bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutation of the SPS1-encoded protein kinase of Saccharomyces cerevisiae leads to defects in transcription and morphology during spore formation.
Authors: Friesen H , Lunz R , Doyle S , Segall J
Published: June 2007

During sporulation of Saccharomyces cerevisiae, meiosis is followed by encapsulation of haploid nuclei within multilayered spore walls. Completion of the late events of the sporulation program requires the SPS1 gene. This developmentally regulated gene, which is expressed as cells are nearing the end of meiosis, encodes a protein with homology to serine/threonine protein kinases. The catalytic domain of Sps1 is 44% identical to the kinase domain of yeast Ste20, a protein involved in the pheromone-induced signal transduction pathway. Cells of a MATa/MAT alpha sps1/sps1 strain arrest after meiosis and fail to activate genes that are normally expressed at a late time of sporulation. The mutant cells do not form refractile spores as assessed by phase-contrast microscopy and do not display the natural fluorescence and ether resistance that is characteristic of mature spores. Examination by electron microscopy reveals, however, that prospore-like compartments form in some of the mutant cells. These immature spores lack the cross-linked surface layer that surrounds wild-type spores and are more variable in size and number than are the spores of wild-type cells. Despite their inability to complete spore formation, sps1-arrested cells are able to resume mitotic growth on transfer to rich medium, generating haploid progeny. Our results suggest that the developmentally regulated Sps1 kinase is required for normal progression of transcriptional, biochemical, and morphological events during the later portion of the sporulation program.
A temperature-sensitive MEK mutation demonstrates the conservation of the signaling pathways activated by receptor tyrosine kinases.
Authors: Hsu J C , Perrimon N
Published: June 2007

MEK, a dual specificity threonine/tyrosine kinase, has been postulated to be a convergent point for signaling from receptor protein tyrosine kinases (RTKs) and G-protein-coupled receptors. In contrast to yeast and mammalian cells where several MEKs have been isolated, only one Drosophila MEK (D-Mek) has been characterized to date. Previous studies have shown that D-Mek acts in the Torso RTK signaling pathway. To demonstrate that D-Mek also operates downstream of other RTKs, we generated a temperature-sensitive allele of D-mek (D-mekts) by site-directed mutagenesis based on the amino acid change of a yeast cdc2ts mutation. Using D-mekts, we show that in addition to its role in Torso signaling, D-Mek operates in the Sevenless and in the Drosophila epidermal growth factor RTK pathways. Because loss-of-function mutations in D-mek and the upstream receptors give rise to similar phenotypes, it suggests that D-mek is the only MEK activated by Drosophila RTKs. In addition, we demonstrate that different RTK pathways respond differently to alteration in D-Mek activity.
Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt's lymphoma cells.
Authors: Madisen L , Groudine M
Published: June 2007

In murine plasmacytoma and human Burkitt's lymphoma cells, one allele of c-myc is translocated into one of the immunoglobulin loci, resulting in a characteristic pattern of deregulated c-myc transcription. Translocation events between c-myc and the IgH locus segregate c-myc and the IgH intron enhancer to different reciprocal products in all plasmacytomas and in most Burkitt's lymphoma cells, suggesting that an additional element(s) capable of affecting c-myc expression over a large and variable distance must exist in the IgH locus. The region 3' of the IgH C alpha gene contains four tissue-specific and cell stage-specific DNase I hypersensitive sites (HSs), two of which map to the late B cell-specific 3' C alpha enhancer. We report here that DNA sequences comprising the two other 3' C alpha HSs contain potential protein-binding motifs for trans-activators commonly associated with immunoglobulin enhancers and that these sites can function as cell stage-specific enhancers in transient B cell assays. A DNA fragment containing all four HSs (HS1234) synergistically activates c-myc transcription in plasmacytoma and Burkitt's lymphoma cells in transient assays and induces high-level transcription, a promoter shift from P2 to P1, and an increase in readthrough transcription in stable transfections. Furthermore, plasmacytoma clones stably transfected with a HS1234-linked c-myc construct express c-myc in a position-independent, copy number-dependent manner. These results suggest that HS1234 may function as a locus control region (LCR), deregulating c-myc expression in t(15;12) plasmacytomas, as well as potentially contributing to aspects of normal IgH chain expression.
Diploidy of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot.
Authors: Fuse N , Hirose S , Hayashi S
Published: June 2007

