bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor.
Authors: Ponzetto C , Bardelli A , Maina F , Longati P , Panayotou G , Dhand R , Waterfield M D , Comoglio P M
Published: October 2015

The pleiotropic effects (mitogenesis, motogenesis, and morphogenesis) elicited by hepatocyte growth factor/scatter factor (HGF/SF) are mediated by the activation of the tyrosine kinase receptor encoded by the MET proto-oncogene. Following autophosphorylation, the receptor associates with the p85/110 phosphatidylinositol (PI) 3-kinase complex in vivo and in vitro. By a combination of two complementary approaches, competition with synthetic phosphopeptides and association with Tyr-Phe receptor mutants, we have identified Y-1349 and Y-1356 in the HGF/SF receptor as the binding sites for PI 3-kinase. Y-1349VHV and Y-1356VNV do not conform to the canonical consensus sequence YXXM for PI 3-kinase binding and thus define YVXV as a novel recognition motif. Y-1349 and Y-1356 are located within the C-terminal portion of the HGF/SF receptor and are phosphorylation sites. The affinity of the N- and C-terminal src homology region 2 (SH2) domains of p85 for the phosphopeptides including Y-1349 and Y-1356 is 2 orders of magnitude lower than that measured for Y-751 in the platelet-derived growth factor receptor binding site. However, the closely spaced duplication of the novel recognition motif in the native HGF/SF receptor may allow binding with both SH2 domains of p85, thus generating an efficient docking site for PI 3-kinase. In agreement with this model, we have observed that a phosphopeptide including both Y-1349 and Y-1356 activates PI 3-kinase in vitro.
Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3.
Authors: Bushman J L , Foiani M , Cigan A M , Paddon C J , Hinnebusch A G
Published: October 2015

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.
Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.
Authors: Johansen F E , Prywes R
Published: October 2015

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.
Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos.
Authors: Rollins M B , Del Rio S , Galey A L , Setzer D R , Andrews M T
Published: October 2015

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.
Ubiquitin pathway involvement in human lymphocyte gamma-irradiation-induced apoptosis.
Authors: Delic J , Morange M , Magdelenat H
Published: October 2015

Apoptosis (the classical type of programmed cell death) can be triggered in many cell types by widely diverse stimuli. gamma rays, at low doses, can induce apoptosis in vitro in interphase human lymphocytes. In this type of apoptosis induction, activated gene expression is necessary for the fulfillment of the death program. In this report, we present evidence for a relationship between ubiquitin gene expression or ubiquitination and gamma-irradiation-mediated apoptosis in normal circulating human lymphocytes. Using in vitro nuclear transcription assays (run-on), Northern (RNA) blot analysis, immunolocalization studies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation, we demonstrate that (i) the ubiquitin mRNA level is increased as a consequence of the activation of ubiquitin gene transcription 15 to 90 min after initiation of apoptosis; (ii) specifically in apoptotic cells, and not in all irradiated cells, nuclear proteins are highly ubiquitinated; and (iii) ubiquitin sequence-specific antisense oligonucleotide inhibition results in a decreased level of ubiquitinated nuclear proteins and considerably diminishes the proportion of cells exhibiting the apoptotic death pattern. Each of these results might be explained by different modifications occurring in irradiated cells. Their convergence strongly suggests that the ubiquitin gene is one of the genes with induced activity in the apoptotic death program and that ubiquitination of nuclear proteins might be involved in chromatin disorganization and oligonucleosomal fragmentation, which are among the key events occurring in apoptosis.
The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.
Authors: Heitman J , Koller A , Kunz J , Henriquez R , Schmidt A , Movva N R , Hall M N
Published: October 2015

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.
A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites.
Authors: Marcote M J , Knighton D R , Basi G , Sowadski J M , Brambilla P , Draetta G , Taylor S S
Published: October 2015

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.
cis-acting components in the replication origin from ribosomal DNA of Saccharomyces cerevisiae.
Authors: Miller C A , Kowalski D
Published: October 2015

The ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae contain an autonomously replicating sequence (ARS) that colocalizes with a chromosomal origin of replication. We show that a minimal sequence necessary for full ARS function corresponds to a 107-bp rDNA fragment which contains three 10-of-11-bp matches to the ARS consensus sequence. Point mutations in only one of the 10-of-11-bp matches, GTTTAT GTTTT, inactivate the rDNA ARS, indicating that this consensus sequence is essential. A perfect match to a revised ARS consensus is present but not essential. Sequences up to 9 bp 5' from the essential consensus are dispensable. A broad DNA region directly 3' to the essential consensus is required and is easily unwound as indicated by: (i) hypersensitivity to nicking of an approximately 100-bp region by mung bean nuclease in a negatively supercoiled plasmid and (ii) helical instability determined by thermodynamic analysis of the nucleotide sequence. A correlation between DNA helical instability and replication efficiency of wild-type and mutated ribosomal ARS derivatives suggests that a broad region 3' to the essential ARS consensus functions as a DNA unwinding element. Certain point mutations that do not stabilize the DNA helix in the 3' region but reduce ARS efficiency reveal an element distinct from, but overlapping, the DNA unwinding element. The nucleotide sequence of the functionally important constituents in the ARS appears to be conserved among the rDNA repeats in the chromosome.
Mutational and functional analysis of dominant SPT2 (SIN1) suppressor alleles in Saccharomyces cerevisiae.
Authors: Lefebvre L , Smith M
Published: October 2015

The Saccharomyces cerevisiae SPT2 gene was identified by genetic screens for mutations which are suppressors of Ty and delta insertional mutations at the HIS4 locus. The ability of spt2 mutations to suppress the transcriptional interference caused by the delta promoter insertion his-4-912 delta correlates with an increase in wild-type HIS4 mRNA levels. The SPT2 gene is identical to SIN1, which codes for a factor genetically defined as a negative regulator of HO transcription. Mutations in SPT2/SIN1 suppress the effects of trans-acting mutations in SWI genes and of partial deletions in the C-terminal domain of the largest subunit of RNA polymerase II. Nuclear localization and protein sequence similarities suggested that the SPT2/SIN1 protein may be related to the nonhistone chromosomal protein HMG1. To assess the significance of this structural similarity and identify domains of SPT2 functionally important in the regulation of his4-912 delta, we have studied recessive and dominant spt2 mutations created by in vitro mutagenesis. We show here that several alleles carrying C-terminal deletions as well as point mutations in the C-terminal domain of the SPT2 protein exhibit a dominant suppressor phenotype. C-terminal basic residues necessary for wild-type SPT2 protein function which are absent from HMG1 have been identified. The competence of these mutant SPT2 proteins to interfere with the maintenance of the His- (Spt+) phenotype of a his4-912 delta SPT2+ strain is lost by deletion of internal HMG1-like sequences and is sensitive to the wild-type SPT2+ gene dosage. Using cross-reacting antipeptide polyclonal antibodies, we demonstrate that the intracellular level of the wild-type SPT2 protein is not affected in presence of dominant mutations and furthermore that the reversion of the dominance by internal deletion of HMG1-like sequences is not mediated by altered production or stability of the mutant polypeptides. Our results suggest that the products of dominant alleles directly compete with the wild-type protein. On the basis of primary sequence similarities, we propose that an HMG-box-like motif is required for SPT2 function in vivo and that this motif also is necessary for the dominant suppressor phenotype exhibited by some mutant SPT2 alleles.
Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA.
Authors: Gilbert E , Del Gatto F , Champion-Arnaud P , Gesnel M C , Breathnach R
Published: October 2015

The fibroblast growth factor receptor 2 gene pre-mRNA can be spliced by using either the K-SAM exon or the BEK exon. The exon chosen has a profound influence on the ligand-binding specificity of the receptor obtained. Cells make a choice between the two alternative exons by controlling use of both exons. Using fibroblast growth factor receptor 2 minigenes, we have shown that in cells normally using the K-SAM exon, the BEK exon is not used efficiently even in the absence of the K-SAM exon. This is because these cells apparently express a titratable repressor of BEK exon use. In cells normally using the BEK exon, the K-SAM exon is not used efficiently even in the absence of a functional BEK exon. Three purines in the K-SAM polypyrimidine tract are at least in part responsible for this, as their mutation to pyrimidines leads to efficient use of the K-SAM exon, while mutating the BEK polypyrimidine tract to include these purines stops BEK exon use.
The conserved ninth C-terminal heptad in thyroid hormone and retinoic acid receptors mediates diverse responses by affecting heterodimer but not homodimer formation.
Authors: Au-Fliegner M , Helmer E , Casanova J , Raaka B M , Samuels H H
Published: October 2015

