position-specific inductive signals. We now report that proper mesodermal cell migration is dependent on the function of a fibroblast growth factor (FGF) receptor encoded by heartless (htl). In htl mutant embryos, the mesoderm forms normally but fails to undergo its usual dorsolateral migration. As a result, cardiac, visceral, and dorsal somatic muscle fates are not induced by Decapentaplegic (Dpp), a transforming growth factor beta family member that is derived from the dorsal ectoderm. Visceral mesoderm can nevertheless be induced by Dpp in the absence of htl function. Ras1 is an important downstream effector of Htl signaling because an activated form of Ras1 partially rescues the htl mutant phenotype. The evolutionary conservation of htl function is suggested by the strikingly similar mesodermal migration and patterning phenotypes associated with FGF receptor mutations in species as diverse as nematode and mouse. These studies establish that Htl signaling provides a vital connection between initial formation of the embryonic mesoderm in Drosophila and subsequent cell-fate specification within this germ layer.
Glossy15, an APETALA2-like gene from maize that regulates leaf epidermal cell identity.
Authors: Moose S P , Sisco P H
Published: June 2007

Vegetative development in many plants progresses through distinct juvenile and adult phases. In maize, the transition from juvenile to adult shoot development affects a variety of leaf epidermal cell traits. These include epicuticular waxes, leaf hairs, and cell wall characteristics. Previous genetic and phenotypic analyses have shown that the maize Glossy15 (Gl15) gene is required for the expression of juvenile epidermal traits after leaf 2. We report here the molecular cloning of the Gl15 gene using a defective Suppressor-Mutator (dSpm) element insertion as a transposon-tag. Consistent with the gl15 mutant phenotype, the pattern of Gl15 mRNA expression was correlated with a juvenile leaf epidermal cell identity and was regulated by upstream factors such as Corngrass1. The Gl15 gene encodes a putative transcription factor with significant sequence similarity to the Arabidopsis regulatory genes APETALA2 and AINTEGUMENTA, which act primarily to regulate floral organ identity and ovule development. This finding expands the known functions of APETALA2-related genes to include the control of both vegetative and reproductive lateral organ identity and provides molecular support for the hypothesis that leaves and floral organs are related structures derived from a common growth plan.
Glucose regulates protein interactions within the yeast SNF1 protein kinase complex.
Authors: Jiang R , Carlson M
Published: June 2007

The SNF1 protein kinase is broadly conserved in eukaryotes and has been implicated in responses to environmental and nutritional stress. In yeast, the SNF1 kinase has a central role in the response to glucose starvation. SNF1 is associated with its activating subunit, SNF4, and other proteins in complexes. Using the two-hybrid system, we show that interaction between SNF1 and SNF4 is strongly regulated by the glucose signal. Moreover, this interaction is appropriately affected by mutations in regulators, including protein phosphatase 1. We show that SNF4 binds to the SNF1 regulatory domain in low glucose, whereas in high glucose the regulatory domain binds to the kinase domain of SNF1 itself. Genetic analysis further suggests that the SNF1 regulatory domain autoinhibits the kinase activity and that in low glucose SNF4 antagonizes this inhibition. Finally, these interactions have been conserved from yeast to plants, indicating that homologs of the SNF1 kinase complex respond to regulatory signals by analogous mechanisms.
Single factors direct the differentiation of stem cells from the fetal and adult central nervous system.
Authors: Johe K K , Hazel T G , Muller T , Dugich-Djordjevic M M , McKay R D
Published: June 2007

Identifying the signals that regulate stem cell differentiation is fundamental to understanding cellular diversity in the brain. In this paper we identify factors that act in an instructive fashion to direct the differentiation of multipotential stem cells derived from the embryonic central nervous system (CNS). CNS stem cell clones differentiate to multiple fates: neurons, astrocytes, and oligodendrocytes. The differentiation of cells in a clone is influenced by extracellular signals: Platelet-derived growth factor (PDGF-AA, -AB, and -BB) supports neuronal differentiation. In contrast, ciliary neurotrophic factor and thyroid hormone T3 act instructively on stem cells to generate clones of astrocytes and oligodendrocytes, respectively. Adult stem cells had remarkably similar responses to these growth factors. These results support a simple model in which transient exposure to extrinsic factors acting through known pathways initiates fate decisions by multipotential CNS stem cells.
SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins.
Authors: Rouvière P E , Gross C A
Published: June 2007

