bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.
Authors: Allshire R C , Nimmo E R , Ekwall K , Javerzat J P , Cranston G
Published: June 2007

The ura4+ gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4+ expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ura4+ gene adjacent to telomeres and at the three centromeric sites tested. The clr4-S5 and rik1-304 mutations cause the most dramatic derepression at two out of three sites within cen1. All six mutations had only slight or intermediate effects on a third site in the center of cen1 or on telomeric repression. Strains with lesions at the clr4, rik1, and swi6 loci have highly elevated rates of chromosome loss. We propose that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere.
The Saccharomyces cerevisiae Msh2 protein specifically binds to duplex oligonucleotides containing mismatched DNA base pairs and insertions.
Authors: Alani E , Chi N W , Kolodner R
Published: June 2007

The yeast Saccharomyces cerevisiae encodes four proteins, Msh1, Msh2, Msh3, Msh4, that show strong amino acid sequence similarity to MutS, a central component of the bacterial mutHLS mismatch repair system. MutS has been shown to recognize base pair mismatches in DNA in vitro. Previous studies have suggested that Msh2 is the major mismatch recognition protein in yeast. In this study, the 109-kD Msh2 polypeptide was overexpressed and purified to analyze its DNA-binding properties. This analysis demonstrated that Msh2 can bind selectively to duplex oligonucleotide substrates containing a G/T mismatch, 1- to 14-nucleotide insertion mismatches, and palindromic (12- to 14-nucleotide) insertion mismatches. A general trend was that the affinity of Msh2 for substrate was proportional to the size of the insertion mispair present (+14 PAL, +12 PAL > +14 > +8 > GT, +6, +4, +2, +1). Kinetic studies indicated that the specificity of Msh2 to mismatch substrates was a function of its ability to form stable complexes with mispair-containing duplex DNAs. These complexes decayed more slowly than Msh2 complexes formed with homoduplex DNA.
The HCF repeat is an unusual proteolytic cleavage signal.
Authors: Wilson A C , Peterson M G , Herr W
Published: June 2007

The herpes simplex virus VP16-associated protein HCF is a nuclear host-cell factor that exists as a family of polypeptides encoded by a single gene. The mature HCF polypeptides are amino- and carboxy-terminal fragments of a large approximately 300-kD precursor protein that arise through cleavage at one or more centrally located sites. The sites of cleavage are the HCF repeats, highly conserved 26-amino-acid sequences repeated six times in the HCF precursor protein. The HCF repeat alone is sufficient to induce cleavage of a heterologous protein, and cleavage occurs at a defined site--PPCE/THET--within the HCF repeat. Alanine-scan mutagenesis was used to identify a large 18-amino-acid segment of the HCF repeat that is important to induce cleavage of a heterologous protein. Even though HCF is cleaved, the majority of amino- and carboxy-terminal cleavage products remain tightly, albeit noncovalently, associated. Modulation of this noncovalent association may provide a mechanism for regulating HCF activity. For example, the cleaved products of an alternative mRNA splicing variant of HCF do not remain associated.
The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast.
Authors: Lafontaine D , Vandenhaute J , Tollervey D
Published: June 2007

The m6(2)A1779m6(2)A1780 dimethylation at the 3' end of the small subunit rRNA has been conserved in evolution from bacteria to eukaryotes. The yeast 18S rRNA dimethylase gene DIM1 was cloned previously by complementation in Escherichia coli and shown to be essential for viability in yeast. A conditional GAL10::dim1 strain was constructed to allow the depletion of Dim1p from the cell. During depletion, dimethylation of the pre-rRNA is progressively inhibited and pre-rRNA processing at cleavage sites A1 and A2 is concomitantly lost. In consequence, the mature 18S rRNA and its 20S precursor drastically underaccumulate. This has the effect of preventing the synthesis of nonmethylated rRNA. To test whether the processing defect is a consequence of the absence of the dimethylated nucleotides or of the Dim1p dimethylase itself, a cis-acting mutation was created in which both dimethylated adenosines are replaced by guanosine residues. Methylation cannot occur on this mutant pre-rRNA, but no clear pre-rRNA processing defect is seen. Moreover, methylation of the wild-type pre-rRNA predominantly occurs after cleavage at sites A1 and A2. This shows that formation of the m6(2)A1779m6(2)A1780 dimethylation is not required for pre-rRNA processing. We propose that the binding of Dim1p to the pre-ribosomal particle is monitored to ensure that only dimethylated pre-rRNA molecules are processed to 18S rRNA.
Misexpression of Hoxa-13 induces cartilage homeotic transformation and changes cell adhesiveness in chick limb buds.
Authors: Yokouchi Y , Nakazato S , Yamamoto M , Goto Y , Kameda T , Iba H , Kuroiwa A
Published: June 2007

