bluuebunny/crossref_metadata_2025 huggingface dataset:
The HAT4 gene of Arabidopsis encodes a developmental regulator.
Authors: Schena M , Lloyd A M , Davis R W
Published: June 2007

The HAT4 gene from the plant Arabidopsis thaliana encodes a homeo domain protein that contains a leucine zipper motif. Homeo domain-leucine zipper (HD-Zip) proteins have not been found in animal systems, suggesting that HAT4 may define a new family of transcription factors that regulate higher plant development. To explore this possibility, functional studies of HAT4 were carried out in yeast and in transgenic plants. Point mutants of HAT4 isolated in yeast define functionally critical residues within the HD-Zip domain, many of which correspond to highly conserved positions in known homeo domains and leucine zippers. Transgenic plants bearing constructs that alter HAT4 expression exhibit a series of interesting developmental phenotypes, including changes in morphology and developmental rate. Thus, the HAT4 gene of Arabidopsis encodes an HD-Zip protein that functions as a novel developmental regulator.
Cloning and characterization of PSF, a novel pre-mRNA splicing factor.
Authors: Patton J G , Porro E B , Galceran J , Tempst P , Nadal-Ginard B
Published: June 2007

Previously, we characterized cDNAs encoding polypyrimidine tract-binding protein (PTB) and showed that a complex between PTB and a 100-kD protein was necessary for pre-mRNA splicing. In this paper we have used two different in vitro-binding assays to confirm and extend the interaction between these two proteins. Peptide sequence information was used to clone and sequence cDNAs encoding alternatively spliced forms of the 100-kD protein. It contains two consensus RNA-binding domains and an unusual amino terminus rich in proline and glutamine residues. The protein is highly basic and migrates anomalously on SDS gels. Owing to its interaction with PTB and its role in pre-mRNA splicing, we have termed the 100-kD protein PTB-associated splicing factor (PSF). The RNA-binding properties of PSF are apparently identical to those of PTB. Both proteins, together and independently, bind the polypyrimidine tract of mammalian introns. Biochemical complementation, antibody inhibition, and immunodepletion experiments demonstrate that PSF is an essential pre-mRNA splicing factor required early in spliceosome formation. Bacterially synthesized PSF is able to complement immunodepleted extracts and restore splicing activity. Despite association with PSF, complementary experiments with antibodies against PTB do not suggest an essential role for PTB in pre-mRNA splicing.
An enhancer screen identifies a gene that encodes the yeast U1 snRNP A protein: implications for snRNP protein function in pre-mRNA splicing.
Authors: Liao X C , Tang J , Rosbash M
Published: June 2007

In an enhancer screen for yeast mutants that may interact with U1 small nuclear RNA (snRNA), we identified a gene that encodes the apparent yeast homolog of the well-studied human U1A protein. Both in vitro and in vivo, the absence of the protein has a dramatic effect on the activity of U1 snRNP containing the mutant U1 snRNA used in the screen. Surprisingly, the U1A gene is inessential in a wild-type U1 RNA background, as growth rate and the splicing of endogenous pre-mRNA transcripts are normal in these strains that lack the U1A protein. Even in vitro, the absence of the protein has little effect on splicing. On the basis of these observations, we suggest that a principal role of the U1A protein is to help fold or maintain U1 RNA in an active configuration.
Dosage-sensitive modifiers of Drosophila abl tyrosine kinase function: prospero, a regulator of axonal outgrowth, and disabled, a novel tyrosine kinase substrate.
Authors: Gertler F B , Hill K K , Clark M J , Hoffmann F M
Published: June 2007

In the absence of the Drosophila abl protein-tyrosine kinase (PTK), loss-of-function mutations in either disabled or prospero have dominant phenotypic effects on embryonic development. Molecular and genetic characterizations indicate that the products of these genes interact with the abl PTK by different mechanisms. The interaction between abl and prospero, which encodes a nuclear protein required for correct axonal outgrowth, is likely to be indirect. In contrast, the product of disabled may be a substrate for the abl PTK. The disabled protein is colocalized with abl in axons, its predicted amino acid sequence contains 10 motifs similar to the major autophosphorylation site of abl, and the protein is recognized by antibodies to phosphotyrosine.
Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation.
Authors: Miller M W , Duhl D M , Vrieling H , Cordes S P , Ollmann M M , Winkes B M , Barsh G S
Published: June 2007

The mouse agouti gene controls the deposition of yellow and black pigment in developing hairs. Several dominant alleles, including lethal yellow (Ay), result in the exclusive production of yellow pigment and have pleiotropic effects that include obesity and increased tumor susceptibility. In an interspecific backcross, we established genetic limits for the agouti gene and found that the Ay and the lethal non-agouti (ax) allele were not separated from a previously identified probe at the breakpoint of the Is1GsO chromosomal rearrangement. Using the Is1GsO probe, we isolated the agouti gene, and find that it has the potential to code for a secreted protein expressed in hair follicles and the epidermis, and that the level of expression correlates with the synthesis of yellow pigment. In the Ay mutation, there is a chromosomal rearrangement that results in the production of a chimeric RNA expressed in nearly every tissue of the body. The 5' portion of this chimeric RNA contains highly expressed novel 5' sequences, but the 3' portion retains the protein-coding potential of the nonmutant allele. We speculate that dominant pleiotropic effects of Ay are caused by ectopic activation of a signaling pathway similar to that used during normal hair growth.
Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells.
Authors: Ma C , Martin S , Trask B , Hamlin J L
Published: June 2007