The Drosophila escargot (esg) gene encodes a C2-H2-type zinc finger protein that is expressed in the imaginal discs and histoblasts. In some esg mutants, the abdominal histoblasts become polyploid. It has therefore been suggested that the role of esg is to maintain diploidy of the imaginal cells. We show that esg encodes a DNA-binding protein with high affinity for G/ACAGGTG, the consensus-binding sequence for the basic helix-loop-helix (bHLH) family of transcription factors (E2 box). This DNA-binding activity is essential for esg function in vivo as the strong embryonic lethal allele esgVS8 is caused by an amino acid change within the zinc finger region, leading to reduced affinity for DNA. In cultured cells, a heterodimer of the bHLH proteins Scute and Daughterless activates transcription from promoters containing E2 boxes. The esg protein strongly inhibits this activation, suggesting that esg may regulate developmental processes dependent on bHLH proteins. In larvae, esg protein expressed by the heat shock promoter can rescue the polyploid phenotype of abdominal histoblasts, demonstrating that the phenotype is attributable to a loss of esg function. esg must be expressed continuously during the larval period for efficient rescue. Ectopic expression of esg in the salivary glands inhibits endoreplication of DNA. These results suggest that esg is involved in transcriptional inhibition of genes required for endoreplication.
Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription.
Authors: Ozer J , Moore P A , Bolden A H , Lee A , Rosen C A , Lieberman P M
Published: June 2007

TFIIA is thought to play an important role in transcriptional regulation in higher eukaryotes, but its precise function is unclear. A human cDNA encoding a protein with 45% identity to the small subunit of yeast TFIIA has been isolated. TFIIA activity could be reconstituted by the mixing of recombinant large (alpha beta) and small (gamma) subunits. TFIIA-depleted HeLa nuclear extracts were used to demonstrate that TFIIA is essential for basal and activated transcription by several distinct classes of activators. Recombinant TFIIA functioned in transcriptional activation whether expressed as a dimer (alpha beta+gamma) or as a trimer (alpha+beta+gamma), which closely resembles the native form. Yeast TFIIA also functioned in transcriptional activation, and the human gamma subunit was functionally interchangeable with TOA2, its yeast homolog. Recombinant TFIIA mediated the stimulation of TFIID binding to the TATA region and downstream promoter sequences by the Zta transcriptional activator. Significantly, we found that TFIIA bound directly to Zta in an activation domain-dependent manner. One consequence of the TFIIA-mediated interaction between Zta and TFIID was the formation of a promoter-bound complex resistant to TATA oligonucleotide competition. These results demonstrate that TFIIA is an evolutionarily conserved general factor critical for activator-regulated transcription.
A genetic link between light response and multicellular development in the bacterium Myxococcus xanthus.
Authors: Nicolás F J , Ruiz-Vázquez R M , Murillo F J
Published: June 2007

The Gram-negative bacterium Myxococcus xanthus responds to blue light by producing carotenoid pigments (Car+ phenotype). Genes for carotenoid synthesis lie at two unlinked chromosomal sites, the carC and the carBA operon, but are integrated in a single "light regulon" by the action of common trans-acting regulatory elements. Three known regulatory genes are grouped together at the (light-inducible) carQRS operon. By screening the Car phenotype of a large collection of transposon-induced mutants, we have identified a new car locus that has been named carD (carD1 for the mutant allele). The carD gene product plays a critical role in the light regulon, as it is required for activation of the carQRS and carC promoters by blue light. The carD1 mutant is impaired in the (starvation-induced) developmental process that allows M. xanthus cells both to form multicellular fruiting bodies and to sporulate. Our results indicate that the carD gene product is also required for the expression of a particular set of development-specific genes that are normally activated through the action of intercellular signals.
A MAP kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in Caenorhabditis elegans.
Authors: Lackner M R , Kornfeld K , Miller L M , Horvitz H R , Kim S K
Published: June 2007

During development of the Caenorhabditis elegans hermaphrodite, the gonadal anchor cell induces nearby Pn.p cells to adopt vulval fates. The response to this signal is mediated by a receptor tyrosine kinase signal transduction pathway that has been remarkably well conserved during metazoan evolution. Because mitogen-activated protein (MAP) kinases are activated by receptor tyrosine kinase pathways in vertebrate cells, we hypothesized that C. elegans MAP kinase homologs may play a role in vulval induction. Two C. elegans MAP kinase genes, mpk-1 and mpk-2 (mpk, MAP kinase), were cloned using degenerate oligonucleotide primers and PCR amplification; in parallel, genes involved in vulval induction were identified by screening for mutations that suppress the vulval defects caused by an activated let-60 ras gene. One such suppressor mutation is an allele of mpk-1. We used a new type of mosaic analysis to show that mpk-1 acts cell autonomously in the Pn.p cells. Our results show that mpk-1 plays an important functional role as an activator in ras-mediated cell signaling in vivo.
Adenovirus E1B oncoprotein tethers a transcriptional repression domain to p53.
Authors: Yew P R , Liu X , Berk A J
Published: June 2007

