bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates.
Authors: Joseph S , Berzal-Herranz A , Chowrira B M , Butcher S E , Burke J M
Published: June 2007

Substrate recognition by the hairpin ribozyme has been proposed to involve two short intermolecular helices, termed helix 1 and helix 2. We have used a combination of three methods (cleavage of mismatched substrates, in vitro selection, and site-specific mutational analysis) to systematically determine the substrate recognition rules for this RNA enzyme. Assays measuring substrate cleavage in trans under multiple turnover conditions were conducted using the wild-type ribozyme and substrates containing mismatches in all sites potentially recognized by the ribozyme. Molecules containing single- and multiple-base mismatches in helix 2 at sites distant from the cleavage site (g-4c, u-5a, g-4c: u-5a) were cleaved with reduced efficiency, whereas those with mismatches proximal to the cleavage site (c-2a, a-3c, c-2a: a-3c) were not cut. Analogous results were obtained for helix 1, where mismatches distal from the cleavage site (u+7a, u+8a, u+9a, u+7a: u+8a: u+9a) were used much more efficiently than those proximal to the cleavage site (c+4a, u-5a, g+6c, c+4a: u+5a: g+6c). In vitro selection experiments were carried out to identify active variants from populations of molecules in which either helix 1 or helix 2 was randomized. Results constitute an artificial phylogenetic data base that proves base-pairing of nucleotides at five positions within helix 1 and three positions within helix 2 and reveals a significant sequence bias at 3 bp (c+4.G6, c-2.G11, and a-3.U12). This sequence bias was confirmed at two sites by measuring relative cleavage rates of all 16 possible dinucleotide combinations at base pairs c+4.G6 and c-2.G11.(ABSTRACT TRUNCATED AT 250 WORDS)
Tissue-specific RNA splicing generates an ankyrin-like domain that affects the dimerization and DNA-binding properties of a bHLH protein.
Authors: Klein E S , Simmons D M , Swanson L W , Rosenfeld M G
Published: June 2007

mRNAs encoding two rat bHLH proteins, referred to as REB alpha and REB beta, have been identified as alternatively spliced transcripts derived from a single genomic locus. Alternative RNA processing events results in tissue-specific differences in the ratios of these two mRNAs. Although it exhibits a highly enriched level of expression in the developing neural tube, the REB gene is expressed at variable levels in many organs of the mature animal. The REB alpha sequence contains a region characterized by a leucine heptad repeat that is situated amino-terminal of the carboxy-terminally located bHLH domain. REB beta is identical to REB alpha except for a 24-amino-acid insertion in the leucine heptad repeat that results from the inclusion of an additional 72-bp exon in the REB beta transcript. As a consequence of this insertion, REB beta exhibits a markedly diminished capacity to bind to cognate E-box-binding sites and to form homodimers and heterodimers with other members of the bHLH gene family. Analysis of the 24-amino-acid REB beta-specific insert revealed that it mediates an inhibitory function and exhibits a significant degree of sequence similarity to ankyrin-like repeats. It is proposed that this tissue-specific pattern of REB RNA splicing is involved in the determination of corresponding tissue-specific combinations of heterodimeric complexes of ubiquitous and tissue-restricted bHLH proteins. Thus, REB alpha and REB beta represent a novel example of a regulated formation of an ankyrin-like domain within a bHLH protein, thereby mediating control of protein-protein interactions.
Circular forms of developmentally excised DNA in Euplotes crassus have a heteroduplex junction.
Authors: Klobutcher L A , Turner L R , LaPlante J
Published: June 2007

Extensive DNA elimination via a DNA breakage and rejoining process occurs during macronuclear development in the hypotrich ciliate Euplotes crassus. The excision process involves the removal of short, unique segments of DNA (internal eliminated sequences; IESs) and at least two highly repetitive families of transposon-like elements (Tec elements). Previous studies have demonstrated that circular forms of both IESs and Tec elements are generated following their developmental excision and that some flanking DNA sequences are retained at the circle junctions. In this study we have further analyzed the circle junctions of IESs. Analysis of polymerase chain reaction (PCR) products derived from IES circle junctions indicates that at least two sequence arrangements can be present. The circle junctions contain both of the direct repeats that define the ends of the IES separated by either 2 bp flanking the right end of the IES and 8 bp from the left-flanking region, or 8 bp from the right and 2 bp from the left. Using a method that we have termed "strand-biased PCR," we obtained evidence that the junctions of free circular IESs have a 6-base heteroduplex at their center, such that one strand of the DNA is derived from the left-flanking region of the IES and the other from the right. Models of IES excision are presented that incorporate these results and those of previous studies on the excision process.
Elimination of Tec elements involves a novel excision process.
Authors: Jaraczewski J W , Jahn C L
Published: June 2007