We have utilized a dihydrofolate reductase (DHFR) probe in combination with selected probes from other positions along the 2q chromosome arm in a two-color fluorescence in situ hybridization analysis of early DHFR gene amplification events in CHO cells. These studies show clearly that the most frequent initiating event is the formation of a giant inverted duplication, resulting from chromosome breakage and terminal fusion or a reverse unequal sister chromatid exchange. The dicentric chromosomes thus formed initiate bridge/breakage/fusion cycles that appear to mediate subsequent amplification steps to higher copy number.
Torso, a receptor tyrosine kinase required for embryonic pattern formation, shares substrates with the sevenless and EGF-R pathways in Drosophila.
Authors: Doyle H J , Bishop J M
Published: June 2007

The maternally expressed Drosophila gene torso (tor) is a receptor tyrosine kinase that, when activated, initiates a signal transduction cascade that is responsible for the proper differentiation of the terminal, nonsegmented regions of the embryo. l(1)pole hole, the Drosophila raf-1 serine-threonine kinase homolog, and corkscrew, a tyrosine phosphatase, have been shown previously to function in this signal transduction pathway. We have identified other products in this pathway by carrying out a mutagenesis screen for dominant suppressors of a tor gain-of-function allele. More than 40 mutations, some of which fall into seven complementation groups, have been characterized genetically. Two of these correspond to mutations in ras-1 and Son of sevenless (Sos), which also function in the sevenless and EGF receptor (Der) tyrosine kinase pathways. The phenotypes of several other Su(tor) mutations suggest that they also function in other receptor tyrosine kinase-activated pathways at different times during Drosophila development.
Association with terminal exons in pre-mRNAs: a new role for the U1 snRNP?
Authors: Wassarman K M , Steitz J A
Published: June 2007

Psoralen cross-linking experiments in HeLa cell nuclear extracts have revealed the binding of U1 snRNA to substrates containing the SV40 late and adenovirus L3 polyadenylation signals. The sites of U1 cross-linking to the substrates map different distances upstream of the AAUAAA sequence to regions with limited complementarity to the 5' end of U1 snRNA. U1 cross-linking to the same site in the SV40 late pre-mRNA is enhanced by the addition of an upstream 3' splice site, which also enhances polyadenylation. Examination of different nuclear extracts reveals a correlation between U1 cross-linking and the coupling of splicing and polyadenylation, suggesting that the U1 snRNP participates in the coordination of these two RNA-processing events. Mutational analyses demonstrate that U1/substrate association cannot be too strong for coupling to occur and suggest that the U1 snRNP plays a similar role in recognition of internal and 3' terminal exons. Possible mechanisms for communication between the splicing and polyadenylation machineries are discussed, as well as how interaction of the U1 snRNP with 3' terminal exons might contribute to mRNA export.
A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly.
Authors: Dammel C S , Noller H F
Published: June 2007

A C-->U substitution at position 23 of 16S rRNA confers a dominant, cold-sensitive phenotype. The mutation changes the G11-C23 base pair of the 5' terminal pseudoknot helix to a G-U pair, which is predicted to cause significant weakening of the helix. Ribosomes containing mutant RNA are impaired in assembly and function at low temperature. Cells expressing the C23U mutation have decreased polysome levels and accumulate free 30S and 50S subunits and particles that resemble those previously observed in cold-sensitive alleles of ribosomal protein S5 and in in vitro reconstitution of 30S subunits carried out at low temperature. Three second-site suppressor mutations suggest that cold sensitivity is caused by competition between the 5' helix and an alternative helix formed by base-pairing of the upstream precursor sequence with one strand of the mature helix. Cold sensitivity appears to be relieved by destabilization of the competing precursor helix relative to the mature helix.
p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction.
Authors: Mercurio F , DiDonato J A , Rosette C , Karin M
Published: June 2007

The Rel/NF-kappa B family of transcription factors is composed of two distinct subgroups, proteins that undergo proteolytic processing and contain SWI6/ankyrin repeats in their carboxyl termini (p105, p98), and those without such repeats that do not require processing (p65, c-Rel, RelB, and Dorsal). We demonstrate that the p105 and p98 precursors share functional properties with the I kappa B proteins, which also contain SWI6/ankyrin repeats. Both p105 and p98 were found to form stable complexes with other Rel/NF-kappa B family members, including p65 and c-Rel. Association with the precursors is sufficient for cytoplasmic retention of either p65 or c-Rel, both of which are otherwise nuclear. These complexes undergo stimulus-responsive processing to produce active p50/c-Rel and p55/c-Rel complexes. These observations suggest a second pathway leading to NF-kappa B induction, in which processing of the precursors rather than phosphorylation of I kappa B plays a major role.
Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression.
Authors: Hagman J , Belanger C , Travis A , Turck C W , Grosschedl R
Published: June 2007