During chick limb development, the Abd-B subfamily of genes in the HoxA cluster are expressed in a region-specific manner along the proximodistal axis. To elucidate the function of Hoxa-13 that is expressed in the autopod during normal limb development, Hoxa-13 was misexpressed in the entire limb bud with a replication-competent retroviral system. Misexpression of Hoxa-13 resulted in a remarkable size reduction of the zeugopodal cartilages as a result of the arrest of cartilage cell growth and differentiation restricted in the zeugopod. This size reduction seems to be attributable to homeotic transformation of the cartilages in the zeugopod to the more distal cartilage, that of the carpus/tarsus. This transformation was specific to Hoxa-13 and was not observed by overexpression of other Hox genes. These results indicate that Hoxa-13 is responsible for switching the genetic code from long bone formation to short bone formation during normal development. When the limb mesenchymal cells were dissociated and cultured in vitro, Hoxa-13-expressing limb mesenchymal cells reassociated and were sorted out from nonexpressing cells. Forced expression of Hoxa-13 at the stage that endogenous Hoxa-13 was not expressed as of yet altered the homophilic cell adhesive property. These findings indicate the involvement of Hoxa-13 in determining homophilic cell-to-cell adhesiveness that is supposed to be crucial for the cartilage pattern formation.
Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity.
Authors: Bailey A M , Posakony J W
Published: June 2007

We have investigated the functional relationships among three loci that are required for multiple alternative cell fate decisions during adult peripheral neurogenesis in Drosophila: Notch (N), which encodes a transmembrane receptor protein, Suppressor of Hairless [Su(H)], which encodes a DNA-binding transcription factor, and the Enhancer of split gene complex [E(spl)-C], which includes seven transcription units that encode basic helix-loop-helix (bHLH) repressor proteins. We describe several lines of evidence establishing that Su(H) directly activates transcription of E(spl)-C genes in response to N receptor activity. Expression of an activated form of the N receptor leads to elevated and ectopic E(spl)-C transcript accumulation and promoter activity in imaginal discs. We show that the proximal upstream regions of three E(spl)-C genes contain multiple specific binding sites for Su(H). The integrity of these sites, as well as Su(H) gene activity, are required not only for normal levels of expression of E(spl)-C genes in imaginal disc proneural clusters, but also for their transcriptional response to hyperactivity of the N receptor. Our results establish Su(H) as a direct regulatory link between N receptor activity and the expression of E(spl)-C genes, extending the known linear structure of the N cell-cell signaling pathway.
The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event.
Authors: Min H , Chan R C , Black D L
Published: June 2007

The proteins and RNA regulatory elements that control tissue-specific pre-mRNA splicing in mammalian cells are mostly unknown. In this study, a set of proteins is identified that binds to a splicing regulatory element downstream of the neuron specific c-src N1 exon. This complex of proteins bound specifically to a short RNA containing the regulatory sequence in neuronal extracts that splice the N1 exon. It was not seen in non-neuronal cell extracts that fail to splice this exon. UV-cross-linking experiments identified a neuron-specific 75-kD protein and several nontissue-specific proteins, including the 53-kD heterogeneous nuclear ribonucleoprotein F (hnRNP F), as components of this complex. Although present in both extracts, hnRNP F binds tightly to the RNA only in the neuronal extracts. A mutation in the regulatory RNA sequence, that inhibits N1 splicing in vivo, abolished formation of the neuron-specific complex and the binding of the neuron-specific 75-kD protein. Competition experiments in the two extracts show that the binding of the neuronal protein complex to the src pre-mRNA is required to activate N1 exon splicing in vitro. Antibody inhibition experiments indicate that the hnRNP F protein is a functional part of this complex. The assembly of regulatory complexes from both constitutive and specific proteins is likely to be a general feature of tissue-specific splicing regulation.
HSF access to heat shock elements in vivo depends critically on promoter architecture defined by GAGA factor, TFIID, and RNA polymerase II binding sites.
Authors: Shopland L S , Hirayoshi K , Fernandes M , Lis J T
Published: June 2007

Chromatin structure can modulate gene expression by limiting transcription factor access to gene promoters. We examined sequence elements of the Drosophila hsp70 promoter for their ability to facilitate the binding of the transcription factor, heat shock factor (HSF), to chromatin. We assayed HSF binding to various transgenic heat shock promoters in situ by measuring amounts of fluorescence at transgenic loci of polytene chromosomes that were stained with an HSF antibody. We found three promoter sequences that influence the access of HSF to its binding sites: the GAGA element, sequences surrounding the transcription start site, and a region in the leader of hsp70 where RNA polymerase II arrests during early elongation. The GAGA element has been shown previously to disrupt nucleosome structure. Because the two other critical regions include sequences that are required for stable binding of TFIID in vitro, we examined the in vivo occupancy of the TATA elements in the transgenic promoters. We found that TATA occupancy correlated with HSF binding for some promoters. However, in all cases HSF accessibility correlated with the presence of paused RNA polymerase II. We propose that a complex promoter architecture is established by multiple interdependent factors, including GAGA factor, TFIID, and RNA polymerase II, and that this structure is critical for HSF binding in vivo.
Amino acid substitutions in the structured domains of histones H3 and H4 partially relieve the requirement of the yeast SWI/SNF complex for transcription.
Authors: Kruger W , Peterson C L , Sil A , Coburn C , Arents G , Moudrianakis E N , Herskowitz I
Published: June 2007