Little is known about either the process of periplasmic protein folding or how information concerning the folding state in this compartment is communicated. We present evidence that SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, is involved in the maturation and assembly of LamB. LamB is a trimeric outer membrane porin for maltodextrins as well as the bacteriophage lambda receptor in Escherichia coli. We demonstrate that SurA is involved in the conversion of unfolded monomers into a newly identified intermediate in LamB assembly, which behaves as a folded monomer. The absence of SurA blocks the assembly pathway and leads to accumulation of species prior to the folded monomer. These species also accumulate when the stress sigma factor sigmaE is induced by LamB overexpression. We suggest that accumulation of species prior to the generation of folded monomer is a stress signal sensed by sigmaE.
Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast.
Authors: Alarcon C M , Cardenas M E , Heitman J
Published: June 2007

In complex with the prolyl isomerase FKBP12, the natural product rapamycin blocks signal transduction in organisms as diverse as yeast and man. The yeast targets of FKBP12-rapamycin, TOR1 and TOR2, are large proteins with homology to lipid and protein kinases. A mammalian FKBP12-rapamycin binding protein, RAFT1, shares 39% and 43% identity with TOR1 and TOR2 proteins, respectively but has not been linked to rapamycin action in vivo. We find that when expressed in yeast, neither wild-type nor mutant RAFT1 complemented tor mutations or conferred rapamycin resistance. In contrast, TOR1-RAFT1 and TOR1-RAFT1 hybrid proteins containing the carboxy-terminal RAFT1 kinase domain complemented tor2 and tor1 mutant strains, respectively. Moreover, TOR2-RAFT1 and TOR1-RAFT1 hybrid proteins mutated at the position corresponding to rapamycin-resistant TOR mutants (S20351) conferred rapamycin resistance. Like the TOR2 protein, the TOR2-RAFT1 proteins were stably expressed, localized to the vacuolar surface, and associated with a phosphatidylinositol-4 kinase activity. These findings directly link the mammalian TOR homolog RAFT1 to rapamycin action in vivo and indicate that the TOR/RAFT1 kinase domain has been functionally conserved from yeast to man.
Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.
Authors: Lutz C S , Murthy K G , Schek N , O'Connor J P , Manley J L , Alwine J C
Published: June 2007

We have previously shown that the U1 snRNP-A protein (U1A) interacts with elements in SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.
U1 snRNA promotes the selection of nearby 5' splice sites by U6 snRNA in mammalian cells.
Authors: Hwang D Y , Cohen J B
Published: June 2007

Communication between exon boundaries is a central feature of the exon definition model of pre-mRNA splice-site selection and an exon-bridging interaction involving U1 small nuclear RNA (snRNA) paired with the 5' splice site (5'ss) has been identified previously. It has become increasingly clear, however, that the 5'ss is not defined relative to the base-pairing interaction with U1, suggesting that a connection in the proposed line of communication between exon boundaries is missing. To explore this issue, we have first sought to characterize the role in mammalian 5'ss selection of a previously suggested base-pairing interaction with U6 snRNA. Using transfection experiments, we show that mutations at positions 5 and 6 of a 5'ss associated with an internal exon can be suppressed by compensatory changes in the first two positions of a conserved hexanucleotide of U6 RNA. The specificity of the effect was established by covariation experiments as well as by experiments with two splice sites arranged in tandem. Suppression of 5'ss mutations by U6 was more efficient when U1 could pair nearby than when pairing was restored further away and individual U1 RNAs stimulated U6-defined proximal sites more efficiently than distal sites. These results are interpreted to suggest that U1 acts to direct 5'ss choice by U6 to matching sequences nearby. Our work supports a central role for base-pairing with U6 snRNA in mammalian 5'ss selection and suggests how the interaction may be established properly despite the limited complementarity involved.
The E1B 19K protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting Bax protein.
Authors: Han J , Sabbatini P , Perez D , Rao L , Modha D , White E
Published: June 2007

The E1B 19K protein is a potent apoptosis inhibitor and the putative adenovirus Bcl-2 homolog. To investigate the mechanism of apoptosis regulation, 19K-interacting cellular proteins were identified using the yeast two-hybrid system, and Bax was one of seven 19-K interacting clones. Residues 50-78 of Bax containing a conserved region designated Bcl-2 homology region 3 (BH3) were sufficient for specific binding to both the E1B 19K and Bcl-2 proteins. The Bax-E1B 19K interaction was detectable in vitro and in lysates from mammalian cells, and Bax expression antagonized E1B 19K protein function. bax mRNA and protein levels were p53-inducible with kinetics identical to that of p21/Waf-1/Cip-1, and E1B 19K and Bcl-2 expression did not affect Bax or p21/Waf-1/Cip-1 accumulation. In cells where p53 was mutant, Bax expression induced apoptosis, suggesting that Bax was sufficient for apoptosis, and acted downstream of p53. p53 may simultaneously activate the transcription of genes required for both growth arrest (p21/Waf-1/Cip-1) and death (bax), and E1B 19K and Bcl-2 may act distally and function through interaction with and antagonism of Bax to prevent apoptosis. With the death pathway disabled, induction of growth arrest by p53 can then be manifested.
Functionally redundant interactions between U2 and U6 spliceosomal snRNAs.
Authors: Field D J , Friesen J D
Published: June 2007