Early B-cell factor (EBF) was identified previously as a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific mb-1 gene. Partial amino acid sequences obtained from purified EBF were used to isolate cDNA clones, which by multiple criteria encode EBF. The recombinant polypeptide formed sequence-specific complexes with the EBF-binding site in the mb-1 promoter. The cDNA hybridized to multiple transcripts in pre-B and B-cell lines, but transcripts were not detected at significant levels in plasmacytoma, T-cell, and nonlymphoid cell lines. Expression of recombinant EBF in transfected nonlymphoid cells strongly activated transcription from reporter plasmids containing functional EBF-binding sites. Analysis of DNA binding by deletion mutants of EBF identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors. DNA-binding assays with cotranslated wild-type and truncated forms of EBF indicated that the protein interacts with its site as a homodimer. Deletions delineated a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins. Together, these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression.
The Cdk-associated protein Cks1 functions both in G1 and G2 in Saccharomyces cerevisiae.
Authors: Tang Y , Reed S I
Published: June 2007

The CKS1 gene of Saccharomyces cerevisiae encodes a small essential protein shown to interact genetically and physically with the Cdc28 protein kinase. To investigate the specific functions of the CKS1 gene product, conditional temperature-sensitive mutant alleles were generated. The mutations were found to impair the ability of cells to undergo both the G1/S-phase and G2/M-phase transitions of the cell cycle, as well as the ability to bud. Mutants were not defective, however, in their ability to activate Cdc28 kinase as assayed in vitro on the substrate histone H1. It is likely, therefore, that Cks1 mediates a more specialized function of the Cdc28 kinase such as its ability to form specific multimeric complexes or to localize properly in cellular compartments.
Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit.
Authors: Fang G , Gray J T , Cech T R
Published: June 2007

A telomere-binding protein heterodimer of 56-kD (alpha) and 41-kD (beta) subunits binds to the single-stranded (T4G4)2 terminus of each Oxytricha nova macronuclear DNA molecule. The alpha-subunit by itself binds to telomeric DNA. The beta-subunit alone does not bind to DNA specifically but interacts with the alpha-subunit to form a very stable ternary complex. We show that the formation of alpha-beta-DNA ternary complex is extremely cooperative. Furthermore, the binary complex (alpha-DNA) has a dissociation half-life of much less than 1 min; addition of the beta-subunit increases the half-life to approximately 100 hrs. Libraries of plasmids with random deletions of the open reading frame for the alpha-subunit were introduced into Escherichia coli, and extracts were subsequently checked for both protein expression and DNA-binding activity with or without added beta-subunit. The alpha-subunit was found to contain two structurally separable domains with distinct functions. The amino-terminal two-thirds is necessary and sufficient for sequence-specific DNA binding. The carboxy-terminal one-third is responsible for alpha/beta-subunit interactions. When expressed separately in E. coli, purified, and mixed together, these two domains reconstitute the activity of the wild-type alpha-subunit (trans-complementation in vitro). The amino-terminal two-thirds of the beta-subunit is necessary and sufficient both for alpha/beta-subunit interactions and for ternary complex formation. We conclude that the alpha-subunit of the telomere-binding protein, like many transcription factors, has separable DNA-binding and protein-protein interaction domains.
Multiple functional domains of human transcription factor IIB: distinct interactions with two general transcription factors and RNA polymerase II.
Authors: Ha I , Roberts S , Maldonado E , Sun X , Kim L U , Green M , Reinberg D
Published: June 2007

Transcription factor IIB (TFIIB) plays a pivotal role in the formation of transcription-competent initiation complexes. TFIIB was found to interact with the TATA-binding protein, the small subunit of TFIIF, and RNA polymerase II. These interactions require distinct domains in TFIIB. Using the gel mobility-shift assay, it was found that the amino terminus of TFIIB was necessary for the formation of complexes containing RNA polymerase II and TFIIF, whereas the carboxy-terminal domain, which is composed of two imperfect direct repeats and includes a putative amphipathic alpha-helix, was sufficient for the formation of complexes containing the TATA-binding protein and TFIIB (DB complex). Protein-protein interaction analyses demonstrate that the amphipathic alpha-helix in TFIIB is important for the interaction with the TATA-binding protein. Specific residues mapping to the carboxyl terminus of the second direct repeat were found to be crucial for the interaction of TFIIB and RNA polymerase II. The interaction with the small subunit of TFIIF was mapped to the amino terminus of TFIIB, which includes a zinc finger.
Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design.
Authors: Vinson C R , Hai T , Boyd S M
Published: June 2007