Transcription of many yeast genes requires the SWI/SNF regulatory complex. Prior studies show that reduced transcription of the HO gene in swi and snf mutants is partially relieved by mutations in the SIN1 and SIN2 genes. Here we show that SIN2 is identical to HHT1, one of the two genes coding for histone H3, and that mutations in either can result in a Sin- phenotype. These mutations are partially dominant to wild type and cause amino acid substitutions in three conserved positions in the structured domain of histone H3. We have also identified partially dominant sin mutations that affect two conserved positions in the histone-fold domain of histone H4. Three sin mutations affect surface residues proposed to interact with DNA and may reduce affinity of DNA for the histone octamer. Two sin mutations affect residues at or near interfaces between (H2A-H2B) dimer and (H3-H4)2 tetramer subunits of the histone octamer and may affect nucleosome stability or conformation. The ability of mutations affecting the structure of the histone octamer to relieve the need for SWI and SNF products supports the proposal that the SWI/SNF complex stimulates transcription by altering chromatin structure and can account for the apparent conservation of SWI and SNF proteins in eukaryotes other than yeast.
A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye.
Authors: Dudley A T , Lyons K M , Robertson E J
Published: June 2007

BMP-7/OP-1, a member of the transforming growth factor-beta (TGF-beta) family of secreted growth factors, is expressed during mouse embryogenesis in a pattern suggesting potential roles in a variety of inductive tissue interactions. The present study demonstrates that mice lacking BMP-7 display severe defects confined to the developing kidney and eye. Surprisingly, the early inductive tissue interactions responsible for establishing both organs appear largely unaffected. However, the absence of BMP-7 disrupts the subsequent cellular interactions required for their continued growth and development. Consequently, homozygous mutant animals exhibit renal dysplasia and anophthalmia at birth. Overall, these findings identify BMP-7 as an essential signaling molecule during mammalian kidney and eye development.
BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning.
Authors: Luo G , Hofmann C , Bronckers A L , Sohocki M , Bradley A , Karsenty G
Published: June 2007

Bone morphogenetic proteins (BMPs) are multifunctional growth factors originally identified by their ability to induce ectopic bone formation. To investigate the function of one of the BMPs, BMP-7, we have generated BMP-7-deficient mice using embryonic stem cell technology. BMP-7-deficient mice die shortly after birth because of poor kidney development. Histological analysis of mutant embryos at several stages of development revealed that metanephric mesenchymal cells fail to differentiate, resulting in a virtual absence of glomerulus in newborn kidneys. In situ hybridization analysis showed that the absence of BMP-7 affects the expression of molecular markers of nephrogenesis, such as Pax-2 and Wnt-4 between 12.5 and 14.5 days postcoitum (dpc). This identifies BMP-7 as an inducer of nephrogenesis. In addition, BMP-7-deficient mice have eye defects that appear to originate during lens induction. Finally, BMP-7-deficient mice also have skeletal patterning defects restricted to the rib cage, the skull, and the hindlimbs.
An in vivo transposase-catalyzed single-stranded DNA circularization reaction.
Authors: Polard P , Chandler M
Published: June 2007

Expression of the bacterial insertion sequence IS911 transposase in vivo leads to excision and circularization of IS911-based transposons. We show here that transposase produces an unusual molecular form generated by single-strand cleavage, transfer, and ligation of one end of the element to the opposite end. When the transposon is carried by a circular plasmid, this results in the formation of a "figure-eight" molecule in which a single strand of the transposon is circularized while the corresponding strand of the vector backbone retains a single-strand interruption at this position. The results show that a 3' end of the transposon is transferred to the opposite target end. Transposase is therefore capable of introducing single-strand cleavages at the ends of the element, an activity similar to that of retroviral integrases with which it shares significant similarities in amino acid sequence. Kinetic studies demonstrate that the figure-eight accumulates earlier than transposon circles after transposase induction and disappears before circles after inhibition of transposase expression, raising the possibility that the figure-eight molecules are precursors to the circles. Therefore, IS911 excision as a circle may not occur by double-strand cleavage leading to its prior separation from the vector backbone in a linear form but could proceed by consecutive circularization of each strand.
Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli.
Authors: Zechiedrich E L , Cozzarelli N R
Published: June 2007

For a cell to complete DNA replication, every link between the Watson-Crick strands must be removed by topoisomerases. Previously, we reported that the inhibition of topoisomerase IV (topo IV) leads to the accumulation of catenated plasmid replicons to a steady-state level of approximately 10%. Using pulse labeling with [3H]thymidine in Escherichia coli, we have found that in the absence of topo IV activity, nearly all newly synthesized plasmid DNA is catenated. Pulse-chase protocols revealed that catenanes are metabolized even in the absence of topo IV and that the residual turnover is carried out by DNA gyrase at a rate of approximately 0.01/sec. Using extremely short pulse-labeling times, we identified significant amounts of replication catenanes in wild-type cells. The rate of catenane unlinking in wild-type cells by the combined activities of topo IV and DNA gyrase was approximately 1/sec. Therefore, gyrase is 100-fold less efficient than topo IV in plasmid replicon decatenation in vivo. This may explain why a fully functional gyrase cannot prevent the catenation of newly synthesized plasmid DNA and the partition phenotype of topo IV mutants. We conclude that catenanes are kinetic intermediates in DNA replication and that the essential role of topo IV is to unlink daughter replicons.
The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability.
Authors: Brachmann C B , Sherman J M , Devine S E , Cameron E E , Pillus L , Boeke J D
Published: June 2007

