bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of synthesis and positioning of a Caulobacter crescentus flagellar protein.
Authors: Loewy Z G , Bryan R A , Reuter S H , Shapiro L
Published: June 2007

The Caulobacter crescentus flagellum is assembled during a defined time period in the cell cycle. Two genes encoding the major components of the flagellar filament, the 25K and the 27.5K flagellins, are expressed coincident with flagellar assembly. A third gene, flgJ, is also temporally regulated. The synthesis of the product of flgJ, the 29K flagellin, occurs prior to the synthesis of the other flagellin proteins. We demonstrate here that the time of initiation of flgJ expression is independent of chromosomal location but is dependent upon cis-acting sequences present upstream of the flgJ structural gene. Evidence that there is transcriptional control of flgJ expression includes the following: (1) The initial appearance of flgJ message was coincident with the onset of 29K flagellin protein synthesis, and (2) expression of an NPT II reporter gene driven by the flgJ promoter was temporally correct. Post-transcriptional regulation might contribute to the control of expression, because the flgJ mRNA persisted for a longer period of time than did the synthesis of the 29K protein. The 29K flagellin was found only in the progeny swarmer cell after cell division. In a mutant strain that failed to assemble a flagellum, the 29K flagellin still segregated to the presumptive swarmer cell, demonstrating that positioning of the protein is independent of filament assembly. Analysis of a chimeric flgJ-NPT II transcriptional fusion showed that the flgJ regulatory sequences do not control the segregation of the 29K flagellin to the swarmer cell progeny, suggesting that correct segregation depends on the protein product.
Adenovirus transforming 19-kD T antigen has an enhancer-dependent trans-activation function and relieves enhancer repression mediated by viral and cellular genes.
Authors: Yoshida K , Venkatesh L , Kuppuswamy M , Chinnadurai G
Published: June 2007

The adenovirus E1b region codes for two major tumor antigens of 19 kD and 55 kD, which are important for cell transformation. Our results indicate that the 19-kD T antigen possesses two enhancer-regulatory functions. It can trans-activate enhancer-linked promoters and relieve enhancer repression mediated by viral and cellular repressors. The 19-kD activation function enhances expression from different promoters linked to SV40, Py, Ela, and immunoglobulin heavy-chain enhancers. Enhancer activation by the 19-kD protein appears to be cell type-specific, since the heavy-chain and SV40 enhancers were not trans-activated in myeloma cells whereas the same enhancers were trans-activated in fibroblasts. The 19-kD enhancer activation function appears to be dominant over the enhancer repression function of E1a, since in cells expressing the 19-kD protein there is no significant repression despite a large increase in E1a expression. The 19-kD T antigen activates the Py enhancer in undifferentiated F9 cells indicating that the activation function of E1b masks enhancer repression by an "E1a-like" cellular gene product. The enhancer activation function of the 19-kD T antigen may be important for cell transformation and cell differentiation.
The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma.
Authors: Nagaya T , Nakamura T , Tokino T , Tsurimoto T , Imai M , Mayumi T , Kamino K , Yamamura K , Matsubara K
Published: June 2007

Nineteen DNA samples that carry integrated hepatitis B virus (HBV) DNA were isolated from seven independent human hepatomas by molecular cloning, and their structures were determined. The results, combined with reported data, were analyzed so that one can obtain insights into the mechanisms of integration of this virus DNA and possible rearrangements that occur subsequently. The distribution of DNA junctions along the virus genome suggests that there are recombination-proficient regions. Thus, about half of the integrants were the Coh type, viz., one of their virus-cell DNA junctions fell within the so-called cohesive end region that lies between two 11-bp direct repeats (DR1 and DR2) in the virus genome where transcription and replication of the genome are initiated. All the integrated virus genomes were defective at least in one site around the cohesive end region, particularly within the X gene. The recombination-proficient regions are used not only for formation of virus-cell but also of virus-virus junctions. Neither virus nor cell DNA show unique sequences at the junctions, and targets for integration lie on many different chromosomes.
Spliceosome assembly in yeast.
Authors: Cheng S C , Abelson J
Published: June 2007

Precursors to mRNA become substrates for splicing by being assembled into a complex multisubunit structure, the spliceosome. To study the assembly of the yeast spliceosome, intermediate complexes were separated by electrophoresis on nondenaturing polyacrylamide gels. Four splicing-dependent complexes, A1, A2-1, A2-2, and B, were observed. The order of assembly of these complexes was determined to be B----A2-1----A1----A2-2. The assembly process can be blocked at complex A1 by addition of 5 mM EDTA or by carrying out the assembly process in heat-inactivated rna2 extracts. The snRNA composition of the complexes was determined by hybridization with probes for five yeast snRNAs. snR14 (U4) was only found in complex A2-1, snR6 (U6) and snR7 (U5) were in complexes A1, A2-1, and A2-2, whereas snR20 (U2) was in all four of the complexes. snR19 (U1) was not present in any of the complexes. Hybridization with these probes was also employed to detect snRNPs present in yeast splicing extracts. We found that snR6, snR7, and snR14 were present together in a large complex. This complex underwent an ATP-dependent dissociation to give snR7 and snR6-snR14 complexes. snR19 and snR20 are present in distinct RNPs but the mobility of these is not affected by ATP. A mechanism for spliceosome assembly is proposed.
Activation of a cryptic 5' splice site in the upstream exon of the phage T4 td transcript: exon context, missplicing, and mRNA deletion in a fidelity mutant.
Authors: Chandry P S , Belfort M
Published: June 2007