We propose an interhelical salt bridge rule to explain the dimerization specificity between the two amphipathic alpha-helices in the leucine zipper structure. Using the bZIP class of DNA-binding proteins as a model system, we predicted and designed novel dimerization partners. We predicted that ATF4, a member of the ATF/CREB family of transcription factors, would preferentially form heterodimers with IGEBP1, a member of the C/EBP superfamily. These predictions were verified using a gel mobility-shift assay. To further test the value of this interhelical salt bridge rule, we modified the bZIP protein C/EBP attempting to design molecules that would form preferentially heterodimers with C/EBP or molecules that would not interact with C/EBP. These designed molecules behaved as predicted. Therefore, we conclude that this interhelical salt bridge rule is useful in understanding the dimerization specificity of bZIP proteins. In addition, we suggest that this rule could be used to design novel "dominant-negative" molecules to specifically inhibit the function of target leucine zipper proteins in vivo.
A tissue-specific enhancer confers Pit-1-dependent morphogen inducibility and autoregulation on the pit-1 gene.
Authors: Rhodes S J , Chen R , DiMattia G E , Scully K M , Kalla K A , Lin S C , Yu V C , Rosenfeld M G
Published: June 2007

Pit-1 is a tissue-specific POU domain factor obligatory for the appearance of three cell phenotypes in the anterior pituitary gland. Expression of the pit-1 gene requires the actions of a cell-specific 390-bp enhancer, located 10 kb 5' of the pit-1 transcription initiation site, within sequence that proves essential for effective pituitary targeting of transgene expression during murine development. The enhancer requires the concerted actions of a cell-specific cis-active element, Pit-1 autoregulatory sites, and atypical morphogen response elements. Pituitary ontogeny in the Pit-1-defective Snell dwarf mouse reveals that pit-1 autoregulation is not required for initial activation or continued expression during critical phases of Pit-1 target gene activation but, subsequently, is necessary for maintenance of pit-1 gene expression following birth. A potent 1,25-dihydroxyvitamin D3-responsive enhancer element defines a physiological site in which a single nucleotide alteration in the sequence of core binding motifs modulates the spacing rules for nuclear receptor response elements. Unexpectedly, the major retinoic acid response element is absolutely dependent on Pit-1 for retinoic acid receptor function. On this DNA element, Pit-1 appears to function as a coregulator of the retinoic acid receptor, suggesting an intriguing linkage between a cell-specific transcription factor and the actions of morphogen receptors that is likely to be prototypic of mechanisms by which other cell-specific transcription factors might confer morphogen receptor responsivity during mammalian organogenesis.
The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway.
Authors: Sturtevant M A , Roark M , Bier E
Published: June 2007

The rhomboid (rho) gene, which encodes a transmembrane protein, is a member of a small group of genes (ventrolateral genes) required for the differentiation of ventral epidermis in the Drosophila embryo. The ventrolateral genes include spitz, which encodes an EGF-like ligand, and Star. The receptor for spitz may be the gene encoding the Drosophila epidermal growth factor-receptor (Egf-r) because the phenotype resulting from partial loss of function of Egf-r is similar to that of ventrolateral group mutants. Among ventrolateral genes encoding cell-surface or secreted proteins, rho is the only member expressed in a localized pattern corresponding to cells requiring the activity of the ventrolateral pathway. In this paper we provide evidence that spatial localization of rho plays an analogous role in establishing vein pattern in the adult wing. rho is expressed in early wing disc cells likely to be wing vein primordia and later is sharply restricted to developing veins. Flies homozygous for the viable rho(ve) allele have missing veins and rho fails to be expressed in rho(ve) mutant wing discs. Ectopic expression of rho during wing development leads to the formation of extra veins. Gene dosage studies among ventrolateral genes suggest that the rho product (Rho) may facilitate Spi-EGF-R signaling, resulting in activation of RAS. We discuss models for how localized expression of Rho may amplify signaling mediated by ubiquitously distributed ligand and receptor components.
A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization.
Authors: Preuss D , Lemieux B , Yen G , Davis R W
Published: June 2007

Plants distinguish among the pollen grains that land on the stigma, permitting only compatible pollen to fertilize egg cells. To investigate these cell-cell interactions, Arabidopsis mutations that affect pollen-pistil communication were isolated. A male-sterile mutation that disrupts pollen-pistil interactions by eliminating the extracellular pollen coat (tryphine) is described here. Stigma cells that contact the mutant pollen produce callose, a carbohydrate synthesized in response to foreign pollen. The mutant pollen fails to germinate because it does not absorb water from the stigma, yet germinates in vitro, indicating it is viable. The defect is also conditional; high humidity results in pollen hydration and successful fertilization. Analysis of mature, mutant pollen indicated that it is deficient in long-chain lipids and has none of the lipoidic tryphine normally present on its surface. Immature mutant pollen grains have aberrant tryphine that disappears during pollen development. The sterile plants also lack stem waxes, and pollen from other wax-defective (eceriferum) mutants with reduced fertility has few of the lipid droplets normally present in tryphine. These results demonstrate that tryphine is critical for pollen-stigma interactions and suggest that tryphine lipids are required for fertilization, either by directly signaling the stigma or by stabilizing other tryphine components.
polyhomeotic regulatory sequences induce developmental regulator-dependent variegation and targeted P-element insertions in Drosophila.
Authors: Fauvarque M O , Dura J M
Published: June 2007