Approximately 60,000 transposon-like elements of the Tec1 and Tec2 families excise en masse from the micronuclear genome during formation of a macronucleus in Euplotes crassus. The circular product has been shown previously to contain the element inverted repeats joined head to head. To elucidate the mechanism of Tec excision, we have further characterized the circular products. DNA sequence analysis of cloned inverted repeat junctions and of population of supercoiled Tec circles shows that the inverted repeat junctions consist of both copies of the target site duplication surrounding 10 additional bases. The 10 bp differs for each junction. We demonstrate that the circles are highly sensitive to S1, mung bean and Bal 31 nucleases, and the site of sensitivity maps to the junction. Alkaline gel electrophoresis indicates that the junction does not contain a nick or gap; thus, a likely explanation for the nuclease sensitivity is the existence of a heteroduplex DNA structure at the junction. On the basis of these results, we present a model of Tec excision and discuss the relationship of Tec excision to IES elimination and chromosome fragmentation in E. crassus.
Heterodimerization of the transcription factors E2F-1 and DP-1 leads to cooperative trans-activation.
Authors: Helin K , Wu C L , Fattaey A R , Lees J A , Dynlacht B D , Ngwu C , Harlow E
Published: June 2007

The E2F transcription factor has been implicated in the regulation of genes whose products are involved in cell proliferation. Two proteins have recently been identified with E2F-like properties. One of these proteins, E2F-1, has been shown to mediate E2F-dependent trans-activation and to bind the hypophosphorylated form of the retinoblastoma protein (pRB). The other protein, murine DP-1, was purified from an E2F DNA-affinity column, and it was subsequently shown to bind the consensus E2F DNA-binding site. To study a possible interaction between E2F-1 and DP-1, we have now isolated a cDNA for the human homolog of DP-1. Human DP-1 and E2F-1 associate both in vivo and in vitro, and this interaction leads to enhanced binding to E2F DNA-binding sites. The association of E2F-1 and DP-1 leads to cooperative activation of an E2F-responsive promoter. Finally, we demonstrate that E2F-1 and DP-1 association is required for stable interaction with pRB in vivo and that trans-activation by E2F-1/DP-1 heterodimers is inhibited by pRB. We suggest that "E2F" is the activity that is formed when an E2F-1-related protein and a DP-1-related protein dimerize.
Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20.
Authors: Swiatek P J , Gridley T
Published: June 2007

Krox20 is a zinc finger gene expressed in rhombomeres 3 and 5 during hindbrain development in vertebrates. Mice homozygous for a targeted mutation that deletes the majority of the Krox20 genes, including the zinc finger DNA-binding domain, died shortly after birth. The primary phenotype of the homozygous mutant animals was the loss of rhombomeres 3 and 5. This resulted in fusions of the trigeminal ganglion with the facial and vestibular ganglia, and of the superior ganglia of the glossopharyngeal and vagus nerves. These fusions resulted in a disorganization of the nerve roots of these ganglia as they entered the brain stem. These data demonstrate that Krox20 plays an essential role during development of the hindbrain and associated cranial sensory ganglia in mice.
Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain.
Authors: Hibi M , Lin A , Smeal T , Minden A , Karin M
Published: June 2007

The activity of c-Jun is regulated by phosphorylation. Various stimuli including transforming oncogenes and UV light, induce phosphorylation of serines 63 and 73 in the amino-terminal activation domain of c-Jun and thereby potentiate its trans-activation function. We identified a serine/threonine kinase whose activity is stimulated by the same signals that stimulate the amino-terminal phosphorylation of c-Jun. This novel c-Jun amino-terminal kinase (JNK), whose major form is 46 kD, binds to a specific region within the c-Jun trans-activation domain and phosphorylates serines 63 and 73. Phosphorylation results in dissociation of the c-Jun-JNK complex. Mutations that disrupt the kinase-binding site attenuate the response of c-Jun to Ha-Ras and UV. Therefore the binding of JNK to c-Jun is of regulatory importance and suggests a mechanism through which protein kinase cascades can specifically modulate the activity of distinct nuclear targets.
The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II.
Authors: Qiu H , Park E , Prakash L , Prakash S
Published: June 2007

The RAD25 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and, in addition, is essential for viability. RAD25 shares a high degree of homology with the human ERCC3/XPBC-encoded protein, and the yeast and human proteins resemble one another in containing the conserved ATPase/DNA helicase sequence motifs. To determine the nature of the essential role of RAD25, we have isolated a recessive temperature-sensitive conditional lethal mutation of the gene and have examined its effect on transcription. Upon shift to the nonpermissive temperature, the rad25 temperature-sensitive (ts) mutant stops growth rapidly and shows a large decrease in the synthesis of poly(A)+ RNA. Transcription of a large number of yeast genes, including HIS3, TRP3, STE2, MET19, RAD23, CDC9, and ACT1 is inhibited at the restrictive temperature in the rad25 ts mutant, and the galactose-inducible synthesis of GAL7 and GAL10 mRNAs is also severely affected by the loss of RAD25 activity. These findings implicate a general requirement of RAD25 in RNA polymerase II transcription.
In vivo cooperation between introns during pre-mRNA processing.
Authors: Neel H , Weil D , Giansante C , Dautry F
Published: June 2007