Genomic silencing is a fundamental mechanism of transcriptional regulation, yet little is known about conserved mechanisms of silencing. We report here the discovery of four Saccharomyces cerevisiae homologs of the SIR2 silencing gene (HSTs), as well as conservation of this gene family from bacteria to mammals. At least three HST genes can function in silencing; HST1 overexpression restores transcriptional silencing to a sir2 mutant and hst3 hst4 double mutants are defective in telomeric silencing. In addition, HST3 and HST4 together contribute to proper cell cycle progression, radiation resistance, and genomic stability, establishing new connections between silencing and these fundamental cellular processes.
Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction.
Authors: Hawley S H , Wünnenberg-Stapleton K , Hashimoto C , Laurent M N , Watabe T , Blumberg B W , Cho K W
Published: June 2007

Bone morphogenetic proteins (BMPs), which have been implicated in the patterning of mesoderm, are members of the transforming growth factor-beta (TGF-beta) superfamily. We have investigated the roles of Xenopus BMP-7 (XBMP-7) and BMP-4 (XBMP-4), and activin (another TGF-beta-related molecule) in early development by generating dominant-negative versions of these growth factors. Mutations were generated by altering the cleavage sites that are required for maturation of the active dimeric forms of XBMP-7, XBMP-4, and activin. These mutant constructs, designated Cm-XBMP-7, Cm-XBMP-4, and Cm-activin, result in polypeptides that allow for dimerization of the subunits, but are incapable of maturation. Expression of Cm-XBMP-7 and Cm-XBMP-4, but not Cm-activin, in the ventral marginal zone of the Xenopus embryo results in the development of a secondary axis, similar to that seen by ectopic expression of the truncated BMP receptor. These results suggest that the cleavage mutants interfere with BMP signaling during mesodermal patterning. We also found that expression of Cm-XBMP-7 or Cm-XBMP-4 in animal cap ectoderm directly induces neuroectoderm. The neural induction was specific for Cm-XBMP-7 and Cm-XBMP-4 because ectopic expression of Cm-activin or Vg-1 did not mimic the same phenotype. Molecular study of neural patterning by Cm-XBMP-7 and Cm-XBMP-4 revealed that only anterior neuroectodermal markers are expressed in response to these Cm-XBMPs. These results suggest that the BMPs are involved in the specification of ectoderm in Xenopus development, and that neural induction requires the removal of BMP signals in the ectoderm. We propose that neural induction occurs by a default mechanism, whereby the inhibition of BMP signaling is required for the conversion of ectoderm to neuroectoderm in the developing Xenopus embryo.
The symmetry of the yeast U6 RNA gene's TATA box and the orientation of the TATA-binding protein in yeast TFIIIB.
Authors: Whitehall S K , Kassavetis G A , Geiduschek E P
Published: June 2007

The central RNA polymerase III (Pol III) transcription factor TFIIIB is composed of the TATA-binding protein (TBP), Brf, a protein related to TFIIB, and the product of the newly cloned TFC5 gene. TFIIIB assembles autonomously on the upstream promoter of the yeast U6 snRNA (SNR6) gene in vitro, through the interaction of its TBP subunit with a consensus TATA box located at base pair -30. As both the DNA-binding domain of TBP and the U6 TATA box are nearly twofold symmetrical, we have examined how the binding polarity of TFIIIB is determined. We find that TFIIIB can bind to the U6 promoter in both directions, that TBP is unable to discern the natural polarity of the TATA element and that, as a consequence, the U6 TATA box is functionally symmetrical. A modest preference for TFIIIB binding in the natural direction of the U6 promoter is instead dictated by flanking DNA. Because the assembly of TFIIIB on the yeast U6 gene in vivo occurs via a TFIIIC-dependent mechanism, we investigated the influence of TFIIIC on the binding polarity of TFIIIB. TFIIIC places TFIIIB on the promoter in one direction only; thus, it is TFIIIC that primarily specifies the direction of transcription. Experiments using TFIIIB reconstituted with the altered DNA specificity mutant TBPm3 demonstrate that in the TFIIIB-U6 promoter complex, the carboxy-terminal repeat of TBP contacts the upstream half of the TATA box. This orientation of yeast TBP in Pol III promoter-bound TFIIIB is the same as in Pol II promoter-bound TFIID and in TBP-DNA complexes that have been analyzed by X-ray crystallography.
A common function for mRNA 5' and 3' ends in translation initiation in yeast.
Authors: Tarun S Z , Sachs A B
Published: June 2007