Variegation of the miniwhite gene is observed in a euchromatic context in transformant lines that contain a P transposon including regulatory sequences of the polyhomeotic (ph) gene upstream of the resident miniwhite gene (P[ph]). This variegated phenotype is not affected by most of the genetic modifiers of heterochromatic position-effect variegation (PEV) nor by removal of the Y chromosome. Interestingly, it is sensitive to ph and Polycomb (Pc) mutations, which are known to affect homeotic gene regulation. Regulatory DNA of ph can also mediate transvection of the miniwhite gene. This transvection is abolished in a ph but not in a zeste mutant background. In addition, P[ph] inserts preferentially in sites corresponding to PH/PC protein-binding sites as defined at the polytene chromosome level. These insertions induce an unusually high proportion of mutations in genes affecting homeotic gene regulation. In particular, one insertion is located within the tramtrack locus, which is thought to regulate fushi tarazu, an Ultrabithorax activator. We suggest that a multimeric complex containing PH and PC proteins, at a minimum, causes a local and clonally inherited heterochromatinization, which maintains the repressed state of transcription of the homeotic genes.
The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.
Authors: Paull T T , Haykinson M J , Johnson R C
Published: June 2007

The mammalian high mobility group proteins HMG1 and HMG2 are abundant, chromatin-associated proteins whose cellular function is not known. In this study we show that these proteins can substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome, an intermediate structure in Hin-mediated site-specific DNA inversion. Formation of this complex requires the assembly of the Hin recombinase, the Fis protein, and three cis-acting DNA sites, necessitating the looping of intervening DNA segments. Invertasome assembly is strongly stimulated by HU or HMG proteins when one of these segments is shorter than 104 bp. By use of ligase-mediated circularization assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently, forming circles as small as 66 bp, and even 59-bp circles at high HMG protein concentrations. In both invertasome assembly and circularization assays, substrates active in the presence of HMG1 contain one less helical turn of DNA compared with substrates active in the presence of HU protein. Analysis of different domains of HMG1 generated by partial proteolytic digestion indicate that DNA-binding domain B is sufficient for both bending and invertasome assembly. We suggest that an important biological function of HMG1 and HMG2 is to facilitate cooperative interactions between cis-acting proteins by promoting DNA flexibility. A general role for HMG1 and HMG2 in chromatin structure is also suggested by their ability to wrap DNA duplexes into highly compact forms.
Hydrophobic coiled-coil domains regulate the subcellular localization of human heat shock factor 2.
Authors: Sheldon L A , Kingston R E
Published: June 2007

HSF2, one of the heat shock transcription factors in mammalian cells, is localized to the cytoplasm during normal growth and moves to the nucleus upon activation. Heat shock transcription factors in metazoans contain four hydrophobic heptad repeat sequences, three in the amino terminus and one in the carboxy terminus, which are predicted to form alpha-helical coiled-coil structures analogous to the leucine zipper. Here, we show that point mutations in either of two amino-terminal zippers or in the carboxy-terminal zipper disrupt normal localization of HSF2 and cause it to be constitutively nuclear. We demonstrate further that two sequences immediately surrounding the amino-terminal zipper domain are required for nuclear localization. These sequences fit the consensus for a bipartite nuclear localization signal (NLS). We suggest that interactions between the amino- and carboxy-terminal zippers normally mask the NLS sequences of HSF2 and that these interactions are disrupted upon activation to expose the NLS sequences and allow transport of HSF2 to the nucleus. We conclude that zipper domains can regulate subcellular localization.
Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts.
Authors: Quelle D E , Ashmun R A , Shurtleff S A , Kato J Y , Bar-Sagi D , Roussel M F , Sherr C J
Published: June 2007

Mammalian D-type cyclins are growth factor-regulated, delayed early response genes that are presumed to control progression through the G1 phase of the cell cycle by governing the activity of cyclin-dependent kinases (cdks). Overexpression of mouse cyclin D1 in serum-stimulated mouse NIH-3T3 and rat-2 fibroblasts increased their rates of G0 to S- and G1- to S-phase transit by several hours, leading to an equivalent contraction of their mean cell generation times. Although such cells remained contact inhibited and anchorage dependent, they manifested a reduced serum requirement for growth and were smaller in size than their normal counterparts. Ectopic expression of cyclin D2 in rodent fibroblasts, either alone or together with exogenous cdk4, shortened their G0- to S-phase interval and reduced their serum dependency, but cyclin D2 alone did not alter cell size significantly. When cells were microinjected during the G1 interval with a monoclonal antibody specifically reactive to cyclin D1, parental rodent fibroblasts and derivatives overexpressing this cyclin were inhibited from entering S phase, but cells injected near the G1/S phase transition were refractory to antibody-induced growth suppression. Thus, cyclin D1, and most likely D2, are rate limiting for G1 progression.
Coupling of cell identity to signal response in yeast: interaction between the alpha 1 and STE12 proteins.
Authors: Yuan Y O , Stroke I L , Fields S
Published: June 2007

