bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoarchitecture and the patterning of fushi tarazu expression in the Drosophila blastoderm.
Authors: Edgar B A , Odell G M , Schubiger G
Published: June 2007

In the Drosophila embryo at the blastoderm stage, the segmentation gene fushi tarazu (ftz) is expressed in a seven-banded pattern. The generation of this pattern, like many other segmentation gene expression patterns, coincides with the formation of cell membranes around the blastoderm nuclei. To test the role of cellularization in resolving the banded ftz pattern, we used cytoskeletal inhibitors (colcemid and cytochalasin B) to block cellularization. We found that banded ftz RNA and protein patterns can form without cellular structure. We also tested the importance of rapid degradation of the ftz RNA, using cycloheximide to block degradation. RNA degradation is essential to maintain the banded ftz pattern in a syncytium, but is not required to maintain the pattern in a cellularized embryo. A latticework of cytoskeletal microtubules that forms during cellularization appears to be a key component in localizing the ftz mRNA. We conclude that RNA degradation and cellular structure normally work together to localize ftz RNA to its sites of synthesis.
Spec3: embryonic expression of a sea urchin gene whose product is involved in ectodermal ciliogenesis.
Authors: Eldon E D , Angerer L M , Angerer R C , Klein W H
Published: June 2007

We have characterized the temporal and spatial expression of Spec3 mRNA in embryos of the sea urchin, Strongylocentrotus purpuratus. This mRNA, 2.0 kb in length, is present at low levels in unfertilized eggs but accumulates rapidly during cleavage, increasing 50-fold by hatching blastula stage. Message levels then decline abruptly, remain constant during mesenchyme blastula and gastrula stages, and increase again during prism and pluteus stages. This accumulation pattern is quite similar to that of the ectodermally expressed beta-tubulin mRNAs described recently by Harlow and Nemer (1987a). In situ hybridization shows that although Spec3 message accumulates in all blastomeres at early blastula stages, it later becomes restricted to ectoderm. By late blastula stage, hybridization is strongest in the animal hemisphere. At gastrula, signals are variable over ectoderm, and by pluteus, grains are concentrated in the ciliary band, though present in other ectodermal cells as well. Deciliation and regeneration of cilia in gastrula-stage embryos results in a four- to fivefold increase in Spec3 mRNA levels, implying that the Spec3 gene product is associated with ciliogenesis. Spec3 mRNA is encoded by a single gene in the haploid genome, and characterization of the gene shows that it contains three exons that encode an open reading frame for a hydrophobic protein of 21.6 kD. The reading frame reveals that the carboxy-terminal part of the protein contains two long hydrophobic stretches, 31 and 37 residues long, separated by short hydrophilic regions of six to eight residues. The presence of these two distinct hydrophobic stretches suggests that the Spec3 protein contains two alpha-helical domains that either span the lipid bilayer or are associated with some other hydrophobic environment.
Coordinate and selective beta-tubulin gene expression associated with cilium formation in sea urchin embryos.
Authors: Harlow P , Nemer M
Published: June 2007

beta-Tubulin mRNAs associated with cilium formation in Strongylocentrotus purpurpatus sea urchin embryos are expressed selectively from a multiple gene family. The accumulations of three beta-tubulin mRNAs (beta 1, beta 2, and beta 3) are temporally coordinated with ciliogenesis during blastula development and with the regeneration of cilia after their amputation. In contrast, another beta-tubulin mRNA, beta 4, is not induced in either case. The zinc-animalized embryo with its exaggerated blastula phenotype forms longer cilia through a protracted period of ciliogenesis, in which the beta-tubulin mRNAs, principally beta 1, accumulate to higher than normal levels. The rate of beta-tubulin transcription per nucleus in the animalized embryo is greater than that of the normal embryo and is not changed through deciliation, although the tubulin mRNAs accumulate to higher levels. However, deciliation raises the beta-tubulin transcription rate in the normal embryo to that in the animalized embryo. Thus, the induction of beta-tubulin mRNA by cilium amputation is regulated transcriptionally in the normal embryo, but post-transcriptionally in the zinc-animalized embryos. Moreover, the beta-tubulin genes that are expressed in association with cilium formation appear to be induced selectively within the framework of ectodermal cell-type specificity.
Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses.
Authors: Johnson P F , Landschulz W H , Graves B J , McKnight S L
Published: June 2007

This report describes the identification and purification of a nuclear protein from rat liver that binds selectively to DNA sequences associated with several animal virus enhancers. The binding activity was tracked by direct DNase I footprinting through four steps of biochemical fractionation. These procedures led to the identification of a polypeptide species exhibiting an apparent molecular weight of 20 kD that accounts for enhancer binding activity. DNase I and dimethyl sulfate footprinting assays were used to examine the manner in which the purified protein binds to enhancer elements associated with SV40, murine sarcoma virus, and polyoma virus. The results of these assays indicate that the initial interaction established between the 20-kD protein and each viral enhancer occurs via a common DNA sequence known as the enhancer core homology.
Developmental and tissue-specific regulation of beta-tubulin gene expression in the embryo of the sea urchin Strongylocentrotus purpuratus.
Authors: Harlow P , Nemer M
Published: June 2007