Base-pairing between U2 and U6 snRNAs to form intermolecular helix II has been demonstrated previously as a requirement for pre-mRNA splicing in mammalian cells. In contrast, deletion and substitution mutation experiments in yeast have indicated that helix II is not essential; instead, other regions of U2 and U6 have been proposed to pair, forming a helix called Ib. To investigate the importance of U2/U6 helices in yeast, we have systematically mutagenized the regions proposed to form helices II and Ib. Allele-specific suppression of certain U6 mutations by complementary substitutions in U2 show that helix II indeed form in yeast but that it is essential only in the presence of additional mutations that disrupt U2 stem I and the proposed helix Ib. Similarly, the proposed helix Ib is essential only when helix II is disrupted. These observations provide an explanation for apparently conflicting data in yeast and mammalian experimental systems, and identify synergistic or functionally redundant interactions between U2 and U6 snRNAs.
Intramolecular inhibition of activating transcription factor-2 function by its DNA-binding domain.
Authors: Li X Y , Green M R
Published: June 2007

ATF-2 is a cellular basic region-leucine zipper (bZIP) transcription factor that can mediate diverse transcriptional responses, including activation by the adenovirus Ela protein. ATF-2 contains an activation domain, required for transcriptional activity, but in the absence of an appropriate inducer, full-length ATF-2 is transcriptionally inactive. Here we have investigated the mechanism underlying this regulated inhibition of ATF-2 transcriptional activity. We show that the region of ATF-2 that suppresses the activation region is the bZIP DNA-binding domain and that maximal inhibition requires both the basic region and leucine zipper subdomains. Inhibition is activation domain specific: The ATF-2 bZIP suppresses the ATF-2 and the related Ela activation domains but not acidic- and glutamine-rich activation domains. In vitro protein interaction assays demonstrate that the ATF-2 activation domain and bZIP specifically bind to one another. Finally, we show that bZIP-mediated inhibition can be modulated in a cell-type-specific manner by another sequence element within ATF-2. On the basis of these and other data, we propose that the ATF-2 bZIP and activation domain are engaged in an inhibitory intramolecular interaction and that inducers of ATF-2 disrupt this interaction to activate transcription.
p53 is required for both radiation-induced differentiation and rescue of V(D)J rearrangement in scid mouse thymocytes.
Authors: Bogue M A , Zhu C , Aguilar-Cordova E , Donehower L A , Roth D B
Published: June 2007

The murine scid mutation affects both V(D)J recombination and DNA repair. This mutation has been mapped to the gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PK), which is activated by DNA damage in normal cells. In scid mice, antigen receptor gene rearrangements are initiated normally, but impaired joining of coding ends prevents assembly of functional receptor genes, resulting in arrest of B- and T-cell development. Others have shown that exposure of scid mice to genotoxic agents such as gamma-irradiation rescues rearrangement at the T-cell receptor (TCR) beta locus and promotes thymocyte development. Here we demonstrate that irradiation rescues rearrangements at multiple TCR loci, suggesting a general effect on the recombination mechanism. Furthermore, our data show that p53 is required for irradiation-mediated rescue of both thymocyte development and V(D)J recombination. We also find that thymocyte proliferation and differentiation in the absence of DNA damage do not require p53 and are not sufficient to rescue V(D)J recombination. These results suggest that exposure to ionizing radiation facilitates a partial bypass of the scid defect, perhaps by inducing p53-dependent DNA damage response pathways.
Epigenetic effects on yeast transcription caused by mutations in an actin-related protein present in the nucleus.
Authors: Jiang Y W , Stillman D J
Published: June 2007