A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments. Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA). Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site. Of these, only one, SC99, appeared to be a specificity mutant. Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant. The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition. Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant. These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.
Erythroid lineage-specific expression and inducibility of the major heat shock protein HSP70 during avian embryogenesis.
Authors: Banerji S S , Laing K , Morimoto R I
Published: June 2007

We have studied the expression of the major heat shock protein HSP70 during maturation of avian erythroid cells. Primitive and definitive erythroid cells were isolated from staged day 3-8 chicken embryos, and the levels of HSP70 mRNA and protein synthesis were examined. The highest levels of HSP70 are detected in polychromatic cells of the day 3-4 primitive erythroid cell. After the initial burst of HSP70 expression the levels of HSP70 mRNA and protein synthesis decline. Although HSP70 is constitutively expressed, neither HSP70 synthesis nor HSP70 mRNA levels were heat shock inducible in primitive red cells. In contrast, definitive red cells respond to heat shock by a 10- to 20-fold increase in HSP70 protein synthesis with little change in HSP70 mRNA levels. These studies reveal that HSP70 expression in erythroid cells is lineage specific, that the levels of HSP70 mRNA are not induced by heat shock, and finally, that the increased expression of HSP70 in definitive cells is due to increased translatability of HSP70 mRNA.
Separate regulatory elements are responsible for the complex pattern of tissue-specific and developmental transcription of the yellow locus in Drosophila melanogaster.
Authors: Geyer P K , Corces V G
Published: June 2007

DNA sequences involved in the control of the developmental and spatial expression of the yellow locus of Drosophila were identified by phenotypic analysis of germline transformants carrying various in vitro modified yellow genes. Two regions, located between -2873 bp and -1868 bp and between -1868 bp and -700 bp, act as tissue-specific enhancers which respectively regulate yellow transcription in the wings and body of the adult flies. Sequences situated closer to the mRNA cap site, between -225 bp and -91 bp from the start of transcription, are responsible for yellow expression in the denticle belts and mouth parts during larval development. Finally, coloration of the adult bristles is regulated by sequences located in the intron of the yellow gene. These results indicate the existence of several separate DNA elements responsible for the different patterns of temporal and spatial expression of the yellow locus.
The c-ros tyrosine kinase receptor controls regionalization and differentiation of epithelial cells in the epididymis.
Authors: Sonnenberg-Riethmacher E , Walter B , Riethmacher D , Gödecke S , Birchmeier C
Published: June 2007

The c-ros gene was originally identified in mutant form as an oncogene. The proto-oncogene encodes a tyrosine kinase receptor that is expressed in a small number of epithelial cell types, including those of the epididymis. Targeted mutations of c-ros in the mouse reveal an essential role of the gene in male fertility. Male c-ros -/- animals do not reproduce, whereas the fertility of female animals is not affected. We demonstrate that c-ros is not required in a cell autonomous manner for male germ cell development or function. The gene, therefore, does not affect sperm generation or function in a direct manner. The primary defect in the mutant animals was located in the epididymis, showing that c-ros controls appropriate development of the epithelia, particularly regionalization and terminal differentiation. The epididymal defect does not interfere with production or storage of sperm but, rather, with sperm maturation and the ability of sperm to fertilize in vivo. Interestingly, sperm isolated from c-ros -/- animals can fertilize in vitro. Our results highlight the essential role of the epididymis in male fertility and demonstrate a highly specific function of the c-ros receptor tyrosine kinase during development of distinct epithelial cells.
Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with AML1.
Authors: Daga A , Karlovich C A , Dumstrei K , Banerjee U
Published: June 2007

The lozenge (lz) gene encodes a transcription factor involved in prepatterning photoreceptor precursors in the developing Drosophila eye. The central region of the predicted Lz protein product is homologous to AML1, a transcription factor associated with human leukemias, and to the Drosophila protein Runt. We show here that Lz plays a crucial role in governing the fate of two groups of cells that are born in a single round of mitosis in the larval eye disc. Lz helps define a subset of these cells as an equipotential group that is competent to respond to the Sevenless developmental signal. This is achieved by negative regulation of seven-up, a member of the steroid hormone receptor superfamily in these cells. In contrast, in a second group of cells, the Lz protein confers proper photoreceptor identity by positively regulating the homeo box gene Bar. Additionally, our genetic analysis suggests that Lz interacts with the Ras pathway to determine photoreceptor cell fate. This study suggests that the strategies involved in cell fate determination in the Drosophila eye are remarkably similar to those utilized during vertebrate hematopoietic development and require the coordinate action of growth factor and AML1-like pathways.
A protein that shuttles between the nucleus and the cytoplasm is an important mediator of RNA export.
Authors: Lee M S , Henry M , Silver P A
Published: June 2007