Four beta-tubulin mRNAs in the embryo of the sea urchin Strongylocentrotus purpuratus are transcribed from at least 3 of the 9-12 beta-tubulin genes. A beta 1 tubulin mRNA of 1.8 kb, transcribed from a unique beta 1 gene, is expressed with high specificity in the pluteus ectoderm. Another 1.8-kb mRNA, beta 2, and a 2.5-kb beta 3 mRNA are moderately ectoderm specific. In contrast, a 3.0-kb beta 4 mRNA is highly specific for the endomesoderm tissue fraction. Certain similarities in developmental and tissue-specific expression suggest that these beta-tubulin genes may be related in their mode of regulation to counterparts among the genes for actin, another cytoskeletal protein. Measurements of absolute amounts revealed a distinct developmental profile for each beta-tubulin mRNA. An increase in the total amount of beta-tubulin mRNA in the early blastula was correlated with an increase in transcription rate per nucleus; whereas, later in the mesenchyme blastula stage, the beta-tubulin mRNA level decreased sharply as the rate of beta-tubulin gene transcription on a per embryo basis remained constant. Thus, during development through the blastula stages, there was a switch to a predominantly posttranscriptional regulation of beta-tubulin mRNA expression, probably through a decrease in mRNA stability.
Investigation of chromosome-mediated gene transfer using the HPRT region of the human X chromosome as a model.
Authors: Pritchard C A , Goodfellow P N
Published: June 2007

A panel of over 50 hybrid cells containing varying portions of the long arm of the human X chromosome have been obtained by chromosome-mediated gene transfer (CMGT) of human chromosomes to mouse cells deficient in HPRT. This panel is used to investigate the size and integrity of transfected human chromosome fragments and also to examine the effect of including a selectable DNA plasmid in the transfection mix. Chromosomal rearrangements are found to be generated in the chromosome transfer process, and the human X centromeric region is detected in the transfected cells at an unusually high frequency. Extensive lengths of X chromosome DNA are transferred intact, suggesting potential uses of CMGT in cloning large genes and loci for which only the chromosomal map position is known.
The first 28 amino acids of mature LamB are required for rapid and efficient export from the cytoplasm.
Authors: Rasmussen B A , Silhavy T J
Published: June 2007

Our laboratory has been utilizing the Escherichia coli outer membrane protein LamB to study the mechanism of protein localization. Various lines of evidence suggest that, in addition to a signal sequence, regions within the mature protein are required for efficient localization. In particular, studies using LamB-LacZ hybrid proteins have identified regions between amino acids 27 and 49 of mature LamB, which may play an important role in localization. To elucidate further the function of these regions, a series of in-frame deletions that remove varying lengths of early lamB sequences was constructed. The effects of these deletions on export of a large LamB-LacZ hybrid protein, 42-1, and on export of an otherwise wild-type LamB protein were determined. We find a strong correlation between the sequences deleted and the export phenotypes these deletions impart to both LamB and the LamB-LacZ42-1 hybrid protein. On the basis of these findings, the deletions can be divided into several distinct classes that define a region within mature LamB that participates in localization. This region extends amino terminally from amino acid 28 of the mature protein and functions in the rapid and efficient localization of LamB from the cytoplasm.
Characterization of the altered form of the c-src gene product in neuronal cells.
Authors: Brugge J , Cotton P , Lustig A , Yonemoto W , Lipsich L , Coussens P , Barrett J N , Nonner D , Keane R W
Published: June 2007

The pp60c-src protein that is expressed at high levels in cultures of neurons from rat embryos displays an altered mobility on SDS-polyacrylamide gels due to a structural difference in the amino-terminal region of the molecule. In this report we show that the expression of this unique form of pp60c-src, designated pp60c-src(+), is not restricted to cultured neuronal cells since the pp60c-src molecules expressed in tissues from avian and rat neural tissues also display a retarded electrophoretic mobility. The amino-terminal region from pp60c-src(+) was found to contain a novel phosphorylated tryptic peptide that contains phosphoserine. However, this phosphorylation does not appear to be responsible for the retarded electrophoretic mobility of pp60c-src(+), since the mobility of this protein is not altered by phosphatase treatment under conditions that remove greater than 95% of the radiolabeled phosphate on pp60c-src(+). The altered electrophoretic form of pp60c-src was also shown to be radiolabeled with [3H]myristate, indicating that pp60c-src is fatty-acylated in neurons, as is pp60c-src in fibroblasts. The pp60c-src molecules synthesized in vitro using rabbit reticulocyte lysates programmed with mRNA from embryonic brain migrated more slowly on SDS-polyacrylamide gels than the pp60c-src protein that was translated in vitro using RNA from embryonic limb tissue. These results suggest the possibility that the c-src mRNA expressed in neurons may undergo a unique form of processing to generate the structurally distinct form of neuronal pp60c-src(+).
Deletion and duplication of DNA sequences is associated with the embryonic lethal phenotype of the t9 complementation group of the mouse t complex.
Authors: Búcan M , Herrmann B G , Frischauf A M , Bautch V L , Bode V , Silver L M , Martin G R , Lehrach H
Published: June 2007