Many DNA tumor viruses express a protein that inhibits transcriptional activation by the tumor-suppressing transcription factor p53. We report that adenovirus E1B 55K represses p53-mediated activation by a mechanism not described previously. E1B 55K binds p53 without displacing it from its DNA-binding site. A fusion of E1B 55K to the GAL4 DNA-binding domain represses transcription from a variety of promoters with engineered upstream GAL4-binding sites. Mutations within E1B 55K that interfere with its transforming activity and its ability to inhibit p53-mediated trans-activation also interfere with transcriptional repression by the GAL4-55K fusion. These results demonstrate that E1B 55K functions as a direct transcriptional repressor that is targeted to p53-responsive genes by binding to p53.
Targeted deletion of the TGF-beta 1 gene causes rapid progression to squamous cell carcinoma.
Authors: Glick A B , Lee M M , Darwiche N , Kulkarni A B , Karlsson S , Yuspa S H
Published: June 2007

To study the contribution of autocrine and paracrine TGF-beta 1 to tumor progression in a well-defined system of multistage carcinogenesis, keratinocytes with a targeted deletion of the TGF-beta 1 gene were initiated in vitro with the v-rasHa oncogene and their in vivo tumorigenic properties were determined by skin grafting initiated cells onto athymic mice in combination with either wild-type or null dermal fibroblasts. Grafts of v-rasHa-initiated null keratinocytes progressed rapidly to multifocal squamous cell carcinomas within dysplastic papillomas irrespective of the fibroblast genotype, whereas the initiated control genotypes formed well-differentiated papillomas. Malignant progression was not associated with mutations in the c-rasHa gene, alterations in p53 protein, or loss of responsiveness to TGF-beta 1. The tumor cell labeling index was elevated in grafts of initiated null keratinocytes with wild-type fibroblasts compared to tumors of other genotypes. However, labeling index in all tumors was reduced when TGF-beta 1 null fibroblasts formed the stroma. The null tumor cells could not accumulate TGF-beta 1 from the host, but grafts of uninitiated null keratinocytes, which formed a normal epidermis, became TGF-beta 1 positive even though they did not express TGF-beta 1 mRNA. These results demonstrate that autocrine TGF-beta 1 suppresses the frequency and rate of malignant progression, and that autocrine and paracrine TGF-beta 1 can have opposing effects on tumor cell proliferation. The lack of paracrine inhibition of tumor cell progression appears to result from the inability of tumor cells to localize host-derived TGF-beta 1 by a mechanism that operates in normal cells.
The alternatively initiated c-Myc proteins differentially regulate transcription through a noncanonical DNA-binding site.
Authors: Hann S R , Dixit M , Sears R C , Sealy L
Published: June 2007

The myc proto-oncogene family has been implicated in multiple cellular processes, including proliferation, differentiation, and apoptosis. The Myc proteins, as heterodimers with Max protein, have been shown to function as activators of transcription through an E-box DNA-binding element, CACGTG. We have now found that the c-Myc proteins regulate transcription through another, noncanonical, DNA sequence. The non-AUG-initiated form of the c-Myc protein, c-Myc 1, strongly and specifically activates transcription of the C/EBP sequences within the EFII enhancer element of the Rous sarcoma virus long terminal repeat. In contrast, comparable amounts of the AUG-initiated form, c-Myc 2, fail to significantly affect enhancer activity. However, both c-Myc proteins trans-activate the CACGTG sequence comparably. In addition, Myc/Max heterodimers, but not Max homodimers, bind to the EFII enhancer sequence in vitro. Finally, c-Myc 1 overexpression, but not c-Myc 2 overexpression, significantly inhibits cell growth. These results reveal new transcriptional activities for the Myc proteins and demonstrate that the different forms of the Myc protein are functionally distinct. These results also suggest an interplay between two different growth regulatory transcription factor families.
The Drosophila trithorax gene encodes a chromosomal protein and directly regulates the region-specific homeotic gene fork head.
Authors: Kuzin B , Tillib S , Sedkov Y , Mizrokhi L , Mazo A
Published: June 2007

The activity of the Drosophila gene trithorax is required to maintain the proper spatial pattern of expression of multiple homeotic genes of the Bithorax and Antennapedia complexes, trithorax encodes two large protein isoforms of > 400 kD. We have detected its products at 16 discrete sites on larval salivary gland polytene chromosomes, 12 of which colocalize with binding sites of several Polycomb group proteins. The intensity of trithorax protein binding is strongly decreased in larvae carrying mutations in another trithorax group gene ash-1, and in the Polycomb group gene pco/E(z). A strong trithorax binding site was found at the cytological location of the fork head gene, a region-specific homeotic gene not located within a homeotic complex. Further analysis showed that trithorax protein binds at ectopic sites carrying fork head sequences in transformed lines. Trithorax binding occurs within an 8.4-kb regulatory region that directs fork head expression in several embryonic tissues including salivary glands. Consistently, expression of endogenous fork head RNA is greatly reduced in trithorax mutant embryos and in larval tissues. These results show that trithorax maintains expression of target genes by interaction with their regulatory regions and that this interaction depends on the presence of at least some of the other trithorax and Polycomb group proteins.
Inhibition of the DNA-binding activity of Drosophila suppressor of hairless and of its human homolog, KBF2/RBP-J kappa, by direct protein-protein interaction with Drosophila hairless.
Authors: Brou C , Logeat F , Lecourtois M , Vandekerckhove J , Kourilsky P , Schweisguth F , Israël A
Published: June 2007