The mRNA poly(A) tail and its associated poly(A) binding protein (Pab1p) are ubiquitous in eukaryotes. The function of the poly(A) tail is to stabilize mRNA and to stimulate its translation. The development of a poly(A)- and cap-dependent yeast in vitro translation system has allowed us to understand how poly(A) stimulates translation. We find that Pab1p but not the cap binding protein eIF-4E is required for poly(A) tail-dependent translation, and that the Pab1p-poly(A) tail complex functions to recruit the 40S ribosomal subunit to the mRNA. These data introduce a new step into the pathway of translation initiation and merge the translational functions of the two ends of mRNA.
Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms.
Authors: Szabó P E , Mann J R
Published: June 2007

Genomic imprinting determines the monoallelic expression of a small number of genes during at least later stages of development. To obtain information necessary for the elucidation of imprinting mechanisms, we assessed the allele-specific expression and total expression level of four imprinted genes during early stages of development of normal F1 hybrid mice utilizing quantitative allele-specific reverse transcription-PCR (RT-PCR) single-nucleotide primer extension assays. The Igf2r and Snrpn genes were activated by the early 4-cell stage and exhibited biallelic and monoallelic expression, respectively, throughout preimplantation development. Thus, with respect to different imprinted genes, epigenetic systems determining monoallelic expression are not uniform in their time of establishment. Biallelic expression of Igf2r was observed in single blastomeres, discounting the possibility of random allelic inactivation at this stage. The closely linked H19 and Igf2 genes were activated after the blastocyst stage and often exhibited biallelic and monoallelic expression respectively in tissues of pregastrulation postimplantation-stage embryos, rather than reciprocal monoallelic modes as observed at later stages. This raises the possibility that imprinting of H19 is involved only in the maintenance and not in the initiation of monoallelic expression of Igf2. Monoallelic expression of Snrpn was observed in each blastomere at the 4-cell stage, demonstrating that the germ line, which exhibits biallelic expression of imprinted genes, must be derived from cells in which imprinting was once manifest.
The Caenorhabditis elegans gene lin-1 encodes an ETS-domain protein and defines a branch of the vulval induction pathway.
Authors: Beitel G J , Tuck S , Greenwald I , Horvitz H R
Published: June 2007

The Caenorhabditis elegans gene lin-1 appears to act after the Ras-Raf-MEK-MAPK signaling cascade that mediates vulval induction. We show that lin-1 is a negative regulator of vulval cell fates and encodes an ETS-domain putative transcription factor containing potential MAPK phosphorylation sites. In lin-1 null mutants, the vulval precursor cells (VPCs) still respond to signaling from the gonadal anchor cell, indicating that lin-1 defines a branch of the inductive signaling pathway. We also provide evidence that the inductive and lateral signaling pathways are integrated to control the 1 degree and 2 degrees vulval cell fates after the point at which lin-1 acts in the inductive pathway and that VPCs can assess the relative rather than absolute levels of inductive and lateral signaling in determining whether to express the 1 degree or 2 degrees vulval cell fates.
Synergistic interactions between two distinct elements of a regulated splicing enhancer.
Authors: Lynch K W , Maniatis T
Published: June 2007

Regulated alternative splicing of doublesex (dsx) pre-mRNA requires a splicing enhancer designated the dsx repeat element (dsxRE) that contains six copies of a 13-nucleotide repeat sequence. Previous studies have shown that the activity of the dsxRE requires the splicing regulators Transformer (Tra) and Transformer 2 (Tra2), and one or more members of the SR family of general splicing factors. In this paper we identify a purine-rich enhancer (PRE) sequence within the dsxRE, and show that this element functionally synergizes with the repeat sequences. In vitro binding studies show that the PRE is required for specific binding of Tra2 to the dsxRE, and that Tra and SR proteins bind cooperatively to the dsxRE in the presence or absence of the PRE. Thus positive control of dsx pre-mRNA splicing requires the Tra- and Tra2-dependent assembly of a multiprotein complex on at least two distinct enhancer elements.
The VS catalytic RNA replicates by reverse transcription as a satellite of a retroplasmid.
Authors: Kennell J C , Saville B J , Mohr S , Kuiper M T , Sabourin J R , Collins R A , Lambowitz A M
Published: June 2007

The mitochondria of certain natural isolates of Neurospora contain both the Varkud plasmid, which encodes a reverse transcriptase, and a small unrelated RNA (VS RNA) that performs RNA-mediated self-cleavage and ligation reactions. Here, we show that VS RNA is transcribed from a VS plasmid DNA template by the Neurospora mitochondrial RNA polymerase using a promoter located immediately upstream of the RNA self-cleavage site that generates monomeric transcripts. VS RNA is then reverse transcribed by the Varkud plasmid reverse transcriptase to yield a full-length (-) strand cDNA, a predicted replication intermediate. Combined with previous genetic evidence, our results indicate that the VS plasmid replicates by reverse transcription as a satellite of the Varkud plasmid. This mode of replication, unprecedented for a satellite RNA, likely reflects the promiscuity of the Varkud plasmid reverse transcriptase, which does not require a specific primer to initiate cDNA synthesis. Our findings indicate how primitive reverse transcriptases with similar relaxed specificity could have facilitated the evolution of new retroelements.
lin-25, a gene required for vulval induction in Caenorhabditis elegans.
Authors: Tuck S , Greenwald I
Published: June 2007