We have analyzed the genomic structure of three mouse t haplotypes of the t9 complementation group. Each of these t haplotypes, tw18, t4, and tks1, is known to have resulted from a rare recombination event between a complete t haplotype and a wild-type chromosome. Using molecular probes that identify sequences in the distal portion of the t complex, we have shown that each of these t haplotypes contains a similar (perhaps identical) deletion of one group of t complex sequences, and duplication of another group. These data suggest that the recombination events that produced these three t haplotypes involved similar unequal crossovers within the distal inversion. The deletion and duplication of genetic material associated with all members of the t9 complementation group tested provides a molecular explanation for the recessive lethal mutation associated with these t haplotypes.
The expression and regulation of Sex combs reduced protein in Drosophila embryos.
Authors: Riley P D , Carroll S B , Scott M P
Published: June 2007

Homeotic genes are expressed in spatially precise patterns during Drosophila development to control segmental differentiation. The Sex combs reduced (Scr) gene of the Antennapedia gene complex is involved in the determination of the labial and prothoracic segments of the embryo. To study both the wild-type pattern of Scr expression and the regulatory relationships of Scr to other regulatory genes, an antibody probe that detects the Scr protein was prepared. We find that the Scr gene product is expressed in a dynamic pattern over the course of embryogenesis, beginning in the ectoderm in parasegment 2 while the germ band is elongated and extending to parasegment 3 during the completion of germ band shortening. The locations of Scr protein correlate well with the part of the embryo that are altered in Scr- mutants. After head involution occurs, Scr protein is also expressed in the ganglion corresponding to parasegment 2 of the ventral nervous system. The precise spatial expression of Scr is attained through regulation by both homeotic genes and segmentation genes. The lack of proper Antennapedia or Polycomb gene function causes ectopic Scr protein expression. Mutations in the segmentation genes fushi tarazu, hunchback, Krüppel, and giant alter the spatial pattern of Scr expression.
Developmental stage specificity of the lymphoid V(D)J recombination activity.
Authors: Lieber M R , Hesse J E , Mizuuchi K , Gellert M
Published: June 2007

We have examined the level of immunoglobulin gene V(D)J recombination activity in a number of cell lines derived from lymphoid or nonlymphoid lineages. The assay we employed uses extrachromosomal DNA as substrate and thereby avoids difficulties associated with the use of chromosomally integrated substrates. The recombination activity decreases during B-lymphoid development. It is highest at the earliest stages of committed B-cell differentiation and then falls progressively, reaching undetectable levels at the mature B-cell stage. The activity is also present in multipotential progenitors of myeloid cells and in pre-T cells but not mature T cells. No activity was found in several nonhematopoietic cell lines. Recombination was seen only among substrate molecules which had replicated in the eukaryotic cells. Several possible interpretations of this result are discussed.
In vitro binding of several cell-specific and ubiquitous nuclear proteins to the GT-I motif of the SV40 enhancer.
Authors: Xiao J H , Davidson I , Macchi M , Rosales R , Vigneron M , Staub A , Chambon P
Published: June 2007

We have investigated the specific in vitro binding of nuclear proteins from several cell lines to the GT-I motif of the SV40 enhancer which overlaps with the canonical enhancer "core" homology. The binding of three proteins (GT-IA, GT-IB, and GT-IC), one of which (GT-IC) exhibits cell specificity, was detected. Competition and direct binding experiments demonstrated that the two ubiquitous proteins also bind to the GC-rich motif III from the 21-bp repeat upstream element of the SV40 early promoter and that protein GT-IA is most probably the transcription factor Sp1. The third, cell-specific protein GT-IC exhibited a high affinity for both the GT-I motif and an upstream element in the promoter of the mouse beta-major-globin gene, suggesting that this protein can act both as an enhancer and an upstream element trans-acting factor. The good correlation between the known cell-specific in vivo activity of the wild-type and mutated GT-I motif and the cell-specific binding of protein GT-IC in vitro strongly supports the conclusion that this protein is an enhancer factor. Interestingly, its cognate recognition sequence does not coincide with the core homology.
The ovo locus is required for sex-specific germ line maintenance in Drosophila.
Authors: Oliver B , Perrimon N , Mahowald A P
Published: June 2007