The connection between RNA and protein export from the nucleus was examined in the budding yeast Saccharomyces cerevisiae. NPL3 encodes an RNA-binding protein that shuttles in and out of the nucleus. Export of poly(A)+ RNA has been shown previously to be blocked in np13-1 mutants. To understand the role of Np13p in RNA export, we have developed a novel assay that effectively uncouples nuclear protein export from reimport. With this assay, we show that Np13p satisfies several of the predicted requirements for a protein carrier for mRNA export. Temperature-sensitive mutations in the RNA recognition motifs of Np13p result in nuclear accumulation of poly(A)+ RNA. One such mutation prevents nuclear export of Np13p. Moreover, Np13p export depends on ongoing RNA polymerase II transcription. Export ceases in either the presence of the RNA synthesis inhibitor thiolutin or in a temperature-sensitive RNA polymerase (rpb1) mutant. Together, these findings support a model in which Np13p exits the nucleus in association with poly(A)+ RNA, deposits the RNA in the cytoplasm, and is rapidly reimported for another cycle of export.
Lineage-specific regulators couple cell lineage asymmetry to the transcription of the Caenorhabditis elegans POU gene unc-86 during neurogenesis.
Authors: Baumeister R , Liu Y , Ruvkun G
Published: June 2007

The POU homeo box gene unc-86 specifies neuroblast and neural identities in the developing Caenorhabditis elegans nervous system. After an asymmetric neuroblast division, unc-86 is expressed in one of two daughter cells in 27 lineage classes that are not obviously related by function or position. We show here that unc-86 transcriptional regulatory regions detect cell lineage asymmetry to activate unc-86 expression in one of two neuroblast daughter cells. Distinct regulatory regions activate unc-86 expression in particular sets of sublineages. Therefore the unc-86 regulatory region integrates distinct cell lineage asymmetry cues to activate unc-86 expression in the many classes of neuroblast cell lineages. In agreement with such lineage-specific regulation of unc-86 asymmetric activation, mutations in lin-11 (LIM homeo box), ham-1, and lin-17 affect the asymmetry of unc-86 expression in particular cell lineages, and mutations in lin-32 (achaete/scute family), vab-3 (Pax-6 homolog) and egl-5 (Abd-B homolog) affect the establishment of unc-86 expression in other cell lineages. Homologs of unc-86 and many of these unc-86 regulators have been implicated in control of neurogenesis in vertebrates and invertebrates. These data suggest that unc-86 acts in a phylogenetically conserved pathway that couples neuroblast cell lineage asymmetry to the generation of diverse neural types.
The SL1 trans-spliced leader RNA performs an essential embryonic function in Caenorhabditis elegans that can also be supplied by SL2 RNA.
Authors: Ferguson K C , Heid P J , Rothman J H
Published: June 2007

Covalent joining of leader RNA exons to pre-mRNAs by trans-splicing has been observed in protists and invertebrates, and can occur in cultured mammalian cells. In the nematode Caenorhabditis elegans, approximately 60% of mRNA species are trans-spliced to the 22-nucleotide SL1 leader, and another approximately 10% of mRNAs receive the 22-nucleotide SL2 leader. We have isolated deletions that remove the rrs-1 cluster, a gene complex that contains approximately 110 tandem copies of a repeat encoding both SL1 RNA and 5S rRNA. An SL1-encoding gene alone rescues the embryonic lethality caused by these deletions. Mutations within the Sm-binding site of SL1 RNA, which is required for trans-splicing, eliminate rescue, suggesting that the ability of the SL1 leader to be trans-spliced is required for its essential activity. We observe pleiotropic defects in embryos lacking SL1 RNA, suggesting that multiple mRNAs may be affected by the absence of an SL1 leader. We found, however, that SL1-receiving messages are expressed without an SL1 leader. Surprisingly, when overexpressed, SL2 RNA, which performs a distinct function from that of SL1 RNA in wild-type animals, can rescue the lethality of embryos lacking SL1 RNA. Moreover, in these mutant embryos, we detect SL2 instead of SL1 leaders on normally SL1-trans-spliced messages; this result suggests that the mechanism that discriminates between SL1 and SL2-trans-splicing may involve competition between SL1 and SL2-specific trans-splicing. Our findings demonstrate that SL1 RNA is essential for embryogenesis in C. elegans and that SL2 RNA can substitute for SL1 RNA in vivo.
Hepatic specification of the gut endoderm in vitro: cell signaling and transcriptional control.
Authors: Gualdi R , Bossard P , Zheng M , Hamada Y , Coleman J R , Zaret K S
Published: June 2007