The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition. Our studies also suggest that the ninth heptad is necessary for the dominant inhibition of wild-type T3Rs by mutant T3Rs, as has been found for the thyroid hormone-resistant syndrome in humans. Thus, the ninth heptad repeat is required for heterodimerization, suppression of basal promoter activity, and dominant negative effects of T3R and RAR. Lastly, the finding that cT3R(L365R) and hRAR(M377R) require ligands for heterodimer formation also raises the possibility that heterodimeric interactions are mediated by the ninth heptad without ligands but by a second region of these receptors with ligands.
Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.
Authors: Perry J R , Basrai M A , Steiner H Y , Naider F , Becker J M
Published: October 2015

We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene.
CDC44: a putative nucleotide-binding protein required for cell cycle progression that has homology to subunits of replication factor C.
Authors: Howell E A , McAlear M A , Rose D , Holm C
Published: October 2015

To investigate the means by which a cell regulates the progression of the mitotic cell cycle, we characterized cdc44, a mutation that causes Saccharomyces cerevisiae cells to arrest before mitosis. CDC44 encodes a 96-kDa basic protein with significant homology to a human protein that binds DNA (PO-GA) and to three subunits of human replication factor C (also called activator 1). The hypothesis that Cdc44p is involved in DNA metabolism is supported by the observations that (i) levels of mitotic recombination suggest elevated rates of DNA damage in cdc44 mutants and (ii) the cell cycle arrest observed in cdc44 mutants is alleviated by the DNA damage checkpoint mutations rad9, mec1, and mec2. The predicted amino acid sequence of Cdc44p contains GTPase consensus sites, and mutations in these regions cause a conditional cell cycle arrest. Taken together, these observations suggest that the essential CDC44 gene may encode the large subunit of yeast replication factor C.
Induction of Drosophila RNA polymerase III gene expression by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is mediated by transcription factor IIIB.
Authors: Garber M E , Vilalta A , Johnson D L
Published: October 2015

We have previously found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces specific transcription of tRNA and 5S RNA genes in Drosophila Schneider S-2 cells (M. Garber, S. Panchanathan, R. F. Fan, and D. L. Johnson, J. Biol. Chem. 266:20598-20601, 1991). Having derived cellular extracts from TPA-treated cells, that are capable of reproducing this stimulation in vitro, we have examined the mechanism for this regulatory event. Using conditions that limit reinitiation and produce single rounds of transcription from active gene complexes, we find that the number of functional transcription complexes is increased in extracts prepared from TPA-induced cells. We have analyzed the activities of the transcription factors TFIIIB and TFIIIC derived from extracts prepared from TPA-induced and noninduced cells. Examination of the relative activities of TFIIIC showed that both its ability to reconstitute transcription with TFIIIB and RNA polymerase III and its ability to stably bind to the DNA template are unchanged. However, the activity of TFIIIB derived from the TPA-induced cells is substantially increased compared with that derived from the noninduced cells. The differences in TFIIIB activity account for the differences in the overall transcriptional activities observed in the unfractionated extracts. Western blot analysis of the TATA-binding protein subunit of TFIIIB revealed that there is an increase in the amount of this polypeptide present in the induced cell extracts and TFIIIB fraction. Together, these results indicate that the TPA response in Drosophila cells stimulates specific transcription of RNA polymerase III genes by increasing the activity of the limiting transcription component, TFIIIB, and thereby increasing the number of functional transcription complexes.
The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor.
Authors: Zhang D E , Hetherington C J , Chen H M , Tenen D G
Published: October 2015

The macrophage colony-stimulating factor (M-CSF) receptor is expressed in a tissue-specific fashion from two distinct promoters in monocytes/macrophages and the placenta. In order to further understand the transcription factors which play a role in the commitment of multipotential progenitors to the monocyte/macrophage lineage, we have initiated an investigation of the factors which activate the M-CSF receptor very early during the monocyte differentiation process. Here we demonstrate that the human monocytic M-CSF receptor promoter directs reporter gene activity in a tissue-specific fashion. Since one of the few transcription factors which have been implicated in the regulation of monocyte genes is the macrophage- and B-cell-specific PU.1 transcription factor, we investigated whether PU.1 binds and activates the M-CSF receptor promoter. Here we demonstrate that both in vitro-translated PU.1 and PU.1 from nuclear extracts bind to a specific site in the M-CSF receptor promoter just upstream from the major transcription initiation site. Mutations in this site which eliminate PU.1 binding decrease M-CSF receptor promoter activity significantly in macrophage cell lines only. Furthermore, PU.1 transactivates the M-CSF receptor promoter in nonmacrophage cells. These results suggest that PU.1 plays a major role in macrophage gene regulation and development by directing the expression of a receptor for a key macrophage growth factor.
Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.
Authors: Deng W P , Nickoloff J A
Published: October 2015