Gene inactivation can result when a delta element of the Ty1 transposon inserted into the yeast HIS4 promoter (his4-912delta) alters the transcription initiation site. Previous work has identified mutations that suppress this transcriptional defect by restoring the transcription start site to the native position, and these mutations have been implicated in transcriptional regulation and chromatin structure. We show that in a sin4 mutant such suppression is incompletely penetrant, such that genetically identical yeast cells (sin4 his4-912delta) show either of two distinct phenotypic states, His+ or His-. To study this type of potential epigenetic control of gene expression, we constructed a strain with ADE2 expression under the control of the his4-912delta promoter, as colony color provides a convenient assay for ADE2 expression. We isolated mutations in the ACT3 gene that show variegated expression of this ADE2 reporter. The act3 his4delta-ADE2 colonies display both white and red sectors, showing that the two different phenotypes are possible in a single colony. The two phenotypic states can be inherited during clonal growth, yet are reversible. Analysis of RNA isolated from individual colonies of either red or white color demonstrates that it is the state of the promoter, as either On or Off, that is inherited and is responsible for the colony color. An act3 mutation also affects expression of the HIS4 and LYS2 genes; thus, Act3p is not a delta element-specific transcriptional regulator. Immunofluorescence microscopy experiments demonstrate that the Act3p protein is present in the nucleus. Act3p shows clear homology to actin, and possible roles for an actin-related protein in transcription are discussed.
shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neurectoderm and other morphogenetically active epithelia.
Authors: Tepass U , Gruszynski-DeFeo E , Haag T A , Omatyar L , Török T , Hartenstein V
Published: June 2007

Adhesion molecules of the cadherin superfamily have an important role during vertebrate development. The DE-cadherin homolog DE-cadherin is the first classic cadherin isolated from invertebrates. We report here that DE-cadherin is encoded by the shotgun (shg) gene. shg is expressed in most embryonic epithelia and decreases in cells that undergo epithelial-mesenchymal transitions like the mesoderm or neural precursors. Removal of both maternal and zygotic shg function leads to severe defects in all epithelia expressing shg, suggesting that DE-cadherin, similar to vertebrate classic cadherins, has a crucial role for the formation and/or maintenance of epithelial tissues. Interestingly, the analysis of different shg alleles indicates that the requirement for shg in a given epithelium depends on the degree of its morphogenetic activity. Only epithelia involved in extensive morphogenetic movements require zygotic shg function in addition to maternal expression. In support of this view we find that suppression of morphogenetic movements rescues the zygotic shg phenotype. We find that in zygotic shg nulls the level of Dalpha-catenin and Armadillo at adherens junctions is dramatically reduced, surprisingly also in epithelia that differentiate normally and possess a zonula adherens.
Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters.
Authors: Burke T W , Kadonaga J T
Published: June 2007

We describe the identification and characterization of a conserved downstream basal promoter element that is present in a subset of Drosophila TATA-box-deficient (TATA-less) promoters by using purified, epitope-tagged TFIID complex (eTFIID) from embryos of transgenic Drosophila. DNase I footprinting of the binding of eTFIID to TATA-less promoters revealed that the factor protected a region that extended from the initiation site sequence (about +1) to approximately 35 nucleotides downstream of the RNA start site. In contrast, there was no apparent upstream DNase I protection or hypersensitivity induced by eTFIID in the -25 to -30 region at which TATA motifs are typically located. Further studies revealed a conserved sequence motif, (A/G)G(A/T)CGTG, termed the downstream promoter element (DPE), which is located approximately 30 nucleotides downstream of the RNA start site of many TATA-less promoters. DNase I footprinting and in vitro transcription experiments revealed that a DPE in its normal downstream location is necessary for transcription of DPE-containing TATA-less promoters and can compensate for the disruption of an upstream TATA box of a TATA-containing promoter. Moreover, a systematic mutational analysis of DNA sequences that encompass the DPE confirmed the importance of the consensus DPE sequence motif for basal transcription and further supports the postulate that the DPE is a distinct, downstream basal promoter element. These results suggest that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters.
Selection of axial growth sites in yeast requires Axl2p, a novel plasma membrane glycoprotein.
Authors: Roemer T , Madden K , Chang J , Snyder M
Published: June 2007

Spa2p and Cdc10p both participate in bud site selection and cell morphogenesis in yeast, and spa2delta cdc10-10 cells are inviable. To identify additional components important for these processes in yeast, a colony-sectoring assay was used to isolate high-copy suppressors of the spa2delda cdc10-10 lethality. One such gene, AXL2, has been characterized in detail. axl2 cells are defective in bud site selection in haploid cells and bud in a bipolar fashion. Genetic analysis indicates that AXL2 falls into the same epistasis group as BUD3. Axl2p is predicted to be a type I transmembrane protein. Tunicamycin treatment experiments, biochemical fractionation and extraction experiments, and proteinase K protection experiments collectively indicate that Axl2p is an integral membrane glycoprotein at the plasma membrane. Indirect immunofluorescence experiments using either Axl2p tagged with three copies of a hemagglutinin epitope or high-copy AXL2 and anti-Axl2p antibodies reveal a unique localization pattern for Axl2p. The protein is present as a patch at the incipient bud site and in emerging buds, and at the bud periphery in small-budded cells. In cells containing medium-sized or large buds, Axl2p is located as a ring at the neck. Thus, Axl2p is a novel membrane protein critical for selecting proper growth sites in yeast. We suggest that Axl2p acts as an anchor in the plasma membrane that helps direct new growth components and/or polarity establishment components to the cortical axial budding site.
The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation.
Authors: Nikolic M , Dudek H , Kwon Y T , Ramos Y F , Tsai L H
Published: June 2007