We have purified the sequence-specific DNA-binding protein KBF2 and cloned the corresponding cDNA, which is derived from the previously described RBP-J kappa gene, the human homolog of the Drosophila Suppressor of Hairless [Su(H)] gene. Deletion studies of the RBP-J kappa and Su(H) proteins allowed us to define a DNA-binding domain conserved during evolution. Because Su(H) mutant alleles exhibit dose-sensitive interactions with Hairless (H) loss-of-function mutations, we have investigated whether the RBP-J kappa or Su(H) proteins directly interact with the H protein in vitro. We show here that H can inhibit the DNA binding of both Su(H) and RBP-J kappa through direct protein-protein interactions. Consistent with this in vitro inhibitory effect, transcriptional activation driven by Su(H) in transfected Drosophila S2 cells is inhibited by H. These results support a model in which H acts, at least in part, as a negative regulator of Su(H) activity. This model offers a molecular view to the antagonistic activities encoded by the H and Su(H) genes for the control of sensory organ cell fates in Drosophila. We further propose that a similar mechanism might occur in mammals.
Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae.
Authors: Proweller A , Butler S
Published: June 2007

The polyadenylate tail of eukaryotic mRNAs is thought to influence various metabolic phenomena including mRNA stability, translation initiation, and nucleo-cytoplasmic transport. We have analyzed the fate of mRNAs following inactivation of poly(A) polymerase in Saccharomyces cerevisiae containing a temperature-sensitive, lethal mutation (pap1-1) in the gene for poly(A) polymerase (PAP1). Inactivation of poly(A) polymerase (Pap1) by shifting cells to the nonpermissive temperature resulted in the loss of at least 80% of measurable poly(A) within 60 min. Northern blot analysis revealed the disappearance of some mRNAs (CYH2 and HIS4) consistent with a role for poly(A) tails in mRNA stability. However, other mRNAs (TCM1, PAB1, ACT1, and HTB2) accumulate as poly(A)-deficient (A < approximately 25) transcripts as defined by an inability to bind oligo(dT)-cellulose. Sucrose density gradient analysis of polyribosomes revealed a twofold reduction in the amount of each size class of polyribosomes in shifted cells and a commensurate increase in free ribosomes. However, poly(A)-deficient mRNAs in shifted cells remain associated with the same size polyribosomes as poly(A)+ mRNAs in unshifted cells, indicating normal initiation of translation. RNase mapping of transcripts from pap1-1 cells revealed PAB1 mRNA to be poly(A)- whereas TCM1 exists as equal amounts of poly(A)- and poly(A)+ mRNA 60 min after shift. Interestingly, both of these classes of TCM1 mRNA appear in similar amounts in each polyribosome fraction indicating that ribosomes may not distinguish between them. These findings suggest that under conditions of excess translational capacity, poly(A)- and poly(A)+ mRNAs may initiate translation with comparable efficiencies.
Role of interactions between SpoIIAA and SpoIIAB in regulating cell-specific transcription factor σF of Bacillus subtilis
Authors: Diederich Bettina , Wilkinson Jonathan F. , Magnin Thierry , Najafi S. Mahmoud A. , Errington Jeffery , Yudkin Michael D.
Published: June 2007

Genetic experiments have suggested that σF, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-σ factor SpoIIAB and an anti-anti-σ factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of σF-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA–phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA–phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-σF activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-σF activity both in vivo and in vitro. It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-σ capacity of SpoIIAA and hence the activity of σF.
E2F-4, a new member of the E2F transcription factor family, interacts with p107.
Authors: Ginsberg D , Vairo G , Chittenden T , Xiao Z X , Xu G , Wydner K L , DeCaprio J A , Lawrence J B , Livingston D M
Published: June 2007

The E2F family of transcription factors has been implicated in the regulation of cell proliferation, and E2F-binding sites are present in the promoters of several growth-regulating genes. E2F family members are functionally regulated, in part, by complex formation with one or more members of the nuclear pocket protein family, RB, p107, and p130. Pocket protein regulation of E2F likely contributes to normal cellular growth control. While the three cloned species of E2F, E2F-1, E2F-2, and E2F-3, are known to be targets of RB interaction, no E2F species has yet been shown to be a specific p107 or p130 target. Here, we describe the cloning of a new member of the E2F family, E2F-4, which forms heterodimers with a member(s) of the DP family and, unlike some family members, is present throughout the cell cycle and appears to be a differentially phosphorylated p107-binding partner. p107 binding not only can be linked to the regulation of E2F-4 transcriptional activity, but also to suppression of the ability of E2F-4 to transform an immortalized rodent cell line.
Hairy function as a DNA-binding helix-loop-helix repressor of Drosophila sensory organ formation.
Authors: Ohsako S , Hyer J , Panganiban G , Oliver I , Caudy M
Published: June 2007