In higher eukaryotes the large number of introns present in most genes implies that the pre-mRNA processing machinery should be efficient and accurate. Although this could be achieved at the level of each intron, an attractive alternative would be that interactions between introns improve the performance of this machinery. In this study we tested this hypothesis by comparing the processing of transcripts of the tumor necrosis factor beta gene, which differ only by their number of introns. We took advantage of the ordered splicing of the three introns present in this gene to design constructs that should generate, as primary transcripts, molecules that are normally produced by splicing. We established that the apparent splicing rate of intron 3 is increased 2.5- and 3.5-fold by the presence of one or two other introns on the primary transcript, respectively. Similarly, the apparent splicing rate of intron 2 is increased by the presence of intron 1. As these effects involve the splice sites of the upstream intron, these observations support the existence of cooperative interactions between introns during pre-mRNA processing.
Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex.
Authors: Yokomori K , Admon A , Goodrich J A , Chen J L , Tjian R
Published: June 2007

The basal factor TFIIA has been shown to act early during initiation in both the mammalian and yeast transcription systems, but a TFIIA-like activity has not been identified in Drosophila. While characterizing the Drosophila TFIID complex, we discovered that a 30-kD protein that cofractionated with dTFIID was homologous to the previously identified, large subunit of yeast TFIIA. Here, we report the cloning and biochemical characterization of Drosophila TFIIA-L. Coimmunoprecipitation studies with anti-dTBP, anti-dTFIIA-L, and anti-TAF antibodies indicated a tight association of the endogenous dTFIIA and dTFIID. However, dTFIIA could be dissociated from dTFIID under conditions that did not elute the TAFs, and the eluted material had mobility shift and transcriptional activities associated with TFIIA. Peptide sequence and Western analysis with antibodies raised against the amino- and carboxy-terminal portions of recombinant dTFIIA-L revealed that a precursor 48-kD species was cleaved in vivo, giving rise to the 30- and 20-kD subunits of dTFIIA that remain associated with each other and with dTFIID. Protein-protein interaction assays identified dTBP and dTAFII110 as targets for binding TFIIA in the TFIID complex. These results suggest that TFIIA may form a specific complex with both TAFs and other components of the transcriptional machinery during formation of the initiation complex.
An everted repeat mediates retinoic acid induction of the gamma F-crystallin gene: evidence of a direct role for retinoids in lens development.
Authors: Tini M , Otulakowski G , Breitman M L , Tsui L C , Giguère V
Published: June 2007

The vertebrate lens is a classical system for examining mechanisms of tissue determination and differentiation, yet little is known about the signaling molecules controlling its development. Here, we report that retinoic acid (RA), a substance known for its teratogenic effects on the eye and as a natural endogenous morphogenetic agent, acts as a regulator of gene expression in the lens. We have identified a novel type of RA response element (RARE) within the lens-specific mouse gamma F-crystallin promoter, consisting of two (A/G)GGTCA motifs in an everted arrangement spaced by 8 nucleotides. This element (gamma F-RARE) mediates activation of the gamma F-crystallin promoter by ligand-activated endogenous lens cell RA receptors (RARs) and confers RA responsiveness when linked to a heterologous promoter. gamma F-RARE is bound in vitro by RAR/RXR heterodimers, and both receptors cooperate in vivo to trans-activate this element. These observations demonstrate a direct effect of RA on lens-specific gene expression and reveal a novel role for retinoids in the development and homeostasis of the mammalian eye.
Identity of GABP with NRF-2, a multisubunit activator of cytochrome oxidase expression, reveals a cellular role for an ETS domain activator of viral promoters.
Authors: Virbasius J V , Virbasius C A , Scarpulla R C
Published: June 2007

The ETS domain proteins are a diverse family of transcriptional activators that have been implicated recently in the expression of a number of cell-specific and viral promoters. Nuclear respiratory factor 2 (NRF-2) is a nuclear transcription factor that activates the proximal promoter of the rat cytochrome c oxidase subunit IV (RCO4) gene through tandem sequence elements. These elements conform to the consensus for high-affinity ETS domain recognition sites. We have now purified NRF-2 to homogeneity from HeLa cells and find that it consists of five polypeptides, only one of which has intrinsic DNA-binding ability. The others participate in the formation of heteromeric complexes with distinct binding properties. NRF-2 also specifically recognizes multiple binding sites in the mouse cytochrome c oxidase subunit Vb (MCO5b) gene. As in the functionally related RCO4 gene, tandemly arranged NRF-2 sites are essential for the activity of the proximal MCO5b promoter, further substantiating a role for NRF-2 in respiratory chain expression. Determination of peptide sequences from the various subunits of HeLa NRF-2 reveals a high degree of sequence identity with mouse GA-binding protein (GABP), a multisubunit ETS domain activator of herpes simplex virus immediate early genes. A cellular role in the activation of nuclear genes specifying mitochondrial respiratory function is thus assigned to an ETS domain activator of viral promoters.
Expression of the developmental I antigen by a cloned human cDNA encoding a member of a beta-1,6-N-acetylglucosaminyltransferase gene family.
Authors: Bierhuizen M F , Mattei M G , Fukuda M
Published: June 2007