We have studied the initial development of pluripotent gut endoderm to hepatocytes using a tissue explant system from mouse embryos. We not only find cellular interactions that specify hepatic differentiation but also those that block hepatogenesis in regions of the endoderm that normally give rise to other tissues. The results implicate both positive and negative signaling in early hepatic specification. In vivo footprinting of the albumin enhancer in precursor gut endoderm shows that the transcriptionally silent but potentially active chromatin is characterized by occupancy of an HNF-3 site. Upon hepatic specification, a host of other factors bind nearby sites as the gene becomes active. Genes in pluripotent cells therefore may be marked for potential expression by entry points in chromatin, where additional factors bind during cell type specification. The findings also provide insight into the evolutionary origin of different endodermal cell types.
Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression.
Authors: Maillet L , Boscheron C , Gotta M , Marcand S , Gilson E , Gasser S M
Published: June 2007

Transcriptional repression at the silent mating-type loci in yeast requires the targeting of silent information regulator (Sir) proteins through specific interactions formed at cis-acting silencer elements. We show here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere. Repression is restored by creation of a new telomere 13 kb from the integrated reporter or by elevated expression of SIR1, SIR3, and/or SIR4. Coupled expression represses in an additive manner, suggesting that all three factors are in limiting concentrations. When overexpressed, Sir3 and Sir4 are dispersed throughout the nucleoplasm, in contrast to wild-type cells where they are clustered in a limited number of foci together with telomeres. Efficient silencer function thus seems to require either proximity to a pool of concentrated Sir proteins, that is, proximity to telomeres, or delocalization of the silencing factors.
Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases.
Authors: Di Como C J , Arndt K T
Published: June 2007

We identified an essential Saccharomyces cerevisiae protein, Tap42, that associates with Sit4, a type 2A-related protein phosphatase, and with the type 2A phosphatase catalytic subunits. The association of Tap42 with the phosphatases does not require the previously identified phosphatase subunits. Genetic analysis suggests that Tap42 functions positively with both phosphatases. Mutations in TAP42 can confer almost complete rapamycin resistance. In addition, Tap42/Sit4 and Tap42/PP2A complex formation is regulated by nutrient growth signals and the rapamycin-sensitive Tor signaling pathway. These findings, combined with the defect in translation of the tap42-11 mutant at the nonpermissive temperature, suggest that Tap42, Sit4, and PP2A are components of the Tor signaling pathway.
Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis.
Authors: Solomon J M , Lazazzera B A , Grossman A D
Published: June 2007

We have purified and characterized an extracellular peptide factor that serves as a cell density signal for both competence development and sporulation in Bacillus subtilis. This competence and sporulation stimulating factor (CSF) was purified from conditioned medium (culture supernatant) based on its ability to stimulate expression of srfA (comS) in cells at low cell density. CSF is a 5-amino-acid peptide, glu-arg-gly-met-thr (ERGMT), that is, the carboxy-terminal 5 amino acids of the 40-amino-acid peptide encoded by phrC. No detectable CSF was produced in a phrC null mutant. The activity of chemically synthesized CSF (ERGMT) was virtually indistinguishable from that of CSF that was purified from culture supernatants. At relatively low concentrations (1-10 nM), CSF stimulated expression of srfA, whereas high concentrations of CSF stimulated the ability of cells at low cell density to sporulate. Stimulation of srfA expression by CSF requires the oligopeptide permease encoded by spo0K, a member of the ATP-binding-cassette family of transporters, and the putative phosphatase encoded by rapC, the gene immediately upstream of phrC. RapC was found to be a negative regulator of srfA expression, suggesting that the target of RapC is the transcription factor encoded by comA. We propose that CSF is transported into the cell by the Spo0K oligopeptide permease and stimulates competence gene expression by inhibiting (either directly or indirectly) the RapC phosphatase.
A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae.
Authors: Bai Y , Symington L S
Published: June 2007

With the use of an intrachromosomal inverted-repeat as a recombination reporter we have previously shown that mitotic recombination is dependent on the RAD52 gene. However, recombination was found to be reduced only 4-fold by mutation of RAD51, which encodes a homolog of bacterial RecA proteins. A rad51, which strain containing the recombination reporter was mutagenized to identify components of the RAD51-independent pathway. One mutation identified, rad59, reduced recombination 1200-fold in the presence of a rad51 mutation, but only 4- to 5-fold in a wild-type background. Thus the rad51 and rad59 mutations reduce recombination synergistically. The rad59 mutation reduced both spontaneous and double-strand-break-induced recombination between inverted repeats. However, the rate of interchromosomal recombination was increased in a rad59 homozygous diploid. These observations suggest that RAD59 functions specifically in intrachromosomal recombination. The rad59 mutant strain was sensitive to ionizing radiation, and this phenotype was used to clone the RAD59 gene by complementation. The gene encodes a protein of 238 amino acids with significant homology to members of the Rad52 family. Overexpression of RAD52 was found to suppress the DNA repair and recombination defects conferred by the rad59 mutation, suggesting that these proteins have overlapping roles or function as a complex.
V(D)J recombination activates a p53-dependent DNA damage checkpoint in scid lymphocyte precursors.
Authors: Guidos C J , Williams C J , Grandal I , Knowles G , Huang M T , Danska J S
Published: June 2007