Sensory organ formation in Drosophila is activated by proneural genes that encode basic-helix-loop-helix (bHLH) transcription factors. These genes are antagonized by hairy and other proline-bHLH proteins. hairy has not been shown to bind to DNA and has been proposed to form inactive heterodimers with proneural activator proteins. Here, we show that hairy does bind to DNA and has novel DNA-binding activity: hairy prefers a noncanonical site, CACGCG, although it also binds to related sites. Mutation of a single CACGCG site in the achaete (ac) proneural gene blocks hairy-mediated repression of ac transcription in cultured Drosophila cells. Moreover, the same CACGCG mutation in an ac minigene transformed into Drosophila creates ectopic sensory hair organs like those seen in hairy mutants. Together these results indicate that hairy represses sensory organ formation by directly repressing transcription of the ac proneural gene.
Multiple regions of TBP participate in the response to transcriptional activators in vivo.
Authors: Tansey W P , Ruppert S , Tjian R , Herr W
Published: June 2007

We used mutant yeast and human TBP molecules with an altered DNA-binding specificity to examine the role of TBP in transcriptional activation in vivo. We show that yeast TBP is functionally equivalent to human TBP for response to numerous transcriptional activators in human cells, including those that do not function in yeast. Despite the extensive conservation of TBP, its ability to respond to transcriptional activators in vivo is curiously resistant to clustered sets of alanine substitution mutations in different regions of the protein, including those that disrupt DNA binding and basal transcription in vitro. Combined sets of these mutations, however, can attenuate the in vivo activity of TBP and can differentially affect response to different activation domains. Although the activity of TBP mutants in vivo did not correlate with DNA binding or basal transcription in vitro, it did correlate with binding in vitro to the largest subunit of TFIID, hTAFII250. Together, these data suggest that TBP utilizes multiple interactions across its surface to respond to RNA polymerase II transcriptional activators in vivo; some of these interactions appear to involve recruitment of TBP into TFIID, whereas others are involved in response to specific types of transcriptional activators.
microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family.
Authors: Hemesath T J , Steingrímsson E , McGill G , Hansen M J , Vaught J , Hodgkinson C A , Arnheiter H , Copeland N G , Jenkins N A , Fisher D E
Published: June 2007

The microphthalmia (mi) gene appears essential for pigment cell development and/or survival, based on its mutation in mi mice. It has also been linked to the human disorder Waardenburg Syndrome. The mi gene was recently cloned and predicts a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) factor with tissue-restricted expression. Here, we show that Mi protein binds DNA as a homo- or heterodimer with TFEB, TFE3, or TFEC, together constituting a new MiT family. Mi can also activate transcription through recognition of the M box, a highly conserved pigmentation gene promoter element, and may thereby determine tissue-specific expression of pigmentation enzymes. Six mi mutations shown recently to cluster in the b-HLH-ZIP region produce surprising and instructive effects on DNA recognition and oligomerization. An alternatively spliced exon located outside of the b-HLH-ZIP region is shown to significantly modulate DNA recognition by the basic domain. These findings suggest that Mi's critical roles in melanocyte survival and pigmentation are mediated by MiT family interactions and transcriptional activities.
Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development.
Authors: Tsai S , Bartelmez S , Sitnicka E , Collins S
Published: June 2007

The lymphohematopoietic progenitors represent < 0.01% of nucleated marrow cells. Here, we describe the immortalization of the murine lymphohematopoietic progenitors by a retroviral vector harboring a dominant-negative retinoic acid receptor. The immortalized progenitors proliferate as a stem-cell-factor-dependent clonal line EML that spontaneously generates pre-pro-B lymphocytes and erythroid and myeloid progenitors. Upon stimulation with interleukin-7 and stromal cells, the pre-pro-B lymphocytes express RAG-1 and undergo D-J rearrangements of the immunoglobulin heavy-chain genes. With erythropoietin the erythroid progenitors proliferate and differentiate into red cells. Generation of the common progenitors for neutrophils and macrophages is suppressed in EML but is inducible by high concentrations of retinoic acid. An additional block in neutrophil differentiation occurs at the promyelocyte stage but can also be overcome by high concentrations of retinoic acid. These studies demonstrate a reproducible way to immortalize lymphohematopoietic progenitors and implicate specific roles for retinoic acid receptors at two distinct stages of hematopoiesis.
The WD repeats of Tup1 interact with the homeo domain protein alpha 2.
Authors: Komachi K , Redd M J , Johnson A D
Published: June 2007