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.
Concerted action of RAS and G proteins in the sexual response pathways of Schizosaccharomyces pombe.
Authors: Xu H P , White M , Marcus S , Wigler M
Published: October 2015

We have shown that the expression of mam2, the gene encoding the Schizosaccharomyces pombe P-factor pheromone receptor, is dependent upon components of the pheromone signal transduction pathway, including Ras1, Gpa1, Byr1 and Byr2, each of which is required for both conjugation and sporulation. Studies of the expression of mam2 in mutant S. pombe cells confirm previous conclusions, based on the ability of cells to sporulate, that the Byr1 protein kinase acts downstream of the Byr2 protein kinase and that both act downstream of Ras1, the S. pombe RAS homolog, and Gpa1, the G alpha component that mediates the occupancy of the mam2 receptor. In addition, our present studies show that Ras1 and Gpa1 each act downstream from the other and hence act in concert. The Spk1 kinase, which is required for conjugation and sporulation and which is a structural and functional homolog of the vertebrate MAP kinases, is not required for mam2 expression.
Alternative 5' splice site selection induced by heat shock.
Authors: Takechi H , Hosokawa N , Hirayoshi K , Nagata K
Published: October 2015

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression.
Binding of TFIID and MEF2 to the TATA element activates transcription of the Xenopus MyoDa promoter.
Authors: Leibham D , Wong M W , Cheng T C , Schroeder S , Weil P A , Olson E N , Perry M
Published: October 2015

Members of the MyoD family of helix-loop-helix proteins control expression of the muscle phenotype by regulating the activity of subordinate genes. To investigate processes that control the expression of myogenic factors and regulate the establishment and maintenance of the skeletal muscle phenotype, we have analyzed sequences necessary for transcription of the maternally expressed Xenopus MyoD (XMyoD) gene. A 3.5-kb DNA fragment containing the XMyoDa promoter was expressed in a somite-specific manner in injected frog embryos. The XMyoDa promoter was active in oocytes and cultured muscle cells but not in fibroblasts or nonmuscle cell lines. A 58-bp fragment containing the transcription initiation site, a GC-rich region, and overlapping binding sites for the general transcription factor TFIID and the muscle-specific factor MEF2 was sufficient for muscle-specific transcription. Transcription of the minimal XMyoDa promoter in nonmuscle cells was activated by expression of Xenopus MEF2 (XMEF2) and required binding of both MEF2 and TFIID to the TATA motif. These results demonstrate that the XMyoDa TATA motif is a target for a cell-type-specific regulatory factor and suggests that MEF2 stabilizes and amplifies XMyoDa transcription in mesodermal cells committed to the muscle phenotype.
Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity.
Authors: Muthuswamy S K , Siegel P M , Dankort D L , Webster M A , Muller W J
Published: October 2015

Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30% of human breast cancers. Although the activation of Neu receptor tyrosine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu. To test whether c-Src was implicated in Neu-mediated signal transduction, we measured the activity of the c-Src tyrosine kinase in tissue extracts from either mammary tumors or adjacent mammary epithelium derived from transgenic mice expressing a mouse mammary tumor virus promoter/enhancer/unactivated neu fusion gene. The Neu-induced mammary tumors possessed six- to eightfold-higher c-Src kinase activity than the adjacent epithelium. The increase in c-Src tyrosine kinase activity was not due to an increase in the levels of c-Src but rather was a result of the elevation of its specific activity. Moreover, activation of c-Src was correlated with its ability to complex tyrosine-phosphorylated Neu both in vitro and in vivo. Together, these observations suggest that activation of the c-Src tyrosine kinase during mammary tumorigenesis may occur through a direct interaction with activated Neu.
Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase.
Authors: Li X , Coffino P
Published: October 2015