Double-stranded DNA breaks (DSBs) trigger p53-mediated cell cycle arrest or apoptosis pathways that limit the oncogenic consequences of exposure to genotoxic agents, but p53-mediated responses to DSB generated by normal physiologic events have not been documented. "Broken" V(D)J coding ends accumulate in scid lymphocyte precursors as a consequence of a mutation in DNA-dependent protein kinase (DNA-PK). The ensuing failure to rearrange efficiently antigen receptors arrests lymphoid development. Here we show that scid thymocytes express high levels of p53 protein, attributable to recombinase activating gene (RAG)-dependent generation of DSB adjacent to V, D, and J gene segments. To examine the functional importance of p53 expression in vivo, we bred p53-/- scid mice. The absence of p53 facilitated production of in-frame V(D)Jbeta coding joints and developmental progression of scid thymocytes, in addition to a dramatic accumulation of pro-B cells. All mice developed disseminated pro-B or immature T cell lymphoma/leukemia by 7-12 weeks of age. We present evidence that p53 deficiency prolongs the survival of scid lymphocyte precursors harboring broken V(D)J coding ends, allowing the accumulation of aneuploid cells. These results demonstrate that a p53-mediated DNA damage checkpoint contributes to the immune deficiency characteristic of the scid mutation and limits the oncogenic potential of DSBs generated during V(D)J recombination.
Mutations in the p53 and SCID genes cooperate in tumorigenesis.
Authors: Nacht M , Strasser A , Chan Y R , Harris A W , Schlissel M , Bronson R T , Jacks T
Published: June 2007

DNA damage can cause mutations that contribute to cellular transformation and tumorigenesis. The p53 tumor suppressor acts to protect the organism from DNA damage by inducing either G1 arrest to facilitate DNA repair or by activating physiological cell death (apoptosis). Consistent with this critical function of p53, mice lacking p53 are predisposed to developing tumors, particularly lymphoma. The severe combined immune deficiency (scid) focus encodes the catalytic subunit of DNA protein kinase (DNA-PKcs), a protein complex that has a role in the cellular response to DNA damage. Cells from scid mice are hypersensitive to radiation and scid lymphocytes fail to develop from precursors because they are unable to properly join DNA-coding ends during antigen receptor gene rearrangement. We examined the combined effect of loss of p53 and loss of DNA-PKcs on lymphocyte development and tumorigenesis by generating p53-/- scid mice. Our data demonstrate that loss of p53 promotes T-cell development in scid mice but does not noticeably affect B lymphopoiesis. Moreover, scid cells are able to induce p53 protein expression and activate G1 arrest or apoptosis in response to ionizing radiation, indicating that DNA-PKcs is not essential for these responses to DNA damage. Furthermore, p53-/- scid double mutant mice develop lymphoma earlier than p53-/- littermates, demonstrating that loss of these two genes can cooperate in tumorigenesis. Collectively, these results provide evidence for an unsuspected role of p53 as a checkpoint regulator in early T-cell development and demonstrate that loss of an additional component of the cellular response to DNA damage can cooperate with loss of p53 in lymphomagenesis.
Sequence-specific DNA binding of the B-cell-specific coactivator OCA-B.
Authors: Cepek K L , Chasman D I , Sharp P A
Published: June 2007

B-cell-specific transcription of immunoglobulin genes is mediated by the interaction of a POU domain containing transcription factor Oct-1 or Oct-2, with the B-cell-specific coactivator OCA-B (Bob-1, OBF-1) and a prototype octamer element. We find that OCA-B binds DNA directly in the major groove between the two subdomains of the POU domain, requiring both an A at the fifth position of the octamer element and contact with the POU domain. An amino-terminal fragment of OCA-B binds the octamer site in the absence of a POU domain with the same sequence specificity. Coactivator OCA-B may undergo a POU-dependent conformational change that exposes its amino terminus, allowing it to recognize specific DNA sequences in the major groove within the binding site for Oct-1 or Oct-2. The recognition of both the POU domain and the octamer sequence by OCA-B provides a mechanism for differential regulation of octamer sites containing genes by the ubiquitous factor Oct-1.
Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer.
Authors: Lynch K W , Maniatis T
Published: June 2007

The Drosophila doublesex female-specific splicing enhancer consists of two classes of regulatory elements, six 13-nucleotide repeat sequences, and a single purine-rich element (PRE). Here, we show that the Drosophila regulatory proteins Transformer (Tra) and Transformer 2 (Tra2) recruit different members of the SR family of splicing factors to the repeats and the PRE. The complexes formed on the repeats in HeLa cell extract consist of Tra, Tra2, and the SR protein 9G8. in Drosophila Kc cell extract, Tra and Tra2 recruit the SR protein RBP1 to the repeats. These proteins are arranged in a specific order on the repeats, with the SR protein at the 5' end of each repeat, and Tra2 at each 3' end. Although Tra did not cross-link strongly to the repeats, its presence was essential for the binding of Tra2 to the 3' end of the repeat. Individual SR proteins were also recruited to the PRE by Tra and Tra2, but in this case they were SF2/ASF and dSRp30 in HeLa and Drosophila cell extracts, respectively. The binding of Tra2, Tra, and the specific SR proteins to the repeats or the PRE was highly cooperative within each complex. Thus, Tra2, which contains a single RNA binding domain, can recognize distinct sequences in the repeats and the PRE in conjunction with specific SR proteins. These observations show that the protein composition of each complex is determined by the RNA recognition sequence and specific interactions between SR proteins and Tra and Tra2.
A hierarchy of SSB protomers in replication protein A.
Authors: Philipova D , Mullen J R , Maniar H S , Lu J , Gu C , Brill S J
Published: June 2007