Cyclin-dependent kinase 5(cdk5) is highly homologous to other members of the cdk family that are known to function in proliferating cells. Despite the structural similarity, cdk5-associated histone H1 kinase activity is only detectable in postmitotic neurons of the central nervous system (CNS). p35 is a neuronal-specific cdk5 regulator that activates cdk5 kinase activity upon association. The cdk5/p35 kinase activity increases during the progression of CNS neurogenesis, suggesting a function of cdk5 in neuronal differentiation. Here we show that both cdk5 and p35 proteins are present in the growth cones of developing neurons. The staining pattern of cdk5 in the growth cones is similar to that of actin filaments but not microtubules. To address the functional significance of the cdk5/p35 kinase in neurogenesis, we ectopically expressed wild-type or mutant kinases in cortical cultures. Expression of dominant-negative mutants of cdk5 (cdk5N144 and cdk5T33) inhibited neurite outgrowth, which was rescued by coexpression of the wild-type proteins. A similar extent of neurite outgrowth inhibition was obtained by transfection of an antisense p35 construct, which in turn was only rescued by p35 but not cdk5 coexpression. In contrast, longer neurites were elaborated in neurons that coexpressed exogenous cdk5 and p35. These observations suggest that the cdk5/p35 kinase plays a critical role in neurite outgrowth during neuronal differentiation.
Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex.
Authors: Keys D A , Lee B S , Dodd J A , Nguyen T T , Vu L , Fantino E , Burson L M , Nogi Y , Nomura M
Published: June 2007

Like most eukaryotic rDNA promoters, the promoter for rDNA in Saccharomyces cerevisiae consists of two elements: a core element, which is essential, and an upstream element, which is not essential but is required for a high level of transcription. We have demonstrated that stimulation of transcription by the upstream element is mediated by a multiprotein transcription factor, UAF (upstream activation factor) which contains three proteins encoded by RRN5, RRN9, and RRN10 genes, respectively, and probably two additional uncharacterized proteins. The three genes were originally defined by mutants that show specific reduction in the transcription of rDNA. These genes were cloned and characterized. Epitope tagging of RRN5 (or RRN9), combined with immunoaffinity purification was used to purify UAF, which complemented all three (rrn5, rrn9, and rrn10) mutant extracts. Using rrn10 mutant extracts, a large stimulation by UAF was demonstrated for template containing both the core element and the upstream element but not for a template lacking the upstream element. In the absence of UAF, the mutant extracts showed the same weak transcriptional activity regardless of the presence or absence of the upstream element. We have also demonstrated that UAF alone makes a stable complex with the rDNA template, committing that template to transcription. Conversely, no such template commitment was observed with rrn10 extracts without UAF. By using a series of deletion templates, we have found that the region necessary for the stable binding of UAF corresponds roughly to the upstream element defined previously based on its ability to stimulate rDNA transcription. Differences between the yeast UAF and the previously studied metazoan UBF are discussed.
Silencers are required for inheritance of the repressed state in yeast.
Authors: Holmes S G , Broach J R
Published: June 2007

Transcriptional silencers in the yeast Saccharomyces induce position-specific, sequence-independent repression by promoting formation of a heterochromatin-like structure across sequences adjacent to them. We have examined the role of silencers in maintenance and inheritance of repression at the silent mating-type cassettes in yeast by monitoring the expression state of one of these cassettes following in vivo deletion of the adjacent silencer. Our experiments indicate that although silencer sequences are dispensable for the maintenance of repression in the absence of cell-cycle progression, silencers are required for the stable inheritance of a repressed state. That is, silenced loci from which the silencer is deleted most often become derepressed within one generation of losing the silencer. Thus, the heritability of a repressed state is not intrinsic to a silenced locus or to the chromatin encompassing it; rather, heritability of repression appears to be a property of the silencer itself.
Repression and activation by multiprotein complexes that alter chromatin structure.
Authors: Kingston R E , Bunker C A , Imbalzano A N
Published: June 2007