The blood group i/I antigens were the first identified alloantigens that display a dramatic change during human development. The i and I antigens are determined by linear and branched poly-N-acetyllactosaminoglycans, respectively. In human erythrocytes during embryonic development, the fetal (i) antigen is replaced by the adult (I) antigen as a result of the appearance of a beta-1,6-N-acetylglucosaminyltransferase, the I-branching enzyme. Here, we report the cDNA cloning and expression of this branching enzyme that converts linear into branched poly-N-acetyllactosaminoglycans, thus introducing the I antigen in transfected cells. The cDNA sequence predicts a protein with type II membrane topology as has been found for all other mammalian glycosyltransferases cloned to date. The Chinese hamster ovary cells that stably express the isolated cDNA acquire I-branched structures as evidenced by the structural analysis of glycopeptides from these cells. Comparison of the amino acid sequence with those of other glycosyltransferases revealed that this I-branching enzyme and another beta-1,6-N-acetylglucosaminyltransferase that forms a branch in O-glycans are strongly homologous in the center of their putative catalytic domains. Moreover, the genes encoding these two beta-1,6-N-acetylglucosaminyltransferases were found to be located at the same locus on chromosome 9, band q21. These results indicate that the I-branching enzyme represents a member of a beta-1,6-N-acetylglucosaminyltransferase gene family of which expression is controlled by developmental programs.
The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit.
Authors: Durfee T , Becherer K , Chen P L , Yeh S H , Yang Y , Kilburn A E , Lee W H , Elledge S J
Published: June 2007

The retinoblastoma protein (p110RB) interacts with many cellular proteins in complexes potentially important for its growth-suppressing function. We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind p110RB. One clone encodes a novel type 1 protein phosphatase catalytic subunit (PP-1 alpha 2), which differs from the originally defined PP-1 alpha by an amino-terminal 11-amino-acid insert. In vitro-binding assays demonstrated that PP-1 alpha isoforms preferentially bind the hypophosphorylated form of p110RB. Moreover, similar p110RB sequences are required for binding PP-1 alpha 2 and SV40 large T antigen. Cell cycle synchrony experiments revealed that this association occurs from mitosis to early G1. The implications of these findings on the regulation of both proteins are discussed.
Transcriptional silencing in yeast is associated with reduced nucleosome acetylation.
Authors: Braunstein M , Rose A B , Holmes S G , Allis C D , Broach J R
Published: June 2007

Two classes of sequences in the yeast Saccharomyces cerevisiae are subject to transcriptional silencing: the silent mating-type cassettes and telomeres. In this report we demonstrate that the silencing of these regions is strictly associated with acetylation of the epsilon-amino groups of lysines in the amino-terminal domains of three of the four core histones. Both the silent mating-type cassettes and the Y domains of telomeres are packaged in nucleosomes in vivo that are hypoacetylated relative to those packaging active genes. This difference in acetylation is eliminated by genetic inactivation of silencing: The silent cassettes from sir2, sir3, or sir4 cells show the same level of acetylation as other active genes. The correspondence of silencing and hypoacetylation of the mating-type cassettes is observed even for an allele lacking a promoter, indicating that silencing per se, rather than the absence of transcription, is correlated with hypoacetylation. Finally, overexpression of Sir2p, a protein required for transcriptional silencing in yeast, yields substantial histone deacetylation in vivo. These studies fortify the hypothesis that silencing in yeast results from heterochromatin formation and argue that the silencing proteins participate in this formation.
Control of cell fate determination by p21ras/Ras1, an essential component of torso signaling in Drosophila.
Authors: Lu X , Chou T B , Williams N G , Roberts T , Perrimon N
Published: June 2007

Determination of cell fate at the posterior termini of the Drosophila embryo is specified by the activation of the torso (tor) receptor tyrosine kinase. This signaling pathway is mediated by the serine/threonine kinase D-raf and a protein tyrosine phosphatase corkscrew (csw). We found that expression of an activated form of Ras1 during oogenesis resulted in embryos with tor gain-of-function phenotypes. To demonstrate that p21ras/Ras1 mediates tor signaling, we injected mammalian p21ras variants into early Drosophila embryos. We found that the injection of activated p21v-ras rescued the maternal-effect phenotypes of both tor and csw null mutations. These rescuing effects of p21v-ras are dependent on the presence of maternally derived D-raf activity. In addition, wild-type embryos show a terminal-class phenotype resembling csw when injected with p21rasN17, a dominant-negative form of p21ras. Furthermore, we have analyzed the maternal-effect phenotype of Son of sevenless (Sos), a positive regulator of Ras1, and showed that embryos derived from germ cells lacking Sos+ activity exhibit a terminal-class phenotype. Our study demonstrates that the Drosophila p21ras, encoded by Ras1, is an intrinsic component of the tor signaling pathway, where it is both necessary and sufficient in specifying posterior terminal cell fates. p21ras/Ras1 operates upstream of the D-raf kinase in this signaling pathway.
An endocrine-specific element is an integral component of an exocrine-specific pancreatic enhancer.
Authors: Kruse F , Rose S D , Swift G H , Hammer R E , MacDonald R J
Published: June 2007

We have analyzed the function of individual elements of the elastase I transcriptional enhancer in transgenic animals. This pancreas-specific enhancer comprises three functional elements, one of which (the B element) plays a dual role. Within the context of the enhancer, the B element contributes to appropriate acinar cell expression. However, when separated from the other enhancer components, the B element selectively directs transcription in islet cells of transgenic animals. This islet-specific activity is normally suppressed by an upstream repressor domain. The B element binds a novel islet-specific factor, and similar B-like elements are present in other pancreatic genes, both exocrine and endocrine specific. We suggest that a principal role of this transcriptional element and its associated factors is to activate many pancreatic genes as part of the program of pancreatic determination prior to the divergence of the acinar and islet cell lineages.
Negative regulation of FAR1 at the Start of the yeast cell cycle.
Authors: McKinney J D , Chang F , Heintz N , Cross F R
Published: June 2007