During vulval development in the Caenorhabditis elegans hermaphrodite, the fates of six vulval precursor cells (VPCs) are influenced by distinct cell signaling events. In one event, a somatic gonadal cell, the anchor cell, induces the three nearest VPCs to adopt vulval cell fates. In another event, lateral signaling between adjacent VPCs specifies one of two different vulval fates, 1 degrees and 2 degrees. Induction of vulval fates by the anchor cell is mediated by a signal transduction pathway involving let-60 Ras, lin-45 Raf, and mpk-1/sur-1 MAP kinase, whereas lateral signaling is mediated by lin-12. We have shown that the mutant phenotype of lin-25, a gene required for VPC fate specification, results from a defect in vulval induction. Genetic epistasis experiments indicate that lin-25 is required in the inductive signaling pathway downstream of let-60 Ras and the Raf/MAP kinase cascade. A decrease in induction also appears to decrease lateral signaling. We have cloned and sequenced the lin-25 gene and shown that it encodes a novel protein that may be a target of the mpk-1/sur-1 MAPK.
Regulation of dorsal in cultured cells by Toll and tube: tube function involves a novel mechanism.
Authors: Norris J L , Manley J L
Published: June 2007

We described previously a transient cotransfection assay that allows us to study regulation of the Drosophila Dorsal protein (dl) in cultured cells. For example, we showed that over-expression of the Toll transmembrane receptor was sufficient to cause relocalization of dl from the cytoplasm to the nucleus. Here we present data that the tube protein, shown previously by genetic studies to act downstream of Toll, can function in a novel way to enhance dl activity. In the absence of dl, or when dl is cytoplasmic, tube is also found in the cytoplasm of transfected cells. But when dl is localized to the nucleus, so is tube. tube can then function to enhance reporter gene expression, either by cooperation with dl or as a GAL4-tube fusion protein. tube thus appears capable of acting both as a chaperon or escort for dl as it moves to the nucleus, and then as a transcriptional coactivator. We also show that the intracytoplasmic domain of Toll, and specifically the region sharing homology with the interleukin-1 receptor, is sufficient to induce dl-tube nuclear translocation.
Reciprocal interferences between nucleosomal organization and transcriptional activity of the yeast SNR6 gene.
Authors: Marsolier M C , Tanaka S , Livingstone-Zatchej M , Grunstein M , Thoma F , Sentenac A
Published: June 2007

Recent work has demonstrated a repressive effect of chromatin on the transcription of the yeast SNR6 gene in vitro. Here, we show the relations between chromatin structure and transcriptional activity of this gene in vivo. Analysis of the SNR6 locus by micrococcal nuclease digestion showed a protection of the TATA box, nuclease-sensitive sites around the A and B blocks, and arrays of positioned nucleosomes in the flanking regions. Analysis of a transcriptionally silent SNR6 mutant containing a 2-bp deletion in the B block showed a loss of TATA-protection and rearrangement or destabilization of nucleosomes in the flanking regions. Hence, SNR6 organizes the chromatin structure in the whole region in a manner dependent on its transcriptional state. Transcriptional analysis was performed by use of maxi-gene SNR6 constructs introduced into histone-mutated strains. Chromatin disruption induced by histone H4 depletion stimulated the transcription of promoter-deficient, but not of wild-type SNR6 genes, revealing a competition between the formation of nucleosomes and the assembly of Pol III transcription complexes that was much in favor of transcription factors. On the other hand, amino-terminal mutations in histone H3 or H4 had no effect (H4) or only a moderate stimulatory effect (H3) on the transcription of promoter-deficient SNR6 genes.
DNA rearrangements located over 100 kb 5' of the Steel (Sl)-coding region in Steel-panda and Steel-contrasted mice deregulate Sl expression and cause female sterility by disrupting ovarian follicle development.
Authors: Bedell M A , Brannan C I , Evans E P , Copeland N G , Jenkins N A , Donovan P J
Published: June 2007

The Steel (Sl) locus is essential for the development of germ cells, hematopoietic cells, and melanocytes and encodes a growth factor (Mgf) that is the ligand for c-kit, a receptor tyrosine kinase encoded by the W locus. We have identified the molecular and germ cell defects in two mutant Sl alleles, Steel-panda (Slpan) and Steel-contrasted (Slcon), that cause sterility only in females. Unexpectedly, both mutant alleles are shown to contain DNA rearrangements, located > 100 kb 5' of Mgf-coding sequences, that lead to tissue-specific effects on Mgf mRNA expression. In Slpan embryos, decreased Mgf mRNA expression in the gonads causes a reduced number of primordial germ cells in both sexes. However, Mgf expression and spermatogenesis in the postnatal mutant tests is normal, and spermatogonial proliferation compensates for deficiencies in germ cell numbers. In Slpan and Slcon homozygous females, decreased Mgf mRNA expression causes sterility by affecting the initiation and maintenance of ovarian follicle development. Thus, regulated expression of Mgf is required for multiple stages of embryonic and postnatal germ cell development. Surprisingly, other areas of the Slcon female reproductive tract displayed ectopic expression of Mgf mRNA. We propose that the Slpan and Slcon rearrangements alter Mgf mRNA abundance through position effects on expression that act at a distance from the Sl gene.
RNA polymerase II subunit RPB9 is required for accurate start site selection.
Authors: Hull M W , McKune K , Woychik N A
Published: June 2007