Recent studies have provided strong evidence that macromolecular complexes are used in the cell to remodel chromatin structure during activation and to create an inaccessible structure during repression, Although there is not yet any rigorous demonstration that modification of chromatin structure plays a direct, causal role in either activation or repression, there is sufficient smoke to indicate the presence of a blazing inferno nearby. It is clear that complexes that remodel chromatin are tractable in vitro; hopefully this will allow the establishment of systems that provide a direct analysis of the role that remodeling might play in activation. These studies indicate that establishment of functional systems to corroborate the elegant genetic studies on repression might also be tractable. As the mechanistic effects of these complexes are sorted out, it will become important to understand how the complexes are regulated. In many of the instances discussed above, the genes whose products make up these complexes were identified in genetic screens for effects on developmental processes. This implies a regulation of the activity of these complexes in response to developmental cues and further implies that the work to fully understand these complexes will occupy a generation of scientists.
Drosophila IRBP/Ku p70 corresponds to the mutagen-sensitive mus309 gene and is involved in P-element excision in vivo.
Authors: Beall E L , Rio D C
Published: June 2007

The P family of transposable elements in Drosophila transpose by a cut-and-paste mechanism involving double-strand gap repair. We report here that a Drosophila mutagen-sensitive mutant, mus3O9, contains a mutation in IRBP (inverted repeat binding protein), the Drosophila homolog of the mammalian Ku p70 gene. We show that the repair of double-strand DNA breaks after P-element excision is severely reduced in mus3O9 mutants using an in vivo assay for P-element transposase activity. In addition, excision products recovered from mus3O9 mutant embryos by use of a plasmid-based P-element mobility assay contain large deletions, suggesting that IRBP is involved in the repair of double-strand DNA breaks. Our findings provide the first demonstration that a mutation in the IRBP gene affects double-strand DNA break repair and suggest that DNA repair functions are conserved between Drosophila and mammals.
Productive T-cell receptor beta-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo.
Authors: Hoffman E S , Passoni L , Crompton T , Leu T M , Schatz D G , Koff A , Owen M J , Hayday A C
Published: June 2007

Productive gene rearrangement at the T-cell receptor (TCR) beta-chain locus facilitates formation of the "pre-TCR," a molecular complex that is important for the subsequent development of alpha beta T cells. The transition of thymocytes from a population of cells undergoing TCRbeta chain genes to a population enriched in cells with productively rearranged TCRbeta chain genes is known as "beta selection." This is the first point in alpha beta T-cell development at which the products of an activated TCR locus define cell phenotype. Toward an understanding of these events, this study has focused on a set of thymocytes defined by cell surface phenotype as HSA+ CD44low CD25+, in which the bulk of TCRbeta gene rearrangement occurs. The analysis of this set, presented here, allows its novel subdivision into two subsets that are respectively strong candidates for cells immediately prior to and immediately following TCRbeta selection. Cells that have passed beta selection differ from the preceding cells by several criteria, including hyperphosphorylation of Rb, increased expression of cyclins A and B, down-regulation of p27, increased CDK2 activity, an induction of cdc2 activity, and progression through DNA synthesis. Consistent with these changes being attributable to productive TCRbeta chain gene rearrangement, the identified "beta-selected" subset is not detected in mutant mice that cannot assemble a pre-TCR. Interestingly, there is a coincident selective and transient down-regulation of the protein RAG2, on which TCR gene rearrangement obligatorily depends. Together, these findings demonstrate that productive TCR gene rearrangement is associated with events that can ensure thymocyte expansion and monoclonality.
Forced expression of E-cadherin in the mouse intestinal epithelium slows cell migration and provides evidence for nonautonomous regulation of cell fate in a self-renewing system.
Authors: Hermiston M L , Wong M H , Gordon J I
Published: June 2007

The adult mouse small intestinal epithelium is self-renewing. Its crypt-villus unit provides a model for studying many of the processes that occur during tissue morphogenesis such as control of proliferative status, specification of cell fate, regulation of differentiation, and induction of death. To assess the contributions of cell-cell and cell-substratum interactions to the coordinated control of these processes, 129/Sv embryonic stem (ES) cells, transfected with a recombinant DNA consisting of a fatty acid-binding protein gene (Fabp1) promoter that functions along the entire length of the crypt-villus axis linked to mouse E-cadherin, were introduced into normal C57Bl/6 (B6) blastocysts. Analyses of adult B6 <--> 129/Sv mice indicated that forced expression of E-cadherin suppresses proliferation and induces apoptosis in the crypt, and slows cell movement up the villus. The slowed migration is not accompanied by a change in distribution of terminal differentiation markers along the crypt-villus axis suggesting that differentiation is largely cell nonautonomous. To determine whether the slowed migration was a direct effect of forced expression of E-cadherin or a secondary effect of reduced crypt cell production, another Fabp promoter was used to restrict overproduction of E-cadherin to the villus epithelium of transgenic mice. Enterocytic migration was slowed, although proliferation and apoptosis were not perturbed in crypts. Augmentation of cellular E-cadherin pools was accompanied by an increase in beta-catenin levels. These findings establish that cadherins and their associated proteins modulate cellular migration, proliferation, and death programs in an adult vertebrate organ.
A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome.
Authors: Ohno M , Shimura Y
Published: June 2007