In budding yeast, a switch between the mutually exclusive pathways of cell cycle progression and conjugation is controlled at Start in late G1 phase. Mating pheromones promote conjugation by arresting cells in G1 phase before Start. Pheromone-induced cell cycle arrest requires a functional FAR1 gene. We have found that FAR1 transcription and protein accumulation are regulated independently during the cell cycle. FAR1 RNA and protein are highly expressed in early G1, but decline sharply at Start. Far1 is phosphorylated just before it disappears at Start, suggesting that modification may target Far1 for degradation. Although FAR1 mRNA levels rise again during late S or G2 phase, reaccumulation of Far1 protein to functional levels is restricted until after nuclear division.
A Xenopus egg factor with DNA-binding properties characteristic of terminus-specific telomeric proteins.
Authors: Cardenas M E , Bianchi A , de Lange T
Published: June 2007

We have identified a Xenopus laevis protein factor that specifically recognizes vertebrate telomeric repeats at DNA ends. This factor, called Xenopus telomere end factor (XTEF), is detected predominantly in extracts of Xenopus eggs and ovaries, which are estimated to contain sufficient XTEF to bind approximately 3 x 10(7) DNA ends. In contrast, XTEF is much less abundant (approximately 90 per cell) in extracts of somatic cell nuclei. Mobility retardation analysis of the XTEF activity in egg extracts indicates that this factor binds the vertebrate telomeric repeat sequence (TTAGGG)2 when present in a single-stranded 3' overhang. Single-stranded 3' extensions of (TTTGGG)2, (AAAGGG)2, (TTACCC)2, or a nonrepetitive sequence fail to bind XTEF efficiently, whereas changes in the double-stranded sequence 5' to the TTAGGG repeat tail are tolerated. TTAGGG repeats are not recognized at internal position, at a 5' protruding end, or in double-stranded DNA. In addition, the factor does not bind RNA with single-stranded UUAGGG repeats at a 3' end. XTEF-DNA complexes form and are stable in high salt. The DNA-binding properties of XTEF resemble the characteristics of a class of terminus-specific telomere proteins identified previously in hypotrichous ciliates.
A Rhizobium meliloti homolog of the Escherichia coli peptide-antibiotic transport protein SbmA is essential for bacteroid development.
Authors: Glazebrook J , Ichige A , Walker G C
Published: June 2007

Alfalfa nodules induced by a Rhizobium meliloti strain carrying the bacA386::TnphoA mutation (formerly fix386::TnphoA) were examined by light and electron microscopy. These ineffective nodules were found to contain bacteria within infection threads, but no mature bacteroids were observed. A closer examination revealed that there were undeveloped senescent bacteroids in the plant cells of the nodule invasion zone, strongly suggesting that the symbiotic defect of the bacA386::TnphoA mutant is attributable to an early block in bacteroid development. The expression of the bacA gene in effective nodules was monitored with a bacA-phoA fusion and found to be strongest in the region where developing bacteroids are found. The bacA+ gene was cloned and sequenced. Sequence analysis indicated that BacA is probably an integral inner membrane protein with seven transmembrane domains and that it is extremely homologous to Escherichia coli SbmA, an inner membrane protein required for the uptake of microcin B17, a peptide antibiotic. Southern blotting experiments indicate that a gene closely related to bacA/sbmA is found in many bacteria, including some that invade eukaryotic cells. Possible roles for BacA in symbiosis are discussed.
Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex activates transcription from snRNA promoters through the PSE.
Authors: Sadowski C L , Henry R W , Lobo S M , Hernandez N
Published: June 2007

In the human small nuclear RNA (snRNA) promoters, the presence of a TATA box recognized by the TATA box-binding protein (TBP) determines the selection of RNA polymerase III over RNA polymerase II. The RNA polymerase II snRNA promoters are, therefore, good candidates for TBP-independent promoters. We show here, however, that TBP activates transcription from RNA polymerase II snRNA promoters through a non-TATA box element, the snRNA proximal sequence element (PSE), as part of a new snRNA-activating protein complex (SNAPc). In contrast to the previously identified TBP-containing complexes SL1, TFIID, and TFIIIB, which appear dedicated to transcription by a single RNA polymerase, SNAPc is also essential for RNA polymerase III transcription from the U6 snRNA promoter. The U6 initiation complex appears to contain two forms of TBP, one bound to the TATA box and one bound to the PSE as a part of SNAPc, suggesting that multiple TBP molecules can have different functions within a single promoter.
Expression of a retinoblastoma transgene results in dwarf mice.
Authors: Bignon Y J , Chen Y , Chang C Y , Riley D J , Windle J J , Mellon P L , Lee W H
Published: June 2007