Tup1 and Ssn6 transcriptionally repress a wide variety of genes in yeast but do not appear to bind DNA. We provide genetic and biochemical evidence that the DNA-binding protein alpha 2, a regulator of cell-type-specific genes, recruits the Tup1/Ssn6 repressor by directly interacting with Tup1. This interaction is mediated by a region of Tup1 containing seven copies of the WD repeat, a 40 amino acid motif of unknown function found in many other proteins. We have found that a single WD repeat will interact with alpha 2, indicating that the WD repeat is a protein-protein interaction domain. Furthermore, a fragment of Tup1 containing primarily WD repeats provides at least partial repression in the absence of Ssn6, suggesting that the repeats also mediate interaction between Tup1 and other components of the repression machinery.
Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality.
Authors: Lau M M , Stewart C E , Liu Z , Bhatt H , Rotwein P , Stewart C L
Published: June 2007

Murine embryos that inherit a nonfunctional insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor (Igf2r) gene from their fathers are viable and develop normally into adults. However, the majority of mice inheriting the same mutated allele from their mothers die around birth, as a consequence of major cardiac abnormalities. These mice do not express IGF2R in their tissues, are 25-30% larger than their normal siblings, have elevated levels of circulating IGF2 and IGF-binding proteins, and exhibit a slight kink in their tails. These results show that Igf2r is paternally imprinted and reveal that the receptor is crucial for regulating normal fetal growth, circulating levels of IGF2, and heart development.
RNA localization along the anteroposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization.
Authors: Lane M E , Kalderon D
Published: June 2007

Microtubule polarity has been implicated as the basis for polarized localization of morphogenetic determinants that specify the anteroposterior axis in Drosophila oocytes. We describe mutation affecting Protein Kinase A (PKA) that act in the germ line to disrupt both microtubule distribution and RNA localization along this axis. In normal oocytes, the site of microtubule nucleation shifts from posterior to anterior immediately prior to polarized localization of bicoid and oskar RNAs. In PKA-deficient oocytes, posterior microtubules are present during this transition, oskar RNA fails to accumulate at the posterior, and bicoid RNA accumulates at both ends of the oocyte. Similar RNA mislocalization patterns previously reported for Notch and Delta mutants suggest that PKA transduces a signal for microtubule reorganization that is sent by posteriorly located follicle cells.
JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation.
Authors: Kallunki T , Su B , Tsigelny I , Sluss H K , Dérijard B , Moore G , Davis R , Karin M
Published: June 2007

The transcriptional activity of c-Jun is augmented through phosphorylation at two sites by a c-Jun amino-terminal kinase (JNK). All cells express two distinct JNK activities, 46 and 55 kD in size. It is not clear which of them is the more important c-Jun kinase and how they specifically recognize c-Jun. The 46-kD form of JNK was identified as a new member of the MAP kinase group of signal-transducing enzymes, JNK1. Here, we report the molecular cloning of the 55-kD form of JNK, JNK2, which exhibits 83% identity and similar regulation to JNK1. Despite this close similarity, the two JNKs differ greatly in their ability to interact with c-Jun. JNK2 binds c-Jun approximately 25 times more efficiently than JNK1, and as a result has a lower Km toward c-Jun than JNK1. The structural basis for this difference was investigated and traced to a small beta-strand-like region near the catalytic pocket of the enzyme. Modeling suggests that this region is solvent exposed and therefore is likely to serve as a docking site that increases the effective concentration of c-Jun near JNK2. These results explain how two closely related MAP kinases can differ in their ability to recognize specific substrates and thereby elicit different biological responses.
Murine FGFR-1 is required for early postimplantation growth and axial organization.
Authors: Deng C X , Wynshaw-Boris A , Shen M M , Daugherty C , Ornitz D M , Leder P
Published: June 2007

We have explored the role of fibroblast growth factor receptor 1 (FGFR-1) in early embryonic development using three experimental systems: genetically deficient mice, in vitro blastocyst culture, and FGFR-1-deficient embryonic stem cells. Using these systems, we demonstrate that FGFR-1 is required for proper embryonic cell proliferation and for the correct axial organization of early postimplantation embryos but not for mesoderm formation. FGFR-1-deficient embryos display severe growth retardation both in vitro and in vivo and die prior to or during gastrulation. Although these mutants can form nonaxial tissues, such as the allantois, amnion, and yolk sac mesoderm, they display defective patterning of the primitive streak and other axial structures, and frequently exhibit truncations or disorganization of posterior embryonic regions. Such abnormalities are unlikely to be caused by intrinsic blocks in mesodermal differentiation, as FGFR-1-deficient ES cell lines form teratomas consisting of many mesodermal cell types.
A murine homolog of the yeast RNA1 gene is required for postimplantation development.
Authors: DeGregori J , Russ A , von Melchner H , Rayburn H , Priyaranjan P , Jenkins N A , Copeland N G , Ruley H E
Published: June 2007