Because the nuclear export of mRNA occurs only after the splicing reaction is completed, intron-containing pre-mRNA does not normally appear in the cytoplasm. As a mechanism to secure this, intron-containing RNA is retained in the nucleus via formation of the spliceosome. Therefore, the process of releasing spliced mRNA from the spliceosome after completion of splicing is an essential step for triggering the nuclear export of the spliced mRNA. In budding yeast, RNA helicase-like protein Prp22 is implicated in this process. Here we demonstrate the function of HRH1, a human protein homologous to Prp22, in mammalian cells using dominant-negative HRH1++ mutants (dn-HRH1). dn-HRH1 protein stalls on the spliceosome and prevents release of the spliced RNA from the spliceosome in vitro. Expression of dn-HRH1 in mammalian cells leads to inhibition of splicing and to extensive nuclear export of unspliced pre-mRNA, probably because of the incapability of recycling spliceosome components that normally retain the pre-mRNA in the nucleus. The arginine/serine-rich domain (RS domain) of HRH1, which is missing in Prp22, confers a nuclear localization signal, and appears to facilitate the interaction of HRH1 with the spliceosome. This is the first report on a bona fide mammalian homolog of yeast Prp splicing factor, and also on a mammalian RNA helicase-like splicing factor.
The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease.
Authors: Xue D , Shaham S , Horvitz H R
Published: June 2007

The Caenorhabditis elegans cell-death gene ced-3 encodes a protein similar to mammalian interleukin-1beta-converting enzyme (ICE), a cysteine protease implicated in mammalian apoptosis. We show that the full-length CED-3 protein undergoes proteolytic activation to generate a CED-3 cysteine protease and that CED-3 protease activity is required for killing cells by programmed cell death in C. elegans. We developed an easy and general method for the purification of CED-3/ICE-like proteases and used this method to facilitate a comparison of the substrate specificities of four different purified cysteine proteases. We found that in its substrate preferences CED-3 was more similar to the mammalian CPP32 protease than to mammalian ICE or NEDD2/ICH-1 protease. Our results suggest that different mammalian CED-3/ICE-like proteases may have distinct roles in mammalian apoptosis and that CPP32 is a candidate for being a mammalian functional equivalent of CED-3.
p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity.
Authors: Lioubin M N , Algate P A , Tsai S , Carlberg K , Aebersold A , Rohrschneider L R
Published: June 2007

The production, survival, and function of monocytes and macrophages is regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF to Fms induces the tyrosine phosphorylation and association of a 150-kD protein with the phosphotyrosine-binding (PTB) domain of Shc. We have cloned p150 using a modified yeast two-hybrid screen. p150 contains one SH2 domain, two potential PTB-binding sites, an ATP/GTP-binding domain, several potential SH3-binding sites, and a domain with homology to inositol polyphosphate-5-phosphatases. p150 antibodies detect this protein in FDC-P1 myeloid cells, but the same protein is not detectable in fibroblasts. The antibodies immunoprecipitate a 150-kD protein from quiescent or M-CSF-stimulated FDC-P1 cells that hydrolyzes PtdIns(3,4,5)P3, to PtdIns(3,4)P2. This activity is observed in Shc immunoprecipitates only after M-CSF stimulation. Retroviral expression of p15O in FD-Fms cells results in strong inhibition of cell growth in M-CSF and a lesser inhibition in IL-3. Ectopic expression of p150 in fibroblasts does not inhibit growth. This novel protein, p150(ship) (SH2-containing inositol phosphatase), identifies a component of a new growth factor-receptor signaling pathway in hematopoietic cells.
ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism.
Authors: Kim J B , Spiegelman B M
Published: June 2007