In Saccharomyces cerevisiae, the STE12 protein mediates transcriptional induction of cell type-specific genes in response to pheromones. STE12 binds in vitro to the pheromone response elements (PREs) present in the control region of a-specific genes. STE12 is also required for transcription of alpha-specific genes, but there is no evidence that it binds directly to these genes. Instead, the MAT alpha-encoded protein alpha 1 and the MCM1 product bind to the DNA element that is responsible for alpha-specific and a-factor-inducible expression. To explore the role of STE12 in the pheromone induction of alpha-specific genes, we cloned STE12 and MAT alpha 1 homologs from the related yeast Kluyveromyces lactis. The K. lactis STE12 protein did not cooperate with the S. cerevisiae alpha 1 protein to promote the overall mating process or the induction of transcription of an alpha-specific gene. However, introduction of both K. lactis STE12 along with K. lactis alpha 1 did restore mating, suggesting that an interaction between STE12 and alpha 1 is important for alpha-specific gene activation. We also show that bacterially expressed STE12 and alpha 1 are able to form a complex in vitro. Thus, we demonstrate a coupling in alpha cells between a protein functioning in cell identity, alpha 1, with a protein responsive to the pheromone-induced signal STE12.
Sequence-specific endonucleolytic cleavage and protection of mRNA in Xenopus and Drosophila.
Authors: Brown B D , Zipkin I D , Harland R M
Published: June 2007

Previously, a small region of the 3'-untranslated region (3' UTR) of Xlhbox2B mRNA was shown to be sufficient for sequence-specific endonucleolytic cleavage after injection into Xenopus oocytes. Here, we report an in vitro RNA degradation reaction that mimics the in vivo result accurately. The reaction also reveals that oocytes contain a sequence-specific RNA-binding factor that inhibits the endoribonuclease. These opposing activities may be regulated during Xenopus oogenesis. Partial purification shows that the endonuclease does not require translation or ribosomes and does not resemble previously described RNA processing complexes. We have isolated another Xenopus cDNA, Xoo1, that contains a long, repetitive destabilizing element similar to the one in Xlhbox2B. Based on a comparison of these natural destabilizing sequences and in vitro mutagenesis experiments, we find that a single destabilizing site is, at most, 19 bases in length and that the endonuclease and protective factor recognition sites may be overlapping subsets of this sequence. Finally, we show that Drosophila embryos contain similar activities, each of which can use Xenopus recognition sites. This level of conservation suggests an important biological function for this system of post-transcriptional regulation.
Spatial restriction of cellular differentiation.
Authors: Sager B , Kaiser D
Published: June 2007

Myxococcus xanthus cells differentiate into myxospores within a fruiting body, an aggregate of approximately 10(5) cells. Previous work had discerned an inner and outer domain within the fruiting body differentiated by cell density and cell alignment. To test whether the two domains might play different roles in spore differentiation, developmentally regulated gene fusions were screened for expression restricted to one domain or the other. Transcriptional lacZ fusions to 80 developmentally regulated genes were examined and eight fusions were found that restricted expression to the inner domain, while one fusion, omega 7621, showed initial expression in the outer domain. Initial omega 7621 expression coincided with patches of spore precursors evident in bright-field microscopy. Later in development, both omega 7621 expression and the patches expanded inward, eventually filling both the inner and outer domains. Previous work had also shown that high cell density and cell alignment are required for transmission of the C-signal, which is needed to initiate spore differentiation. Evidence is presented for a novel morphogenetic mechanism in which C-signaling in the outer (high density) domain initiates spore differentiation. It is proposed that spore precursors are passively transported from the outer to the inner domain by the movements of undifferentiated rod cells. Reconstruction experiments showed that developing rod cells move with sufficient force to displace spores. Spore precursors thus accumulate in the inner domain where they express spore-specific genes at high levels and account for inner domain specific expression.
Interactions between dorsal and helix-loop-helix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophila.
Authors: González-Crespo S , Levine M
Published: June 2007

The dorsal (dl) morphogen has been implicated in the establishment of the embryonic mesoderm, neuroectoderm, and dorsal ectoderm in Drosophila. Here we show that the simultaneous reduction in the levels of dl and any one of several helix-loop-helix (HLH) proteins results in severe disruptions in the formation of mesoderm and neuroectoderm. Certain triple heterozygous combinations essentially lack mesoderm as a result of a block in ventral furrow formation during gastrulation. HLH proteins that have been implicated previously in sex determination and neurogenesis (daughterless, achaete, and scute) are shown to be required for the formation of these embryonic tissues. Evidence is also presented that dl-HLH interactions involve the direct physical association of these proteins in solution mediated by the rel and HLH domains. We discuss the striking parallels in mesoderm formation and sex determination.
Masking mRNA from translation in somatic cells.
Authors: Ranjan M , Tafuri S R , Wolffe A P
Published: June 2007

We present evidence for a role for Y-box proteins in both mRNA transcription and translation. We have expressed an oocyte-specific RNA-binding protein (FRGY2) in somatic cells. This protein is normally found together with mRNA in large amounts within the masked maternal mRNA storage particles of Xenopus laevis oocytes. Masked maternal mRNA is believed to be translationally repressed during oogenesis and is stored within ribonucleoprotein storage particles for subsequent use during embryogenesis. Surprisingly, FRGY2 is not only found to direct the accumulation of mRNA from promoters containing binding sites for the protein but also to selectively repress translation of the same mRNA. Thus, the role of FRGY2 in the transcription and storage of maternal mRNA is similar to that of the transcription factor TFIIIA in the transcription and storage of 5S rRNA from the oocyte type 5S rRNA genes.
Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52.
Authors: Milne G T , Weaver D T
Published: June 2007