Mutations at the ovo locus result in a defective female germ line. The male germ line is not affected. Adult females homozygous for loss-of-function alleles have no germ line stem cells. The sex-specific phenotype is evident at late blastoderm and early gastrula stages when the pole cells of embryos homozygous for a loss-of-function allele begin to die. This is the only zygotically acting gene known that is required specifically for embryonic germ line survival. Females heterozygous for dominant alleles or homozygous for alleles reducing gene activity exhibit a range of defects in oogenesis. We have mapped the ovo locus to position 4E1-2 of the salivary gland X chromosome by using a set of cytologically visible deletions.
Complementary patterns of even-skipped and fushi tarazu expression involve their differential regulation by a common set of segmentation genes in Drosophila.
Authors: Frasch M , Levine M
Published: June 2007

We have examined the position-dependent expression of two segmentation genes that contain a homeo box, even-skipped (eve) and fushi tarazu (ftz). Products encoded by both genes accumulate in a series of seven transverse stripes along the anterior-posterior body axis of developing embryos. Double-staining experiments show that eve and ftz proteins accumulate in complementary sets of embryonic cells. A total of eight zygotically active segmentation genes are required for the establishment and refinement of the eve and ftz expression patterns during development. Mutations that affect one of the genes were always found to produce a reciprocal alteration in the other. These results suggest that the eve and ftz promoters independently "interpret" the same positional cues to give differential patterns of expression. We propose that a combination of segmentation gene products that activates the expression of eve represses the expression of ftz, and vice versa. This proposal is discussed in the context of a hierarchy of interactions among segmentation genes, whereby the gap genes establish asymmetric patterns of eve and ftz expression in blastoderm-stage embryos, and interactions with other pair-rule genes serve to refine further the complementarity of eve and ftz expression during gastrulation and germ band elongation.
The Dictyostelium MAP kinase ERK2 regulates multiple, independent developmental pathways.
Authors: Gaskins C , Clark A M , Aubry L , Segall J E , Firtel R A
Published: June 2007

We showed previously that the MAP kinase ERK2 is essential for aggregation. erk2 null cells lack cAMP stimulation of adenylyl cyclase and thus cannot relay the cAMP chemotactic signal, although the cells chemotax to cAMP (Segall et al. 1995). In this paper we have examined the role of ERK2 in controlling developmental gene expression and morphogenesis during the multicellular stages, making use of a temperature-sensitive ERK2 mutation. Using suspension assays, we show that ERK2 is not essential for aggregation-stage, cAMP pulse-induced gene expression, or for the expression of postaggregative genes, which are induced at the onset of mound formation in response to cAMP in wild-type cells. In contrast, the prespore-specific gene SP60 is not induced and the prestalk-specific gene ecmA is induced but at a significantly reduced level. Chimeric organisms, comprised of wild-type and erk2 null cells expressing the prestalk-specific ecmA/lacZ reporter, show an abnormal spatial patterning, in which Erk2ts/erk2 cells are excluded from the very anterior prestalk A region. To further examine the function of ERK2 during the multicellular stages, we bypassed the requirement of ERK2 for aggregation by creating an ERK2 temperature-sensitive mutant. erk2 null cells expressing the ERK2ts mutant develop normally at 20 degrees C and express cell-type-specific genes but do not aggregate at temperatures above 25 degrees C. Using temperature shift experiments, we showed that ERK2 is essential for proper morphogenesis and for the induction and maintenance of prespore but not prestalk gene expression. Our results indicate that ERK2 functions at independent stages during Dictyostelium development to control distinct developmental programs: during aggregation, ERK2 is required for the activation of adenylyl cyclase and during multicellular development, ERK2 is essential for morphogenesis and cell-type-specific gene expression. Analysis of these results and other supports the conclusion that the requirement of ERK2 for cell-type differentiation is independent of its role in the activation of adenylyl cyclase.
Drosophila TFIIA-S is up-regulated and required during Ras-mediated photoreceptor determination.
Authors: Zeidler M P , Yokomori K , Tjian R , Mlodzik M
Published: June 2007

Photoreceptor induction in the developing Drosophila eye is triggered by the activation of the Ras pathway. Subsequently, the Ras-mediated activation of site-specific transcription factors leads to the expression of putative "effector" genes. The coactivator function of the basal transcription factor TFIIA has been shown previously to enhance the trans-activation potential of site-specific transcription factors in vitro. Here, we show that the expression of the small subunit of TFIIA (dTFIIA-S) is specifically up-regulated in a transient manner during Ras-mediated photoreceptor induction. Furthermore, although null mutations in dTFIIA-S are cell lethal, a hypomorphic dTFIIA-S allele demonstrates an increased requirement for this factor during photoreceptor development. In addition, the cone cell to R7 photoreceptor transformation caused by ectopic activation of the Ras pathway during eye development is suppressed by the removal of one functional copy of the dTFIIA-S locus revealing the sensitivity of this process to reductions in dTFIIA-S activity. These results are the first in vivo evidence for the coactivator function in transcriptional enhancement proposed for TFIIA.
Abnormal spermatogenesis in RXR beta mutant mice.
Authors: Kastner P , Mark M , Leid M , Gansmuller A , Chin W , Grondona J M , Décimo D , Krezel W , Dierich A , Chambon P
Published: June 2007