The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12. Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown. We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection. Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo. Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9. In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro. A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro. RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.
flbD encodes a Myb-like DNA-binding protein that coordinates initiation of Aspergillus nidulans conidiophore development.
Authors: Wieser J , Adams T H
Published: June 2007

The timing of asexual fruiting body formation during Aspergillus nidulans colony development is precisely regulated so that conidiophores are typically produced 1-2 mm behind the growing edge of the colony. Mutations in any of four A. nidulans genes, flbB, flbC, flbD, or flbE, result in colonies that are delayed at least 24 hr in their ability to initiate conidiophore development resulting in fluffy colonies with conidiophores forming in the center, at least 12-15 mm behind the growing edge. The requirement for each of these four genes in determining the timing of developmental initiation precedes transcriptional activation of the primary developmental regulatory gene brlA, indicating a possible role for each gene in developmentally regulated activation of brlA expression. The wild-type flbD gene was isolated and shown to encode an approximately 1.6-kb mRNA that is present throughout the A. nidulans life cycle. The deduced FlbD protein sequence predicts a 314-amino-acid polypeptide with significant identity at its amino terminus to the DNA-binding domain of the Myb family of transcription factors indicating that FlbD probably functions as a sequence-specific transcriptional activator. Although conidiophore development does not normally occur in submerged culture, forced overexpression of flbD in submerged hyphae caused inappropriate activation of brlA expression and resulted in production of complex conidiophores that produced all of the distinct cell types observed in wild-type conidiophores including viable spores. This ability of flbD overexpression to activate conidiation requires brlA, flbB, and flbA (another early developmental regulator) but does not require flbC or flbE. We propose that FlbD functions during normal development by activating transcription of other genes required for development (such as brlA) and that FlbD activity is normally controlled post-transcriptionally by an unknown mechanism.
A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis.
Authors: Stoler S , Keith K C , Curnick K E , Fitzgerald-Hayes M
Published: June 2007

The centromere, a differentiated region of the eukaryotic chromosome, mediates the segregation of sister chromatids at mitosis. In this study, a Saccharomyces cerevisiae chromosome mis-segregation mutant, cse4-1, has been isolated and shown to increase the nondisjunction frequency of a chromosome bearing a mutant centromere DNA sequence. In addition, at elevated temperatures the cse4-1 allele causes a mitosis-specific arrest with a predominance of large budded cells containing single G2 nuclei and short bipolar mitotic spindles. The wild-type gene, CSE4, is essential for cell division and encodes a protein containing a domain that is 64% identical to the highly conserved chromatin protein, histone H3. Biochemical experiments demonstrate that CSE4p has similar DNA-binding characteristics as those of histone H3 and might form a specialized nucleosome structure in vivo. Interestingly, the human centromere protein, CENP-A, also contains this H3-like domain. Data presented here indicate that CSE4p is required for proper kinetochore function in yeast and may represent an evolutionarily conserved protein necessary for assembly of the unique chromatin structure associated with the eukaryotic centromere.
Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate.
Authors: Zhou P , Byrne C , Jacobs J , Fuchs E
Published: June 2007

T cell-specific transcription factor (TCF-1) and lymphoid enhancer factor 1 (LEF-1) have been implicated exclusively in the regulation of T cell-specific genes. The only adult tissue other than thymus known to express these factors is spleen and lymph node, which contain low levels of LEF-1 and no TCF-1. We noticed that genes involved in hair-specific gene expression possess LEF-1/TCF-1 consensus motifs located in similar positions relative to their TATA box. We show that of the two factors only LEF-1 is expressed in hair follicles; it can be cloned in both splice forms from human skin keratinocytes and it can bind to these sites in the hair promoters. We show that LEF-1 mRNA is present in pluripotent ectoderm, and it is up-regulated in a highly restricted pattern just before the formation of underlying mesenchymal condensates and commitment of overlying ectodermal cells to invaginate and become hair follicles. New waves of ectodermal LEF-1 spots appear concomitant with new waves of follicle morphogenesis. To test whether LEF-1 patterning might be functionally important for hair patterning and morphogenesis, we used transgenic technology to alter the patterning and timing of LEF-1 over the surface ectoderm. Striking abnormalities arose in the positioning and orientation of hair follicles, leaving a marked disruption of this normally uniform patterning. This provides the first direct evidence that ectodermal cues are critical in establishing these developmental processes, which at later stages are known to be influenced by underlying mesenchyme. Remarkably, elevated LEF-1 in the lip furrow epithelium of developing transgenic animals triggered these cells to invaginate, sometimes leading to the inappropriate adoption of hair follicle and tooth cell fates. Collectively, our findings demonstrate that ectodermal expression of LEF-1 plays a central role in gene expression, pattern formation, and other developmental processes involving epithelial-mesenchymal associations.
The Caenorhabditis elegans gene mek-2 is required for vulval induction and encodes a protein similar to the protein kinase MEK.
Authors: Kornfeld K , Guan K L , Horvitz H R
Published: June 2007