Replication Protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (SSB) found in all eukaryotic cells. RPA is known to be required for many of the same reactions catalyzed by the homotetrameric SSB of bacteria, but its origin, subunit functions, and mechanism of binding remain a mystery. Here we show that the three subunits of yeast RPA contain a total of four domains with weak sequence similarity to the Escherichia coli SSB protomer. We refer to these four regions as potential ssDNA-binding domains (SBDs). The p69 subunit, which is known to bind ssDNA on its own, contains two SBDs that together confer stable binding to ssDNA. The p36 and p13 subunits each contain a single SBD that does not bind stably, but corresponds to the minimal region required for viability in yeast. Photocross-linking of recombinant protein to ssDNA indicates that an SBD consists of approximately 120 amino acids with two centrally located aromatic residues. Mutation of these aromatic residues inactivates ssDNA binding and is a lethal event in three of the four domains. Finally, we present evidence that the p36 subunit binds ssDNA, as part of the RPA complex, in a salt-dependent reaction similar to the wrapping of ssDNA about E. coli SSB. The results are consistent with the notion that RPA arose by duplication of an ancestral SSB gene and that tetrameric ssDNA-binding domains and higher order binding are essential features of cellular SSBs.
Gene activation by recruitment of the RNA polymerase II holoenzyme.
Authors: Farrell S , Simkovich N , Wu Y , Barberis A , Ptashne M
Published: June 2007

The single amino acid "P" (potentiator) mutation in the holoenzyme component GAL11 creates an interaction between that protein and the dimerization region of GAL4. That interaction triggers strong gene activation when the GAL4 fragment is tethered to DNA. Here we show that, among a series of variants of the GAL4 dimerization region and different GAL11P alleles, the strength of the interaction as quantitated in vitro correlates with the degree of activation in vivo; swapping the protein fragments bearing the GAL4 dimerization region and the GAL11P mutation such that the latter is tethered to DNA and the former is attached to the holoenzyme does not diminish gene activation; gene activation in this system is squelched by overproduction of either a fragment bearing the GAL4 dimerization region or a fragment of GAL11 bearing a P mutation; and neither GAL11 nor GAL11P is a target of an acidic activating region. These results argue that the GAL4-GAL11P interaction triggers gene activation simply by recruiting the holoenzyme to DNA. Consistent with this view, we also show that fusion of LexA to another holoenzyme component, SRB2, creates an activator, and that an SRB2 mutant predicted on genetic grounds to interact especially efficiently with a holoenzyme containing a specific mutant form of polymerase also activates more efficiently when tethered to DNA.
The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes.
Authors: Keegan K S , Holtzman D A , Plug A W , Christenson E R , Brainerd E E , Flaggs G , Bentley N J , Taylor E M , Meyn M S , Moss S B , Carr A M , Ashley T , Hoekstra M F
Published: June 2007

A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia- and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are approximately 300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination.
p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells.
Authors: Chen X , Ko L J , Jayaraman L , Prives C
Published: June 2007

It is well established that induction of the p53 tumor suppressor protein in cells can lead to either cell cycle arrest or apoptosis. To further understand features of p53 that contribute to these cell responses several p53-null Saos2 and H1299 cell lines were generated that express wild-type or mutant forms of p53, or the cyclin-dependent kinase inhibitor p21/WAF1, under a tetracycline-regulated promoter. Our results show that the cellular level of p53 can dictate the response of the cell such that lower levels of p53 result in arrest whereas higher levels result in apoptosis; nevertheless, DNA damage can heighten the apoptotic response to p53 without altering the protein level of p53 in cells. We also demonstrate that arrest and apoptosis are two genetically separable functions of p53 because a transcriptionally incompetent p53 can induce apoptosis but not arrest, whereas induction of p21/WAF1, which is a major transcriptional target of p53, can induce arrest but not apoptosis. Finally, we show that a full apoptotic response to p53 requires both its amino and carboxyl terminus, and our data suggest that there is synergism between transcription-dependent and -independent functions of p53 in apoptosis. Thus, there are multiple independent cellular responses to p53 that together may account for the extraordinarily high frequency of p53 mutations in diverse types of human tumors. The implications of these results are discussed and a model is proposed.
Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation.
Authors: Eckner R , Yao T P , Oldread E , Livingston D M
Published: June 2007