Saccharomyces cerevisiae rad52 mutants are characterized by severe defects in double-strand break (DSB) repair and recombination. In this study we have identified several regions of RAD52 that are required for these biological functions. We cloned and characterized a RAD52 homolog from Kluyveromyces lactis that partially complemented S. cerevisiae rad52 mutants while exhibiting negative dominance in wild-type (RAD52) strains. The dominant negative effect was suppressed by overexpression of RAD51, an additional gene known to be required for DSB repair and recombination, indicating a genetic interaction between these loci. Furthermore, GAL4 two-hybrid analysis revealed a physical interaction between Rad51 and the carboxy-terminal one-third of Rad52. Deletion alleles of rad52 (with or without the Rad51 association domain) also produced dominant negative defects, suggesting the disruption of repair through nonfunctional interactions with other DSB repair and recombination proteins. RAD51 relieved the negative dominance of each of these alleles either by competitive titration or functional activation of mutant or heterologous Rad52 proteins. These results demonstrate the importance of Rad52-Rad51 interactions and point to the formation of a higher order repair/recombination complex potentially containing other yet unidentified components.
Transcription-dependent recombination induced by triple-helix formation.
Authors: Kohwi Y , Panchenko Y
Published: June 2007

The homologous recombination between direct repeat sequences separated by either 200 or 1000 bp was induced by active transcription of the downstream gene when poly(dG)-poly(dC) sequences exist between the two direct repeats. This dG tract-mediated and transcription-induced recombination was RecA independent, and the frequency of recombination was dependent on both the length and the orientation of the poly(dG)-poly(dC) sequences relative to the gene. An intramolecular dG.dG.dC triplex formation was detected in Escherichia coli cells in a length-dependent manner when the transcription of the downstream gene was activated. We suggest that the negative superhelical strain generated by active transcription of the downstream gene induces poly(dG)-poly(dC) sequences to adopt a triple-helix structure in vivo and that this structure brings two remote sequences together to stimulate homologous recombination.
odd-paired: a zinc finger pair-rule protein required for the timely activation of engrailed and wingless in Drosophila embryos.
Authors: Benedyk M J , Mullen J R , DiNardo S
Published: June 2007

The pair-rule gene, odd-paired (opa), is essential for parasegmental subdivision of the Drosophila embryo. In addition to its previously defined role in the activation of wingless (wg) in odd parasegments, we find that opa is required for the timely activation of wg in the remaining parasegments and for the timely activation of engrailed (en) in all parasegments. opa encodes a zinc finger protein with fingers homologous to those of the Drosophila segment polarity gene ciD, the human glioblastoma gene GLI and the Caenorhabditis elegans sex determination gene tra-1. Previous work showed that opa activity was essential for the establishment of alternate parasegments, suggesting opa expression or activity would be spatially restricted like other pair-rule genes. Instead, opa mRNA and protein are found throughout all segment primordia. Thus, opa does not act in a spatially restricted manner to establish the position of en and wg expression. Rather, opa must cooperate with other spatially restricted proteins to achieve proper subdivision of the Drosophila embryo.
The Drosophila segment polarity gene dishevelled encodes a novel protein required for response to the wingless signal.
Authors: Klingensmith J , Nusse R , Perrimon N
Published: June 2007

The Drosophila Wnt-1 homolog, wingless (wg), is involved in the signaling of patterning information in several contexts. In the embryonic epidermis, Wg protein is secreted and taken up by neighboring cells, in which it is required for maintenance of engrailed transcription and accumulation of Armadillo protein. The dishevelled (dsh) gene mediates these signaling events as well as wg-dependent induction across tissue layers in the embryonic midgut. dsh is also required for the development processes in which wg functions in adult development. Overall, cells lacking dsh are unable to adopt fates specified by Wg. dsh functions cell autonomously, indicating that it is involved in the response of target cells to the Wg signal. dsh is expressed uniformly in the embryo and encodes a novel protein with no known catalytic motifs, although it shares a domain of homology with several junction-associated proteins. Our results demonstrate that dsh encodes a specific component of Wg signaling and illustrate that Wnt proteins may utilize a novel mechanism of extracellular signal transduction.
A novel POU domain gene, zebrafish pou2: expression and roles of two alternatively spliced twin products in early development.
Authors: Takeda H , Matsuzaki T , Oki T , Miyagawa T , Amanuma H
Published: June 2007