Introduction of the normal retinoblastoma gene (RB) into different tumor cells possessing inactivated RB genes suppresses their tumorigenicity in nude mice. These results suggest that RB replacement is a potential strategy for developing future clinical treatments of cancer. In a transgenic mouse model, we found that the quantity of RB protein in a given cell may play an important role in dictating its effect. Four founder mice containing 1-7 copies of a human RB cDNA transgene under the transcriptional control of the human RB promoter were generated. Most of the transgenic mice were smaller than nontransgenic littermates. This effect was found as early as embryonic day 15. The degree of dwarfism correlated roughly with the copy number of the transgene and the corresponding level of RB protein. The expression pattern of the transgene products was similar to that of the endogenous mouse RB gene with regard to tissue and temporal distribution. Transferring the transgene to RB deficient mice, which are nonviable, resulted in the development of normal, healthy mice, indicating that the human RB gene can functionally complement the mouse homolog. These studies demonstrate that the effect of RB on overall mouse development is closely dependent upon its dosage.
mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.
Authors: Peltz S W , Brown A H , Jacobson A
Published: June 2007

Nonsense mutations in a gene can accelerate the decay rate of the mRNA transcribed from that gene, a phenomenon we describe as nonsense-mediated mRNA decay. Using amber (UAG) mutants of the yeast PGK1 gene as a model system, we find that nonsense-mediated mRNA decay is position dependent, that is, nonsense mutations within the initial two-thirds of the PGK1-coding region accelerate the decay rate of the PGK1 transcript < or = 12-fold, whereas nonsense mutations within the carboxy-terminal third of the coding region have no effect on mRNA decay. Moreover, we find that this position effect reflects (1) a requirement for sequences 3' to the nonsense mutation that may be necessary for translational reinitiation or pausing, and (2) the presence of an additional sequence that, when translated, inactivates the nonsense-mediated mRNA decay pathway. This stabilizing element is positioned within the coding region such that it constitutes the boundary between nonsense mutations that do or do not affect mRNA decay. Rapid decay of PGK1 nonsense-containing transcripts is also dependent on the status of the UPF1 gene. Regardless of the position of an amber codon in the PGK1 gene, deletion of the UPF1 gene restores wild-type decay rates to nonsense-containing PGK1 transcripts.
Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly.
Authors: Kamakaka R T , Bulger M , Kadonaga J T
Published: June 2007

Purified, reconstituted chromatin templates containing regular, physiological nucleosome spacing were transcribed in vitro by RNA polymerase II along with the Gal4-VP16 activator. When Gal4-VP16 was prebound to DNA before reconstitution of either H1-deficient or H1-containing chromatin, the resulting templates were transcribed with a similar efficiency. Under such conditions, we observed long-range (1000 bp) activation of transcription in vitro with H1-containing chromatin, but not naked DNA templates. When Gal4-VP16 was added to preassembled chromatin, the H1-deficient chromatin was transcriptionally active, whereas the H1-containing chromatin, which possessed properties similar to native chromatin, was transcriptionally inert. We then mimicked DNA replication and chromatin assembly at a replication fork and found that Gal4-VP16 could potentiate transcription during, but not after, replication and assembly of histone H1-containing chromatin. These experiments provide biochemical data that support a DNA replication-dependent mechanism for reconfiguration of chromatin structure and activation of transcription by Gal4-VP16.
Rapid transcriptional autoregulation of a yeast metalloregulatory transcription factor is essential for high-level copper detoxification.
Authors: Zhou P , Thiele D J
Published: June 2007

Copper detoxification in the yeast Candida glabrata is carried out in large part by a family of metallothionein (MT) genes: a unique MT-I gene, a tandemly amplified MT-IIa gene, and a single unlinked MT-IIb gene. In response to elevated environmental copper levels, members of this MT gene family are transcriptionally activated by a copper-dependent, sequence-specific DNA-binding transcription factor, AMT1. AMT1 shares several structural and functional features with the Saccharomyces cerevisiae copper metalloregulatory transcription factor ACE1, which is constitutively expressed and poised for rapid transcriptional responses to the toxic metal copper. In this paper, we demonstrate that AMT1 is subject to positive transcriptional autoregulation, which is exerted through binding of copper-activated AMT1 to a single copper responsive element in the AMT1 promoter. A nonautoregulatory amt1 mutant displayed a marked decrease in both copper tolerance and expression of the MT-II genes, which are critical for high-level copper detoxification in Candida glabrata. Kinetic analysis demonstrated the remarkably rapid AMT1 mRNA accumulation in the presence of copper, which is followed by increased expression of the metallothionein gene products. These results demonstrate that AMT1-positive autoregulation plays a critical role in metal detoxification and suggest that the rapid autoactivation of the AMT1 metalloregulatory transcription factor biosynthesis is essential for C. glabrata to quickly build up a cellular defense line to protect cells upon exposure to high environmental copper levels.
Overcoming telomeric silencing: a trans-activator competes to establish gene expression in a cell cycle-dependent way.
Authors: Aparicio O M , Gottschling D E
Published: June 2007