A gene has been characterized that is required for postimplantation mouse development. The gene, designated fug1, was disrupted in embryonic stem cells by the U3Neo gene trap retrovirus, and the disrupted allele was introduced into the germ line. Homozygous mutant embryos arrest at the egg cylinder stage at about embryonic day 6 and are mostly resorbed by day 8.5. The appearance of the proamniotic cavity is delayed, and epiblast cells that surround the cavity are disorganized. fug1 transcripts are undetectable at E6 but are induced throughout the embryo after E6.5. The gene is expressed at low levels in all adult tissues examined, maps to chromosome 15, and is conserved among mammals. The cDNA sequence encodes a protein of 589 amino acids, the first 400 of which are 38% identical to the Saccaromyces cerevisiae RNA1 gene. Regions of greatest similarity include a long acidic domain and 11 leucine-rich motifs, thought to mediate high affinity protein-protein interactions. These similarities suggest that Fug1 may be required for developmental changes in RNA processing or chromatin structure prior to gastrulation.
Upstream introns influence the efficiency of final intron removal and RNA 3'-end formation.
Authors: Nesic D , Maquat L E
Published: June 2007

For all intron-containing pre-mRNAs of higher eukaryotes that have been examined using either living cells or cell-free extracts, a functional 3' splice site within the 3'-terminal intron is required for efficient RNA 3'-end formation. The mechanism by which intron sequences facilitate RNA 3'-end formation, which is achieved by endonucleolytic cleavage and polyadenylation, is not understood. We report here that in intact cells the efficiency of RNA 3'-end formation correlates with the efficiency of final intron removal, even when the intron is normally a 5'-terminal or internal intron. Therefore, the influence of the 3'-terminal intron on 3'-end formation is likely to be attributable to the determinants of splicing efficiency, which include but are not limited to the 3' splice site. Quantitative RNase mapping and methods that couple reverse transcription and the polymerase chain reaction were used to assess the consequence to RNA 3'-end formation of intron deletions within the human gene for triosephosphate isomerase (TPI). Results indicate that the formation of TPI RNA 3' ends requires TPI gene introns in addition to the last intron, intron 6, to proceed efficiently. These additional TPI gene introns are also required for the efficient removal of intron 6. When introns 1 and 5 were engineered to be the final intron, they were found, as was intron 6, to function in RNA 3'-end formation with an efficiency that correlated with their efficiency of removal. The simultaneous deletion of the 5' and 3' splice sites of intron 6 reduced the efficiencies of both RNA 3'-end formation and the removal of intron 5, which constituted the 3'-most functional intron. Deletion of only the 3' splice site of intron 6 precluded RNA 3'-end formation but had no effect on the efficiency of intron 5 removal. Deletion of only the 5' splice site of intron 6, which resulted in exon 6 skipping (i.e., the removal of intron 5, exon 6, and intron 6 as a single unit), had no effect on the efficiencies of either RNA 3'-end formation or the removal of intron 5-exon 6-intron 6. These results indicate that sequences within the 3'-terminal intron are functionally coupled to both RNA 3'-end formation and removal of the penultimate intron via a network of interactions that form across the last two exons and, most likely, between RNA processing factors.
Distinct molecular mechanism regulate cell cycle timing at successive stages of Drosophila embryogenesis.
Authors: Edgar B A , Sprenger F , Duronio R J , Leopold P , O'Farrell P H
Published: June 2007

The conserved regulators of cell cycle progression--Cyclins, Cdc2 kinase, and String phosphatase (Cdc25)--accommodate multiple modes of regulation during Drosophila embryogenesis. During cell cycles 2-7, Cdc2/Cyclin complexes are continuously present and show little fluctuation in abundance, phosphomodification, or activity. This suggests that cycling of the mitotic apparatus does not require cytoplasmic oscillations of known regulatory activities. During cycles 8-13 a progressive increase in the degradation of Cyclins at mitosis leads to increasing oscillations of Cdc2 kinase activity. Mutants deficient in cyclin mRNAs suffer cell cycle delays during this period, suggesting that Cyclin accumulation times these cycles. During interphase 14, programmed degradation of maternal String protein leads to inhibitory phosphorylation of Cdc2 and cell cycle arrest. Subsequently, mitoses 14-16 are triggered by pulses of zygotic string transcription.
A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif.
Authors: Duncan R , Bazar L , Michelotti G , Tomonaga T , Krutzsch H , Avigan M , Levens D
Published: June 2007