We have generated mouse lines in which the RXR beta gene was disrupted by homologous recombination. Approximately 50% of the RXR beta homozygous mutants died before or at birth, but those that survived appeared normal except that the males were sterile, owing to oligo-astheno-teratozoospermia. Failure of spermatid release occurred within the germinal epithelium, and the epididymis contained very few spermatozoa that, in addition, exhibited abnormal acrosomes and tails. There was a progressive accumulation of lipids within the mutant Sertoli cells, which were histochemically characterized as unsaturated triglycerides. In old mutant males, progressive degeneration of the germinal epithelium occurred, ending with the formation of acellular lipid-filled tubules. The selective expression of RXR beta in Sertoli cells, together with the timing of appearance of the histological abnormalities, suggests that the primary defect resulting from the mutation resides in these cells.
Repression domain of the yeast global repressor Tup1 interacts directly with histones H3 and H4.
Authors: Edmondson D G , Smith M M , Roth S Y
Published: June 2007

Repression of yeast a cell-specific genes by the global repressor Ssn6/Tup1 has been linked to a specific organization of chromatin. We report here that Tup1 directly interacts with the amino-terminal tails of histones H3 and H4, providing a molecular basis for this connection. This interaction appears to be required for Tup1 function because amino-terminal mutations in H3 and H4 that weaken interactions with Tup1 cause derepression of both a cell-specific and DNA damage-inducible genes. Moreover, the Tup1 histone-binding domain coincides with the previously defined Tup1 repression domain. Tup1/histone interactions are negatively influenced by high levels of histone acetylation, suggesting a mechanism whereby the organization of chromatin may be modulated in response to changing environmental signals.
Intracellular receptors use a common mechanism to interpret signaling information at response elements.
Authors: Starr D B , Matsui W , Thomas J R , Yamamoto K R
Published: June 2007

The glucocorticoid receptor (GR) activates transcription in certain glucocorticoid response element (GRE) contexts, and represses or displays no activity in others. We isolated point mutations in one GRE, plfG, at which GR activated transcription under conditions in which the wild-type element was inactive or conferred repression, implying that GREs may carry signals that are interpreted by bound receptors. Consistent with this notion, we identified a mutant rat GR, K461A, which activated transcription in all GRE contexts tested, implying that this residue is important in interpretation of GRE signals. In a yeast screen of 60,000 GR mutants for strong activation from plfG, all 13 mutants isolated contained substitutions at K461. This lysine residue is highly conserved in the zinc-binding region (ZBR) of the intracellular receptor (IR) superfamily; when it was mutated in MR and RARbeta, the resulting receptors similarly activated transcription at response elements that their wild-type counterparts repressed or were inactive. We suggest that IR response elements serve in part as signaling components, and that a critical lysine residue serves as an allosteric "lock" that restricts IRs to inactive or repressing configurations except in response element contexts that signal their conversion to transcriptional activators. Therefore, mutation of this residue produces altered receptors that activate in many or all response element contexts.
Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein.
Authors: Marcand S , Buck S W , Moretti P , Gilson E , Shore D
Published: June 2007

Rap1p binds to silencer elements and telomeric repeats in yeast, where it appears to initiate silencing by recruiting Sir3p and Sir4p to the chromosome through interactions with its carboxy-terminal domain. Sir3p and Sir4p interact in vitro with histones H3 and H4 and are likely to be structural components of silent chromatin. We show that targeting of these Sir proteins to the chromosome is sufficient to initiate stable silencing either at a silent mating-type locus lacking a functional silencer element or at a telomere in a strain in which the Rap1p carboxy-terminal silencing domain has been deleted. Silencing by Sir protein targeting can also be initiated at a telomere-proximal site (ADH4), but is much weaker at an internal chromosomal locus (LYS2). Strikingly, deletion of the Rap1p silencing domain, which abolishes telomeric silencing, improves targeted silencing at LYS2 by both Sir3p and Sir4p, while weakening the silencing activity of these proteins at or near a telomere. This effect may result from the release of Sir proteins from the telomeres, thus increasing their effective concentration at other chromosomal sites. We suggest that telomeres and Rap1p serve a regulatory role in sequestering Sir proteins at telomeres, controlling silencing at other loci in trans and preventing indiscriminate gene silencing throughout the genome.
The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing.
Authors: Zuo P , Maniatis T
Published: June 2007