Genes located near telomeres in yeast are subject to position-effect variegation. To better understand the mechanism of this variegation, we investigated how a telomeric URA3 gene switches from a silent to an expressed state. We found that silencing of a telomeric URA3 gene was attributable to the elimination of its basal transcription. The reversal of that silencing was dependent on the presence of PPR1, the trans-activator protein of URA3. Maximum expression of URA3 required a higher concentration of PPR1 when URA3 was telomeric compared with when it was at a nontelomeric location. The ability of PPR1 to overcome silencing varied at different points in the cell cycle. In cells arrested in G2/metaphase, PPR1 was able to activate transcription of a telomeric URA3, but in cells arrested in G0, G1, or early S phase it was not. In comparison, a nontelomeric URA3 could be activated by PPR1 at all times. We conclude that once established, telomeric silent chromatin is a relatively stable structure, making a gene recalcitrant to activation. Following the disassembly of silent chromatin during DNA replication, competition of assembly ensues between components of telomeric chromatin, to establish a silent state, and the trans-activator, to establish gene expression. These results help explain the stochastic nature of phenotypic switching in variegated gene expression.
Specific regulation of Xenopus chromosomal 5S rRNA gene transcription in vivo by histone H1.
Authors: Bouvet P , Dimitrov S , Wolffe A P
Published: June 2007

The incorporation of histone H1 into chromatin during embryogenesis directs the specific repression of the Xenopus oocyte 5S rRNA genes. An increase in histone H1 content specifically restricts TFIIIA-activated transcription, and a decrease in histone H1 within chromatin facilitates the activation of the oocyte 5S rRNA genes by TFIIIA. Variation in the amount of histone H1 in chromatin does not significantly influence somatic 5S rRNA gene transcription. Thus, the regulated expression of histone H1 during Xenopus development has a specific and dominant role in mediating the differential expression of the oocyte and somatic 5S rRNA genes. This example demonstrates that histones can exert dominant repressive effects on the transcription of a gene in vivo in spite of an abundance of transcription factors for that gene.
The DCC gene product in cellular differentiation and colorectal tumorigenesis.
Authors: Hedrick L , Cho K R , Fearon E R , Wu T C , Kinzler K W , Vogelstein B
Published: June 2007

The analysis of human colorectal tumors has revealed frequent loss of heterozygosity (LOH) of the long arm of chromosome 18. A novel gene, DCC (deleted in colorectal cancer), located within the region of LOH on chromosome 18q was identified and has been implicated as a tumor suppressor gene. We have now shown that DCC encodes a membrane-bound protein of the immunoglobulin-CAM family, as demonstrated by cell-surface labeling, immunohistochemical analysis, and sequencing of cDNA clones. The DCC protein was found in axons of the central and peripheral nervous system and in differentiated cell types of the intestine. Colorectal tumors that lost their capacity to differentiate into mucus producing cells uniformly lacked DCC expression and loss of a chromosome 18q allele was often accompanied by loss of DCC expression in colon tumors. These results provide evidence that DCC encodes a cell surface-localized protein and emphasize the inverse relationship between differentiation and tumorigenesis.
The Sep1 strand exchange protein from Saccharomyces cerevisiae promotes a paranemic joint between homologous DNA molecules.
Authors: Chen J , Kanaar R , Cozzarelli N R
Published: June 2007

Strand exchange protein 1 (Sep1) from the yeast Saccharomyces cerevisiae promotes the transfer of one strand of a linear duplex DNA to a homologous single-stranded DNA circle. Using a nitrocellulose filter binding assay and electron microscopy, we find that Sep1 promotes the pairing of homologous DNA molecules via a paranemic joint. In this joint there is no net intertwining of the parental DNA molecules, as in the standard plectonemic double helix. The paranemic joints form with as little as 41 bp of homology between the parental DNA molecules. The substrates used were a circular molecule (either single-stranded DNA or duplex supercoiled DNA) and a linear duplex with heterologous regions at both ends to bar duplex plectonemic intertwining. We excluded the possibility that the exonuclease activity of Sep1 exposes complementary single-stranded regions that constitute the joint. The paranemic joint is the key intermediate in the search for homologous DNA by the RecA protein of Escherichia coli. Our results imply that the search process in a eukaryote such as yeast can be mechanistically similar.
Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.
Authors: Turner D L , Weintraub H
Published: June 2007

In Drosophila, the proneural genes of the achaete-scute complex encode transcriptional activators that can commit cells to a neural fate. We have isolated cDNAs for two Xenopus achaete-scute homologs, ASH3a and ASH3b, which are expressed in a subset of central nervous system (CNS) neuroblasts during early neurogenesis. After expressing either ASH3 protein in developing Xenopus embryos, we find enlargement of the CNS at the expense of adjacent non-neural ectoderm. Analysis of molecular markers for neural, epidermal, and neural crest cells indicates that CNS expansion occurs as early as neural plate formation. ASH3-dependent CNS enlargement appears to require neural induction, as it does not occur in animal cap explants. Inhibition of DNA synthesis shows that additional CNS tissue does not depend on cell division--rather it reflects conversion of prospective neural crest and epidermal cells to a neural fate. The differentiation of the early forming primary neurons also seems to be prevented by ASH3 expression. This may be secondary to the observed activation of Xotch transcription by ASH3.
Dorsal midline fate in Drosophila embryos requires twisted gastrulation, a gene encoding a secreted protein related to human connective tissue growth factor.
Authors: Mason E D , Konrad K D , Webb C D , Marsh J L
Published: June 2007