Differentiation of skeletal muscle cells and B lymphocytes is regulated by basic helix-loop-helix (bHLH) proteins. Both differentiation programs are inhibited by the adenovirus E1A oncoprotein. Analysis of E1A mutants has implicated two of its cellular-binding proteins, p300 and CBP, in controlling certain aspects of differentiation. We find that p300 can cooperate with tissue-specific bHLH proteins in activating target genes and requires only the bHLH domain of such proteins to stimulate E box-directed transcription. Importantly, the ability of bHLH proteins to activate transcription correlates with the presence of p300/CBP in E box-dependent DNA-binding complexes, because both phenomena require at least two adjacent E-box motifs. Microinjection of p300/CBP antibodies into myoblasts blocks terminal differentiation, cell fusion, and transcriptional activity of myogenic bHLH proteins. These results suggest that the function of p300/CBP is essential for the execution of key aspects of cellular differentiation.
An intron enhancer recognized by splicing factors activates polyadenylation.
Authors: Lou H , Gagel R F , Berget S M
Published: June 2007

Alternative processing of the pre-messenger RNA encoding calcitonin/calcitonin gene-related peptide (CT/CGRP) involves alternative inclusion of a 3'-terminal exon (exon 4) embedded within a six exon primary transcript. Expression of CT/CGRP in transgenic mice indicates that inclusion of exon 4 occurs in a wide variety of tissues, suggesting that the factors responsible for exon 4 inclusion are widely distributed. Inclusion of exon 4 requires an enhancer sequence located within the intron downstream of the poly(A) site of exon 4. Here we show that the intron enhancer activated in vitro polyadenylation cleavage of precursor RNAs containing the CT/CGRP exon 4 poly(A) site or heterologous poly(A) sites. To our knowledge this is the first example of an intron-located enhancer that facilitates polyadenylation. Within the enhancer sequence is a 5' splice site sequence immediately preceded by a pyrimidine tract. This 5' splice site sequence was required for enhanced polyadenylation and was recognized by both U1 small nuclear ribonucleoproteins (snRNPs) and alternative splicing factor/splicing factor 2 (ASF/SF2). Enhancement of polyadenylation required U1 RNA, suggesting that the 5' splice site sequence within the enhancer mediates enhancement via interaction with factors normally associated with functional 5' splice sites. Mutation of the polypyrimidine track of the enhancer also inhibited in vitro polyadenylation cleavage. Oligonucleotide competitions and UV cross-linking indicated that the enhancer pyrimidine track binds the polypyrimidine tract binding protein (PTB), but not U2 snRNP auxiliary factor (U2AF), and that binding of PTB was required for maximal enhancer-mediated polyadenylation. These results suggest that the enhancer binds known splicing factors, and that binding of these factors activates polyadenylation cleavage. Furthermore, these results suggest that regulation of alternative processing of CT/CGRP could occur at the level of polyadenylation, rather than splicing.
The role of TBP in rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae: TBP is required for upstream activation factor-dependent recruitment of core factor.
Authors: Steffan J S , Keys D A , Dodd J A , Nomura M
Published: June 2007

Transcription of Saccharomyces cerevisiae rDNA by RNA polymerase I involves at least two transcription factors characterized previously: upstream activation factor (UAF) consisting of Rrn5p, Rrn9p, Rrn10p, and two more uncharacterized proteins; and core factor (CF) consisting of Rrn6p, Rrn7p, and Rrn11p. UAF interacts directly with an upstream element of the promoter and mediates its stimulatory function, and CF subsequently joins a stable preinitiation complex. The TATA-binding protein (TBP) has been known to be involved in transcription by all three nuclear RNA polymerases. We found that TBP interacts specifically with both UAF and CF, the interaction with UAF being stronger than that with CF. Using extracts from a TBP (I143N) mutant, it was shown that TBP is required for stimulation of transcription mediated by the upstream element, but not for basal transcription directed by a template without the upstream element. By template competition experiments, it was shown that TBP is required for UAF-dependent recruitment of CF to the rDNA promoter, explaining the TBP requirement for stimulatory activity of the upstream element. We also studied protein-protein interactions and found specific interactions of TBP with Rrn6p and with Rrn9p both in vitro and in the yeast two-hybrid system in vivo. Thus, these two interactions may be involved in the interactions of TBP with CF and UAF, respectively, contributing to the recruitment of CF to the rDNA promoter. Additionally, we observed an interaction between Rrn9p and Rrn7p both in vitro and in the two-hybrid system; thus, this interaction might also contribute to the recruitment of CF.
Expression of Bcl-xL and loss of p53 can cooperate to overcome a cell cycle checkpoint induced by mitotic spindle damage.
Authors: Minn A J , Boise L H , Thompson C B
Published: June 2007