The splicing factor U2AF (U2 snRNP auxiliary factor) is a heterodimer with subunits of 65 and 35 kD (U2AF65 and U2AF35). U2AF65 binds specifically to 3' splice sites, but previous studies failed to demonstrate a function for U2AF35. Here, we report that U2AF35 is required for constitutive splicing and also functions as a mediator of enhancer-dependent splicing. Nuclear extracts deficient in U2AF35 were inactive; however, both constitutive and enhancer-dependent splicing could be restored by the addition of purified recombinant U2AF35. In vitro protein-RNA interaction studies with pre-mRNAs containing either a constitutive or regulated splicing enhancer revealed that U2AF35 directly mediates interactions between U2AF65 and proteins bound to the enhancers. Thus, U2AF35 functions as a bridge between U2AF65 and the enhancer complex to recruit U2AF65 to the adjacent intron.
TAF-like function of SV40 large T antigen.
Authors: Damania B , Alwine J C
Published: June 2007

The simian virus 40 (SV40) early gene product large T antigen promiscuously activates simple promoters containing a TATA box or initiator element and at least one upstream transcription factor-binding site. Previous studies have suggested that promoter activation requires that large T antigen interacts with both the basal transcription complex and the upstream-bound factor. This mechanism of activation is similar to that proposed for TBP-associated factors (TAFs). We report genetic and biochemical evidence suggesting that large T antigen performs a TAF-like function. In the ts13 cell line, large T antigen can rescue the temperature-sensitive (ts) defect in TAF(II)250. In contrast, neither E1a, small t antigen, nor mutants of large T antigen defective in transcriptional activation were able to rescue the ts defect. These data suggest that transcriptional activation by large T antigen is attributable, at least in part, to an ability to augment or replace a function of TAF(II)250. In addition, we show that large T antigen interacts in vitro with the Drosophila TAFs (dTAFs) dTAF(II)150, dTAF(II)110, and dTAF(II)40, as well as TBP. The relevance of these in vitro results was established in coimmunoprecipitation experiments using extracts of SV40-infected alpha3 cells that express an epitope-tagged TBP. Large T antigen was coimmunoprecipitated by antibodies to epitope-tagged TBP, endogenous TBP, hTAF(II)100, hTAF(II)130, and hTAF(II)250, under conditions where holo-TFIID would be precipitated. In addition, large T antigen copurified and coimmunoprecipitated with phosphocellulose-purified TFIID from SV40-infected alpha3 cells. Large T antigen also coprecipitated with anti-TBP antibody from extracts of ts13 cells expressing wild-type large T antigen under conditions where the ts defect in TAF(II)250 was rescued. In contrast, a transactivation mutant of large T antigen, which was unable to rescue the ts defect, failed to coprecipitate. We conclude from these data that transcriptional activation of many promoters by large T antigen results from its performing a TAF-like function in a complex with TFIID.
The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3.
Authors: Yost C , Torres M , Miller J R , Huang E , Kimelman D , Moon R T
Published: June 2007

The serine/threonine kinase Xgsk-3 and the intracellular protein beta-catenin are necessary for the establishment of the dorsal-ventral axis in Xenopus. Although genetic evidence from Drosophila indicates that Xgsk-3 is upstream of beta-catenin, direct interactions between these proteins have not been demonstrated. We demonstrate that phosphorylation of beta-catenin in vivo requires an in vitro amino-terminal Xgsk-3 phosphorylation site, which is conserved in the Drosophila protein armadillo. beta-catenin mutants lacking this site are more active in inducing an ectopic axis in Xenopus embryos and are more stable than wild-type beta-catenin in the presence of Xgsk-3 activity, supporting the hypothesis that Xgsk-3 is a negative regulator of beta-catenin that acts through the amino-terminal site. Inhibition of endogenous Xgsk-3 function with a dominant-negative mutant leads to an increase in the steady-state levels of ectopic beta-catenin, indicating that Xgsk-3 functions to destabilize beta-catenin and thus decrease the amount of beta-catenin available for signaling. The levels of endogenous beta-catenin in the nucleus increases in the presence of the dominant-negative Xgsk-3 mutant, suggesting that a role of Xgsk-3 is to regulate the steady-state levels of beta-catenin within specific subcellular compartments. These studies provide a basis for understanding the interaction between Xgsk-3 and beta-catenin in the establishment of the dorsal-ventral axis in early Xenopus embryos.
Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast.
Authors: Piatti S , Böhm T , Cocker J H , Diffley J F , Nasmyth K
Published: June 2007

In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G1 is necessary for subsequent initiation of DNA replication triggered by activation in late G1 of Cdc28/Cdk1 kinases associated with B-type cyclins Clb1-Clb6. The absence of pre-RCs during G2 and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase. Formation of pre-RCs and their maintenance during G1 depend on the synthesis and activity of an unstable protein encoded by CDC6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and Clb6/Cdk1 in late G1. The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G2 and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once C1b/Cdk1 kinases have been activated. We show that Cdc6p associates with Clb/Cdk1 kinases from late G1 until late anaphase, which might be important for inhibiting pre-RC assembly during S, G2, and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.
Multiple determinants controlling activation of yeast replication origins late in S phase.
Authors: Friedman K L , Diller J D , Ferguson B M , Nyland S V , Brewer B J , Fangman W L
Published: June 2007