The twisted gastrulation (tsg) gene is one of seven known zygotic genes that specify the fate of dorsal cells in Drosophila embryos. Mutations in these genes cause at least some of the cells on the dorsal half of the embryo to adopt more ventral cell fates leading to the proposal that most of these genes participate in establishing, maintaining, or modulating a gradient of a single signaling molecule DECAPENTAPLEGIC (DPP). We have examined the effects of tsg mutations on the development of cuticule elements, expression of a region specific enhancer trap, and patterns of mitotic domains. Mutations of tsg only affect the fate of a narrow strip of dorsal midline cells and do not affect dorsal ectoderm cells. However, the pattern of tsg expression is not coincident with the territories affected by tsg mutations. Structural analysis of the tsg gene reveals features of a secreted protein suggesting an extracellular site of action. The TSG protein bears a weak resemblance to human connective tissue growth factor (CTGF), a TGF-beta-induced protein. We propose that dorsal midline cell fate is specified by the combination of both a TSG and a DPP signal to which the dorsal midline cells are uniquely competent to respond.
Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element.
Authors: Huh G S , Hynes R O
Published: June 2007

The alternatively spliced exon EIIIB is regulated in a cell type-specific manner in the rat fibronectin gene. Splicing of EIIIB into fibronectin mRNA is dependent on sequences in the intron immediately downstream of EIIIB. We show that a short, highly repeated TGCATG motif in this intron is important for cell type-specific recognition of EIIIB as an exon. This motif enhances usage of the EIIIB 5' splice site; furthermore, this repeated TGCATG sequence can activate an alternatively spliced exon in the unrelated rat preprotachykinin pre-mRNA. Interestingly, this sequence can also be found within cis-acting elements identified previously in other alternatively spliced genes. This short repeated TGCATG motif is therefore a cell type-specific element that, in addition to controlling fibronectin alternative splicing, may participate in the regulation of other alternative RNA processing events.
Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme.
Authors: Kumar S , Kinoshita M , Noda M , Copeland N G , Jenkins N A
Published: June 2007

By subtraction cloning we previously identified a set of mouse genes (named Nedd1 through Nedd10) with developmentally down-regulated expression in brain. We now show that one such gene, Nedd2, encodes a protein similar to the mammalian interleukin-1 beta-converting enzyme (ICE) and the product of the Caenorhabditis elegans cell death gene ced-3 (CED-3). Both ICE and CED-3 are known to encode putative cysteine proteases and induce apoptosis when overexpressed in cultured cells. Overexpression of Nedd2 in cultured fibroblast and neuroblastoma cells also resulted in cell death by apoptosis, which was suppressed by the expression of the human bcl-2 gene, indicating that Nedd2 is functionally similar to the ced-3 gene in C. elegans. We also show that during embryonic development, Nedd2 is highly expressed in several types of mouse tissue undergoing high rates of programmed cell death such as central nervous system and kidney. Our data suggest that Nedd2 is an important component of the mammalian programmed cell death machinery.
P40SDB25, a putative CDK inhibitor, has a role in the M/G1 transition in Saccharomyces cerevisiae.
Authors: Donovan J D , Toyn J H , Johnson A L , Johnston L H
Published: June 2007

The Saccharomyces cerevisiae protein kinase Dbf2 carries out an essential function in late mitosis, and its kinase activity is cell-cycle regulated around anaphase/telophase. We have isolated SDB25, a high copy suppressor of temperature-sensitive dbf2 mutants, and genetic analysis suggests that the two proteins may function in parallel pathways in late mitosis. SDB25 encodes p40, a previously characterized substrate and potent inhibitor of Cdc28 kinase activity. Sdb25 is a phosphoprotein, and Sdb25 immunoprecipitates have a histone H1 kinase activity that is CDC28-dependent. Remarkably, Sdb25 transcript levels, protein levels, and associated kinase activity are precisely cell-cycle regulated, sharing a common peak in late mitosis. Moreover, Sdb25 protein levels and associated kinase activity are sharply up-regulated at the peak of Dbf2 kinase activity in cells released from a dbf2 ts block. The Sdb25 protein then disappears around Start in the next cell cycle. This indicates that SDB25 function is confined to M/G1, and morphological analysis of sdb25 delta cells supports this conclusion. Our data suggest that Sdb25 functions in a pathway in late mitosis leading to the down-regulation of Cdc28 kinase activity as cells traverse the M/G1 boundary.
A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.
Authors: Wahls W P , Smith G R
Published: June 2007

Homologous recombination hot spots are DNA sites that increase the frequency of recombination in their vicinity. The M26 allele of the ade6 gene in Schizosaccharomyces pombe is the first meiotic hot spot with an identified unique nucleotide sequence. We have purified 40,000-fold a heteromeric protein, containing polypeptides Mts1 (70 kD) and Mts2 (28 kD), that binds to the M26 site. Binding in vitro strictly correlates with hot spot activity in vivo for numerous single base pair substitutions in the vicinity of the M26 site, indicating that Mts1/Mts2 activates the M26 site and promotes a rate-limiting step of meiotic recombination. These and other data suggest that homologous recombination may be regulated primarily by discrete DNA sites and proteins that interact with those sites.