An evolutionarily conserved signal transduction pathway that utilizes a receptor tyrosine kinase and a Ras protein mediates the induction of vulval cell fates in the nematode Caenorhabditis elegans. We sought new genes that function in this pathway by screening for suppressors of the Multivulva phenotype caused by a mutation that activates the let-60 ras gene. Seven such suppressor mutations defined a new gene involved in vulval induction. We named this gene mek-2, because its predicted protein product is most similar to MEK, a protein-serine/threonine and tyrosine kinase. mek-2 mutations can be arranged in an allelic series. A probable null mutation eliminated vulval induction, and the strongest mutations alter codons conserved in most or all protein kinases. Our genetic analysis showed that mek-2 functions downstream of let-60 ras and is required for ras-mediated signal transduction in vivo. The MEK-2 protein may interact with the products of the lin-45 raf and mpk-1 MAP kinase genes, which also mediate vulval induction.
Cactus protein degradation mediates Drosophila dorsal-ventral signaling.
Authors: Belvin M P , Jin Y , Anderson K V
Published: June 2007

Dorsal-ventral patterning in the Drosophila embryo relies on a signal transduction pathway that is similar to a signaling pathway leading to the activation of the mammalian transcription factor NF-kappa B. Stimulation of this Drosophila pathway on the ventral side of the embryo causes the nuclear translocation of Dorsal, the Drosophila NF-kappa B homolog. Cactus, like its mammalian homolog I kappa B, inhibits nuclear translocation by binding Dorsal and retaining it in the cytoplasm. We show that Cactus, like I kappa B, is rapidly degraded in response to signaling. More importantly, signal-dependent degradation of Cactus does not require the presence of Dorsal, indicating that Cactus degradation is a direct response to signaling, and that disruption of the Dorsal/Cactus complex is a secondary result of Cactus degradation. Mutant alleles of cactus that encode more stable forms of the protein block signaling, showing that efficient degradation is necessary for signaling. We find that Cactus protein stability is regulated by two independent processes that rely on different regions within the protein: signal-dependent degradation requires sequences in the amino terminus or ankyrin repeats, whereas signal-independent degradation of free Cactus requires the carboxy-terminal region of the protein that includes a PEST sequence.
Distinct TPR motifs of Cyc8 are involved in recruiting the Cyc8-Tup1 corepressor complex to differentially regulated promoters.
Authors: Tzamarias D , Struhl K
Published: June 2007

The yeast Cyc8(Ssn6)-Tup1 complex is required for transcriptional repression of distinct sets of genes that are regulated by glucose, oxygen, cell type, and DNA damage. It has been proposed that the Cyc8-Tup1 complex is a corepressor that is recruited to promoters by interacting with pathway-specific DNA-binding proteins. Previously, we showed that a specific region of Tup1 mediates the general transcriptional repression function of the complex. Here, we define functional domains of Cyc8, a protein consisting primarily of 10 tandem copies of a TPR motif. Distinct combinations of TPR motifs are required specifically for direct interaction with Tup1, repression of oxygen-regulated genes, and repression of glucose-regulated genes. In contrast, the WD motifs of Tup1 are not essential for repression of genes regulated by glucose and oxygen, but they are required for those regulated by cell type and DNA damage. In addition, we show that the Cyc8-Tup1 complex functions both as a corepressor and an inhibitor of Mig1, a protein that binds to promoters of glucose-repressible genes. These observations suggest that different Cyc8 TPR motifs and the Tup1 WD domain mediate distinct protein-protein interactions that link the Cyc8-Tup1 corepressor to structurally dissimilar DNA-binding proteins required for pathway-specific regulation.
TFIIIC determines RNA polymerase III specificity at the TATA-containing yeast U6 promoter.
Authors: Roberts S , Colbert T , Hahn S
Published: June 2007

The gene encoding yeast U6 snRNA that is transcribed by RNA polymerase III (Pol III) contains both a TATA box upstream of the transcription start site and a downstream binding site for the factor TFIIIC. This juxtaposition of elements typical of both Pol II- and Pol III-transcribed genes raises the question of how polymerase specificity is determined. The upstream U6 promoter containing the TATA box and transcription start site was shown previously to be transcribed by Pol III in vitro. We therefore tested whether the upstream promoter of yeast U6 encodes Pol III specificity. One model is that polymerase specificity is conferred by the homologous Pol II and Pol III transcription factors TFIIB and BRF1. However, we found no specificity in the binding of BRF1 or TFIIB to TATA-containing promoters of genes specifically transcribed by Pol III or Pol II. Yeast strains deficient for Pol II or Pol III transcription were employed to examine U6 polymerase specificity in vivo. We find that the U6 upstream promoter is Pol II-specific in vivo and is converted to Pol III specificity by TFIIIC. Thus, preferential recruitment of TFIIIB by TFIIIC probably excludes the Pol II general factors and promotes Pol III transcription, thereby determining polymerase specificity.