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.
Differential splicing-in of a proline-rich exon converts alphaNAC into a muscle-specific transcription factor.
Authors: Yotov W V , St-Arnaud R
Published: June 2007

NAC (nascent polypeptide-associated complex) was recently purified as an alpha/beta heterodimeric complex binding the newly synthesized polypeptide chains as they emerge from the ribosome. We have identified, cloned, and characterized a muscle-specific isoform of alphaNAC. The 7.0-kb mRNA arises from differential splicing-in of a 6.0 kb-exon giving rise to a proline-rich isoform that we termed skNAC. The skNAC protein was specifically expressed in differentiated myotubes but not in myoblasts. We have identified a specific DNA binding site for skNAC and shown that it can activate transcription through that element. The murine myoglobin promoter contains three putative skNAC-binding sites. skNAC was shown to activate transcription from the myoglobin promoter, and site-specific mutation of the skNAC response elements abrogated skNAC-dependent activation. We also examined the role of the NAC isoforms in the myogenic program. Whereas overexpression of alphaNAC prevented C2C12 differentiation and myotube fusion, the overexpression of skNAC in C2C12 myoblasts led to early fusion of the cells into gigantic myosacs, suggesting that skNAC may be involved in normal differentiation along the myogenic lineage and in the regulation of myoblast fusion. Our data demonstrate that differential splicing converts alphaNAC into a tissue-specific DNA-binding activator and suggest that this regulation may be an important event in the proper control of gene expression during myogenic differentiation.
Cap-prevented recombination between terminal telomeric repeat arrays (telomere CPR) maintains telomeres in Kluyveromyces lactis lacking telomerase.
Authors: McEachern M J , Blackburn E H
Published: June 2007

Deletion of the telomerase RNA gene (TER1) in the yeast Kluyveromyces lactis results in gradual loss of telomeric repeats and progressively declining cell growth capability (growth senescence). We show that this initial growth senescence is characterized by abnormally large, defectively dividing cells and is delayed when cells initially contain elongated telomeres. However, cells that survive the initial catastrophic senescence emerge relatively frequently, and their subsequent growth without telomerase is surprisingly efficient. Survivors have lengthened telomeres, often much longer than wild type, but that are still subject to gradual shortening. Production of these postsenescence survivors is strongly dependent on the RAD52 gene. We propose that shortened, terminal telomeric repeat tracts become uncapped, promoting recombinational repair between them to regenerate lengthened telomeres in survivors. This process, which we term telomere cap-prevented recombination (CPR) may be a general alternative telomere maintenance pathway in eukaryotes.
A widespread transposable element masks expression of a yeast copper transport gene.
Authors: Knight S A , Labbé S , Kwon L F , Kosman D J , Thiele D J
Published: June 2007

The trace element copper (Cu) is essential for cell growth. In this report we describe the identification of a new component of the high-affinity Cu transport machinery in yeast, encoded by the CTR3 gene. Ctr3p is a small intracellular cysteine-rich integral membrane protein that restores high-affinity Cu uptake, Cu, Zn superoxide dismutase activity, ferrous iron transport, and respiratory proficiency to strains lacking the CTR1 (Cu transporter 1) gene. In most commonly used Saccharomyces cerevisiae laboratory strains, expression of CTR3 is abolished by a Ty2 transposon insertion that separates the CTR3 promoter from the transcriptional start sites by 6 kb. In strains that do not possess a Ty2 transposon at the CTR3 locus, expression of CTR3 is repressed by copper and activated by copper starvation. In such strains inactivation of both CTR1 and CTR3 is required to generate lethal copper-deficient phenotypes. Although Ctr1p and Ctr3p can function independently in copper transport, the expression of both proteins provides maximal copper uptake and growth rate under copper-limiting conditions. These results underscore the importance of mobile DNA elements in the alteration of gene function and phenotypic variation.
Genetic determinants of p53-induced apoptosis and growth arrest.
Authors: Polyak K , Waldman T , He T C , Kinzler K W , Vogelstein B
Published: June 2007

Previous studies have suggested that expression of p53 in cancer cells can result in either growth arrest or apoptosis. Accordingly, expression of p53 in a series of colorectal cancer cell lines yielded growth arrest in some lines (A-lines) and apoptosis in others (D-lines). To investigate the basis of this difference, we evaluated the role of p21WAF1/Cip1, a known mediator of p53-induced growth arrest. Inactivation of p21 by homologous recombination converted an A-line to a D-line, suggesting that p21 could protect cells from apoptosis. However, examination of p53-induced p21 expression in naturally occurring D-lines and A-lines demonstrated that the induction of p21 could not account for the differential response to p53. Moreover, when a D-line was fused to an A-line, the resulting hybrid cells underwent apoptosis in response to p53, indicating that the apoptosis pathway was dominant over the growth arrest pathway. Therefore, the apoptotic response to p53 in colorectal cancer cells is modulated by at least two factors: p21-mediated growth arrest that can protect cells from apoptosis in A-cells, and trans-acting factors in D-cells that can overcome this protection, resulting in cell death.
DNA hypomethylation can activate Xist expression and silence X-linked genes.
Authors: Panning B , Jaenisch R
Published: June 2007