Adipocyte determination and differentiation-dependent factor 1 (ADD1) is a member of the basic helix-loop-helix leucine zipper (bHLH-LZ) family of transcription factors that binds to two distinct DNA sequences and has been associated with both adipocyte development and cholesterol homeostasis (where it has been termed SREBP1). To investigate the biological role of ADD1, we expressed wild-type and dominant negative forms of this protein with retroviral vectors in preadipocytes and nonadipogenic cells. A dominant-negative form of ADD1 with a point mutation in the DNA-binding domain sharply represses the differentiation of 3T3-L1 cells as observed morphologically or by the expression of adipocyte-specific mRNAs. When NIH-3T3 cells ectopically expressing ADD1 are cultured under hormonal conditions not favoring differentiation, they do not overtly differentiate but still activate expression of mRNAs for fatty acid synthase (FAS) and lipoprotein lipase (LPL), two key genes that regulate fatty acid metabolism. Under culture conditions permissive for differentiation including a PPAR activator, 15%-20% of the cells expressing ADD1 undergo adipogenesis while 2%-3% of cells containing a control vector differentiate. Simultaneous expression of ADD1 with PPARgamma increases the transcriptional activity of this adipogenic nuclear hormone receptor, suggesting involvement of ADD1 in this pathway. These data indicate that ADD1 plays an important role in fat cell gene expression and differentiation, and suggest that it may function by augmenting a step in PPARgamma-mediated transcription.
Activation of transcription in Drosophila embryos is a gradual process mediated by the nucleocytoplasmic ratio.
Authors: Pritchard D K , Schubiger G
Published: June 2007

We have observed that zygotic transcription does not initiate at a single point in Drosophila embryos. Rather, a gene initiates transcription in a few nuclei of a fraction of embryos. During succeeding cycles, the frequency of transcribing embryos, and of nuclei transcribing in those embryos, gradually increases. For the fushi tarazu (ftz) gene, the timing of this process is regulated by the concentration of the maternally loaded, repressing transcription factor tramtrack (ttk). Altering the dose of Ttk protein in embryos shifts the activation of ftz transcription either forward or backward during development but does not effect Krüppel (Kr) activation. We have observed that the transcription of several genes, including ftz, is triggered in embryos at a critical nuclear density; therefore, we suggest that titration of transcription factors like ttk by the nucleocytoplasmic ratio triggers zygotic transcription in Drosophila.
The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli.
Authors: Muffler A , Fischer D , Hengge-Aronis R
Published: June 2007

The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S). Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli.
Restriction of the activity of the recombination site dif to a small zone of the Escherichia coli chromosome.
Authors: Cornet F , Louarn J , Patte J , Louarn J M
Published: June 2007

The recombination site dif is the target on the Escherichia coli chromosome of the site-specific recombinases XerC and XerD. The dif/XerC-D system plays a role during the cell cycle, probably by favoring sister chromosome monomerization or separation. A phenomenon of regional control over dif activity, also analyzed in this issue, is demonstrated here by translocation of dif to a series of loci close to the normal locus. We found that the site is physiologically active only within a narrow zone around its natural position. Competence for dif activity does not depend on the sequence of the normal dif activity zone (DAZ), because delta(dif) deletions larger than the DAZ result in Dif+ bacteria when dif is reinserted at the junction point. Although dif maps where replication normally terminates, termination of replication is not the elicitor. A strain with a large inversion that places dif and its surrounding region close to oriC remains Dif+, even when a Tus- mutation allows replication to terminate far away from it. Preliminary data suggest the possibility that specialized sequences separate the competent zone from the rest of the chromosome. We suspect that these sequences are members of a set of sequences involved in a polarized process of postreplicative reconstruction of the nucleoid structure. We propose that this reconstruction forces catenation links between sister chromosomes to accumulate within the DAZ, where they eventually favor recombination at dif.
Use of a transposon (Tndif) to obtain suppressing and nonsuppressing insertions of the dif resolvase site of Escherichia coli.
Authors: Kuempel P , Høgaard A , Nielsen M , Nagappan O , Tecklenburg M
Published: June 2007

The dif locus is a RecA-independent recombination site, located in the terminus region of the chromosome of Escherichia coli. This site functions to reduce circular dimer chromosomes to monomers before cell division. Strains lacking this site exhibit the Dif phenotype, in which a fraction of the cells form extended filaments with abnormal nucleoids, and the SOS system is induced. We have used a transposon (Tndif), as well as linear transformation, to position dif in 19 locations around the chromosome. All of the suppressing insertions that we obtained were within 10 kb of the normal site, even in strains in which the normal symmetry, between the origin of replication and dif had been altered by 200 kb. We also observed that the nonsuppressing insertions in the terminus region became suppressing if a deletion occurred that extended from the ectopic site up to or past the normal location of dif. We propose that dif is normally located at the center of converging polarities in the terminus region and that deletions that restore suppression do so by placing ectopic sites once again at the center of this polarity. Similar results and conclusions are described in this issue.