During somatic cell division, faithful chromosomal segregation must follow DNA replication to prevent aneuploidy or polyploidy. Damage to the mitotic spindle is one potential mechanism that interferes with chromosomal segregation. The accumulation of aneuploid or polyploid cells resulting from a disrupted mitotic spindle is presumably prevented by cell cycle checkpoint controls. In the course of studying cells that overexpress the apoptosis-inhibiting protein Bcl-xL, we found that these cells have an increased rate of spontaneous tetraploidization, suggesting that apoptosis may play an important role in eliminating cells that fail to complete mitosis properly. When cells expressing Bcl-xL are treated with mitotic spindle inhibitors, a significant percentage reinitiate DNA replication and become polyploid. Nevertheless, the majority of cells expressing Bcl-xL undergo a prolonged p53-dependent cell cycle arrest following mitotic spindle damage. Unexpectedly, p53 expression is not induced in mitosis, nor does it influence M-phase arrest. Instead, cells with mitotic spindle damage only transiently arrest in M phase, and despite failing to complete mitosis, appear to proceed to G1. During this subsequent growth factor-dependent phase, p53 is induced and mediates cell cycle arrest. In cells that do not overexpress Bcl-xL, elimination of the p53-dependent growth arrest with a dominant negative mutant also results in polyploidy after mitotic spindle damage, but under these conditions most cells die by apoptosis. Expression of Bcl-xL and abrogation of p53 cooperate to allow rapid and progressive polyploidization following mitotic spindle damage. Our results suggest that suppression of apoptosis by bcl-2-related genes and loss of p53 function can act cooperatively to contribute to genetic instability.
The dmf1/mid1 gene is essential for correct positioning of the division septum in fission yeast.
Authors: Sohrmann M , Fankhauser C , Brodbeck C , Simanis V
Published: June 2007

Little is known about the mechanisms that establish the position of the division plane in eukaryotic cells. Wild-type fission yeast cells divide by forming a septum in the middle of the cell at the end of mitosis. Dmf1 mutants complete mitosis and initiate septum formation, but the septa that form are positioned at random locations and angles in the cell, rather than in the middle. We have cloned the dmf1 gene as a suppressor of the cdc7-24 mutant. The dmf1 mutant is allelic with mid1. The gene encodes a novel protein containing a putative nuclear localization signal, and a carboxy-terminal PH domain. In wild-type cells, Dmf1p is nuclear during interphase, and relocates to form a medial ring at the cell cortex coincident with the onset of mitosis. This relocalization occurs before formation of the actin ring and is associated with increased phosphorylation of Dmf1p. The Dmf1p ring can be formed in the absence of an actin ring, but depends on some of the genes required for actin ring formation. When the septum is completed and the cells separate, Dmf1p staining is once again nuclear. These data implicate Dmf1p as an important element in assuring correct placement of the division septum in Schizosaccharomyces pombe cells.
A dominant interfering Myb mutant causes apoptosis in T cells.
Authors: Taylor D , Badiani P , Weston K
Published: June 2007

The c-Myb transcription factor is required for the production of most hemopoietic lineages, but information is sparse about its mode of action and the key genes it regulates. We have made an inducible dominant interfering Myb protein, by creating a chimera comprising the DNA binding domain of c-Myb, the Drosophila Engrailed repressor domain, and a modified estrogen receptor hormone binding domain. When expressed in the murine thymoma cell line EL4, activation of this mutant results in a significant proportion of the cell population undergoing apoptosis, as assessed by nuclear breakdown and DNA fragmentation, but has no apparent effect on cell-cycle progression. The apoptotic phenotype is mirrored during thymopoiesis in transgenic mice expressing dominant interfering Myb mutants; their T cells are fragile both in vivo and in vitro. Induction of the Myb dominant interfering mutant in EL4 cells correlates with down-regulation of bcl-2, but does not affect transcription of other bcl-2 family members; conversely, overexpression of bcl-2 in the transgenic mouse model rescues thymocytes from death. Analysis of the bcl-2 promoter by run-on transcription, bandshifting, and transient expression assays shows that it is a direct target of Myb. These data suggest a new and important role for Myb proteins as regulators of cell survival during hemopoiesis.
Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs.
Authors: Chen P L , Riley D J , Chen Y , Lee W H
Published: June 2007

To define a mechanism by which retinoblastoma protein (Rb) functions in cellular differentiation, we studied primary fibroblasts from the lung buds of wild-type (RB+/+) and null-mutant (RB-/-) mouse embryos. In culture, the RB+/+ fibroblasts differentiated into fat-storing cells, either spontaneously or in response to hormonal induction; otherwise syngenic RB-/- fibroblasts cultured in identical conditions did not. Ectopic expression of normal Rb, but not Rb with a single point mutation, enabled RB-/- fibroblasts to differentiate into adipocytes. Rb appears in murine fibroblasts to activate CCAAT/enhancer-binding proteins (C/EBPs), a family of transcription factors crucial for adipocyte differentiation. Physical interaction between Rb and C/EBPs was demonstrated by reciprocal coimmunoprecipitation, but occurred only in differentiating cells. Wild-type Rb also enhanced the binding of C/EBP to cognate DNA sequences in vitro and the transactivation of a C/EBPbeta-responsive promoter in cells. Taken together, these observations establish a direct and positive role for Rb in terminal differentiation. Such a role contrasts with the function of Rb in arresting cell cycle progression in G1 by negative regulation of other transcription factors like E2F-1.