Xist and other X-linked gene expression was examined by fluorescence in situ hybridization in cells of wild type and DNA methyltranferase (Dnmt) mutant embryos and embryonic stem (ES) cells to determine whether demethylation-induced Xist expression leads to inappropriate X chromosome inactivation. In undifferentiated ES cells low-level Xist expression was detected from the single active X chromosome (Xa) in male cells and on both Xa's in female cells. Upon differentiation Xist expression was detected only in female cells, in which Xist RNA colocalized with the entire inactive X chromosome (Xi). Differentiated Dnmt mutant ES cells or cells of mutant postgastrulation embryos showed aberrant patterns of Xist expression: Xist transcripts colocalized with the single X chromosome in male cells and with both X chromosomes in female cells. X-linked gene expression was not detected from chromosomes coated with Xist RNA. These results suggest that ectopic Xist expression, induced by DNA hypomethylation, may lead to the inactivation of X-linked genes. We conclude that Xist-mediated X chromosome inactivation can occur in the absence of DNA methylation, arguing that DNA methylation may be required to repress Xist expression for the maintenance of a transcriptionally active Xa. In differentiated Dnmt mutant ES cells the activation of Xist expression correlated with a dramatic increase in apoptotic bodies, suggesting that Xist-mediated X chromosome inactivation may result in cell death and contribute to the embryonic lethality of the Dnmt mutation.
Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins.
Authors: Alexandre C , Jacinto A , Ingham P W
Published: June 2007

Members of the Hedgehog (Hh) family of secreted proteins have been identified recently as key signaling molecules that regulate a variety of inductive interactions central to the development of both Drosophila and vertebrates. Despite their widespread importance, the way in which Hh signals are transduced inside the cell remains poorly understood. The best candidate for a transcription factor that mediates Hh signaling in Drosophila is the product of the cubitus interruptus (ci) gene, a zinc finger protein that exhibits significant homology to protein products of the vertebrate GLI gene family. Here, we show that elevated levels of Ci are sufficient to activate patched (ptc) and other hh target genes, even in the absence of hh activity. We also show that Ci can function as a transcriptional activator in yeast and demonstrate that the zinc finger domain of the protein is sufficient for its target specificity. Finally, we identify sequences in the promoter region of the ptc gene, a primary target of Hh signaling, that are identical to the consensus-binding sequence of the GLI protein and are required for reporter gene expression in response to Hh activity. Taken together, our results strongly support the role for Ci as the transcriptional activator that mediates hh signaling.
The rise and fall of apoptosis during multistage tumorigenesis: down-modulation contributes to tumor progression from angiogenic progenitors.
Authors: Naik P , Karrim J , Hanahan D
Published: June 2007

In a mouse model of multistage tumorigenesis of islet beta-cells, apoptosis was activated concomitant with T-antigen oncogene-induced cell proliferation, further increased in the angiogenic stage, and markedly reduced in solid tumors. Crosses to p53-null mice confirmed this stage-specific variation as a p53-independent apoptotic process. Several apoptosis regulators were expressed, of which bcl-xL was up-regulated in tumors. When overexpressed throughout the pathway, bcl-xL protected most oncogene-expressing cells from apoptosis, enhancing progression from angiogenic progenitor to tumor without affecting earlier transitions. Further, two classes of solid tumor are described, distinguished by size and apoptotic incidence, implicating apoptosis regulation in expansive tumor growth. Thus, down-modulation of apoptosis selectively contributes to late steps in a tumorigenesis pathway.
Oskar protein interaction with Vasa represents an essential step in polar granule assembly.
Authors: Breitwieser W , Markussen F H , Horstmann H , Ephrussi A
Published: June 2007

The posterior pole plasm of the Drosophila egg contains the determinants of abdominal and germ-cell fates of the embryo. Pole plasm assembly is induced by oskar RNA localized to the posterior pole of the oocyte. Genetics has revealed three additional genes, staufen, vasa, and tudor, that are also essential for pole plasm formation. Staufen protein is required for both oskar RNA localization and translation. Vasa and Tudor are localized dependent on Oskar protein and are required to accumulate Oskar protein stably at the posterior pole. We have explored interactions between these gene products at the molecular level and find that Oskar interacts directly with Vasa and Staufen, in a yeast two-hybrid assay. These interactions also occur in vitro and are affected by mutations in Oskar that abolish pole plasm formation in vivo. Finally, we show that in the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures. These results suggest that the Oskar-Vasa interaction constitutes an initial step in polar granule assembly. In addition, we discuss the possible biological role of the Oskar-Staufen interaction.