The far upstream element (FUSE) of the human c-myc proto-oncogene stimulates expression in undifferentiated cells. A FUSE-binding protein (FBP) is present in undifferentiated but not differentiated cells. Peptide sequences from the purified protein allowed cloning of cDNAs encoding FBP. Expression of FBP mRNA declined upon differentiation, suggesting transcriptional regulation of FBP. Features in the FBP cDNA suggest that FBP is also regulated by RNA processing, translation, and post-translational mechanisms. Both cellular and recombinant FBP form sequence-specific complexes with a single strand of FUSE. Transfection of FBP into human leukemia cells stimulated c-myc-promoter-driven expression from a reporter plasmid in a FUSE-dependent manner. Deletion and insertion mutagenesis of FBP defined a novel single-strand DNA-binding domain. Analysis of the primary and predicted secondary structure of the amino acid sequence reveals four copies of a reiterated unit comprised of a 30-residue direct repeat and an amphipathic alpha-helix separated by an 18- to 21-residue spacer. The third and fourth copies of this repeat-helix unit constitute the minimum single-stranded DNA-binding domain. To determine whether the FUSE site, in vivo, possesses single-strand conformation, and therefore could be bound by FBP, cells were treated with potassium permanganate (KMnO4) to modify unpaired bases. Modification of genomic DNA in vivo revealed hyperreactivity associated with single-stranded DNA in the FUSE sequence and protection on the strand that binds FBP in vitro. The role of single-stranded DNA and single-strand binding proteins in c-myc regulation is discussed.
A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes.
Authors: Matthews K R , Tschudi C , Ullu E
Published: June 2007

In trypanosomes, the generation of monocistronic mRNAs from polycistronic precursors is achieved via RNA processing, namely trans-splicing of the spliced leader sequence at the 5' end and cleavage/polyadenylation at the 3' end of the mRNA coding region. Recent evidence raised the intriguing possibility that these two reactions are coupled. To begin a dissection of the signals required for mRNA 5'-end and 3'-end formation and to uncover potential interactions between trans-splicing and polyadenylation, we mutagenized the intergenic region between the beta- and alpha-tubulin genes of Trypanosoma brucei. Block substitutions identified the pyrimidine-rich sequences at the alpha-tubulin 3'-splice-acceptor site as a major determinant for accurate trans-splicing downstream and 3'-end formation upstream. In addition to the utilization of cryptic 3'-splice sites, obliteration of the polypyrimidine tracts led to aberrant poly(A)+ site choice, even in the presence of the wild-type poly(A)+ site and neighboring sequences. Taken together, these results indicate that the polypyrimidine-rich sequences act as a bifunctional element that affects RNA processing both upstream and downstream from itself. This is consistent with the possibility that the polypyrimidine tract is recognized by both the trans-splicing and polyadenylation machineries, either sequentially or simultaneously.
Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance.
Authors: Maxon M E , Goodrich J A , Tjian R
Published: June 2007

The basal factor TFIIE is an important component of the RNA polymerase II transcription machinery. In our efforts to determine the role of TFIIE in the transcription process, we have begun to define the interactions between TFIIE and other basal transcription factors. Here we report that TFIIE binds selectively to the nonphosphorylated form of RNA polymerase II (IIa) and that this interaction is mediated by the 56-kD subunit of TFIIE. Additional binding studies reveal that TFII can interact with TBP as well as TFIID and that this interaction is mediated primarily via the 56-kD subunit. Our studies indicate that TFIIE also interacts with both subunits of TFIIF and with TFIIH, a multisubunit basal factor reported to catalyze RNA polymerase II CTD phosphorylation. Protein affinity assays demonstrate that TFIIE binds directly to ERCC-3, a DNA repair protein associated with TFIIH. More importantly, TFIIE affinity resin can selectively isolate transcriptionally competent TFIIH from a partially purified preparation and thereby may recruit TFIIH to the transcription complex in vivo. These multiple interactions between TFIIE, Pol II and TFIIH support a model in which TFIIE plays a role in promoter clearance as well as in the recruitment of proteins required for transcription-coupled DNA repair.
Activation of a yeast replication origin near a double-stranded DNA break.
Authors: Raghuraman M K , Brewer B J , Fangman W L
Published: June 2007

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.
Functional reconstitution of wild-type and mutant Tetrahymena telomerase.
Authors: Autexier C , Greider C W
Published: June 2007

Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)n to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats.
Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model.
Authors: Query C C , Moore M J , Sharp P A
Published: June 2007

Selection of the nucleophile for the first step of nuclear pre-mRNA splicing was probed by site-specific incorporation into splicing substrates of nucleotides modified at the 2' position. The differing abilities of ribose, 2'-deoxyribose, and arabinose nucleotides to base-pair within an RNA.RNA duplex and to contribute a nucleophilic 2'-OH group were exploited to analyze the paired/unpaired disposition of the branch site nucleotide. The results provide direct evidence for a bulged duplex model in which either of two adjacent purines within the consensus branch site sequence may shift into a bulged position and contribute the 2'-OH group for the first step of splicing. Furthermore, the presence of a consensus branch site that cannot present a reactive nucleophile suppresses splicing, including the use of cryptic branch sites elsewhere. We conclude that the branch site region base-pairing with U2 snRNA determines the first step nucleophile and persists at the time of the first transesterification reaction.