POU domain proteins are a large family of transcriptional regulatory proteins, many of which are implicated in the control of gene expression during early development. We describe here the cloning and expression of zebrafish pou2, a novel POU domain gene related to the mouse germ-line-specific transcription factor oct-3. Zebrafish pou2 is maternally expressed, and the transcripts are present from the one-cell stage to the gastrula stage. In situ hybridization analyses revealed that the transcripts were present in all blastomeres until the midblastula stage and that the expression was restricted to the epiblast during gastrulation. We found that alternatively spliced transcripts, t-pou2 RNAs, were also expressed in the embryos. In contrast to the Pou2 product, the t-Pou2 product lacks DNA-binding activity because of its incomplete POU domain structure. To examine the roles of the Pou2 and t-Pou2 products, we increased their expression in the embryo by microinjection of synthetic pou2 and t-pou2 RNAs into the fertilized eggs at the one-cell stage. Most embryos that developed from the eggs injected with pou2 RNA did not show any obvious developmental defects. In contrast, overexpression of the t-Pou2 product greatly affected the embryonic development: There was strong developmental retardation or arrest due to the incomplete gastrulation. In the affected embryos, expression of zebrafish T gene was reduced and the hypoblast formation was disturbed. Temporal and spatial expression patterns and the effects of overexpression of these products on development are consistent with the idea that the Pou2 and t-Pou2 proteins are involved in early development of zebrafish embryos. They may be involved in the proliferation of blastomeres in undetermined state at the blastula stage and/or the early cell commitment events at the gastrula stage. Also, our results indicate that different products generated as a result of alternative splicing from the same gene possess distinct functional capacities.
Anabaena xisF gene encodes a developmentally regulated site-specific recombinase.
Authors: Carrasco C D , Ramaswamy K S , Ramasubramanian T S , Golden J W
Published: June 2007

Two DNA elements are excised from the chromosome during Anabaena heterocyst differentiation. We have identified the gene xisF which encodes the site-specific recombinase responsible for the excision of a 55-kb element from within the fdxN gene. The cloned xisF gene is sufficient to cause site-specific rearrangement of an artificial substrate in Escherichia coli. Inactivation of xisF in the Anabaena chromosome prevents excision of the fdxN element and growth in nitrogen-deficient medium but does not alter the development of heterocysts. Forced transcription of xisF in vegetative cells did not result in excision of the fdxN element, suggesting that other factors may be involved in cell-type specificity. The predicted XisF protein shows significant similarity to the Bacillus subtilis SpoIVCA recombinase.
A weak germ-line excision mutation blocks developmentally controlled amplification of the rDNA minichromosome of Tetrahymena thermophila.
Authors: Kapler G M , Blackburn E H
Published: June 2007

During development of the somatic macronucleus of Tetrahymena thermophila, the rDNA is excised from its germ-line chromosome, rearranged into a palindrome, and amplified to 10(4) copies. We have identified a cis-acting germ-line mutation, rmm11/6, that prevents amplification of the rDNA in all but approximately 1 in 10(5) cells when it is the only rDNA allele in the developing macronucleus. The rmm11/6 mutation resides in a conserved element required for excision, the chromosome breakage sequence (Cbs) flanking the 3' end of the rDNA. Surprisingly, the rmm11/6 mutation only weakly affects excision of the rDNA from its germ-line location; at least 25% of cells heterozygous for this mutation correctly excise the affected rDNA allele. In heterozygotes, when this rDNA allele is excised, it is also poorly amplified. The rDNA amplification defect caused by this mutation is not overcome by delaying amplification with the DNA synthesis inhibitor aphidicolin, indicating that rDNA excision and amplification are not experimentally separable. Our experiments provide the first evidence that the capacity to amplify the rDNA is restricted in the developing macronucleus. We propose that the rmm11/6 mutation delays excision of the rDNA and that the developmental progression of the macronucleus past a restricted window for amplification is responsible for the severe amplification defect caused by this weak rDNA excision mutation.
The Doa locus encodes a member of a new protein kinase family and is essential for eye and embryonic development in Drosophila melanogaster.
Authors: Yun B , Farkas R , Lee K , Rabinow L
Published: June 2007

Mutations at the Darkener of apricot (Doa) locus of Drosophila cause roughened eyes and increase transcript accumulation from the retrotransposon copia up to fourfold. Cloning of the gene and sequencing of cDNAs reveals that it encodes a putative serine/threonine protein kinase. Sequence data base searches identify it is a member of a novel highly conserved protein kinase family, with homologs in humans, mice, and Saccharomyces cerevisiae, not related to each other previously. Family members are characterized by a peptide motif reading EHLAMMERILG at kinase subdomain X, which is virtually 100% identical in all homologs. We therefore refer to this new family as the LAMMER protein kinases. As predicted from its primary sequence, Doa protein possess intrinsic protein kinase activity when expressed in bacteria, as assayed via autophosphorylation. The gene is expressed throughout development, and both stage and tissue-specific RNAs are found. Its function is essential, because maternally deposited or zygotically transcribed mRNA is required for development to larval stages, and defects in segmentation and development of the nervous system are observed in embryos derived from heteroallelic mothers. Doa function is also critical to Drosophila eye development, because the organization and development of pigment cells, bristles, and photoreceptors are affected in various mutant classes. In the most extreme cases that survive to adulthood, retinal photoreceptors degenerate prior to eclosion. These results demonstrate that the kinase encoded by Doa is required at multiple stages of development, for both differentiation and maintenance of specific cell types.