Selective degradation by proteasomes of ornithine decarboxylase, the initial enzyme in polyamine biosynthesis, is mediated by the polyamine-inducible protein antizyme. Antizyme binds to a region near the N terminus of ornithine decarboxylase (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-3562, 1992). This interaction induces a conformational change in ornithine decarboxylase that exposes its C terminus and inactivates the enzyme (X. Li and P. Coffino, Mol. Cell. Biol. 13:1487-1492, 1993). Here we show that the C-terminal half of antizyme alone can inactivate ornithine decarboxylase and alter its conformation, but it cannot direct degradation of the enzyme, either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote degradation.
Efficient homologous recombination of Ty1 element cDNA when integration is blocked.
Authors: Sharon G , Burkett T J , Garfinkel D J
Published: October 2015

Integration of the yeast retrotransposon Ty1 into the genome requires the self-encoded integrase (IN) protein and specific terminal nucleotides present on full-length Ty1 cDNA. Ty1 mutants with defects in IN, the conserved termini of Ty1 cDNA, or priming plus-strand DNA synthesis, however, were still able to efficiently insert into the genome when the elements were expressed from the GAL1 promoter present on a multicopy plasmid. As with normal transposition, formation of the exceptional insertions required an RNA intermediate, Ty1 reverse transcriptase, and Ty1 protease. In contrast to Ty1 transposition, at least 70% of the chromosomal insertions consisted of complex multimeric Ty1 elements. Ty1 cDNA was transferred to the inducing plasmid as well as to the genome, and transfer required the recombination and repair gene RAD52. Furthermore, multimeric insertions occurred without altering the levels of total Ty1 RNA, virus-like particle-associated RNA or cDNA, Ty1 capsid proteins, or IN. These results suggest that Ty1 cDNA is utilized much more efficiently for homologous recombination when IN-mediated integration is blocked.
Identification of a C/EBP-Rel complex in avian lymphoid cells.
Authors: Diehl J A , Hannink M
Published: October 2015

Protein-protein interactions between the CCAAT box enhancer-binding proteins (C/EBP) and the Rel family of transcription factors have been implicated in the regulation of cytokine gene expression. We have used sequence-specific DNA affinity chromatography to purify a complex from avian T cells that binds to a consensus C/EBP motif. Our results provide evidence that Rel-related proteins are components of the C/EBP-DNA complex as a result of protein-protein interactions with the C/EBP proteins. A polyclonal antiserum raised against the Rel homology domain of v-Rel and antisera raised against two human RelA-derived peptides specifically induced a supershift of the C/EBP-DNA complex in mobility shift assays using the affinity-purified C/EBP. In addition, several kappa B-binding proteins copurified with the avian C/EBP complex through two rounds of sequence-specific DNA affinity chromatography. The kappa B-binding proteins are distinct from the C/EBP proteins that directly contact DNA containing the C/EBP binding site. The identification of a protein complex that binds specifically to a consensus C/EBP site and contains both C/EBP and Rel family members suggests a novel mechanism for regulation of gene expression by Rel family proteins.
Hot-spot p53 mutants interact specifically with two cellular proteins during progression of the cell cycle.
Authors: Chen Y , Chen P L , Lee W H
Published: October 2015

Inactivation of both alleles of the p53 gene is commonly found in human cancers. In contrast to mutations of the retinoblastoma gene, certain altered forms of p53 gain growth-promoting functions. To explore the mechanisms underlying this gain of function, we have identified two nuclear proteins, with molecular masses of 42 and 38 kDa, respectively, that are specifically associated with p53 mutated within the simian virus 40 T-antigen-binding domain, "hot spots" found in many human tumors. These mutants transactivate the multiple-drug resistance gene promoter and cause cells to grow to higher density. Both the mutated p53 complex with p42 and p38 increase when cells enter S phase of the cell cycle but decrease in G1 and M phases, suggesting that they may have a role in promoting cell growth.
Two alternative pathways of transcription initiation in the yeast negative regulatory gene GAL80.
Authors: Sakurai H , Ohishi T , Fukasawa T
Published: October 2015

The yeast GAL80 gene, encoding a negative regulatory protein of galactose-inducible genes, shows both constitutive and galactose-inducible expression. The inducible transcription is under the control of Gal4p, a common activator for the galactose-inducible genes, which binds to an upstream activation sequence, called UASG, spanning between -105 and -89 in the 5'-flanking region of GAL80. Here we demonstrate that the constitutive transcription started at +1, whereas the inducible transcription occurs from a set of downstream sites at +37, +47, +56, and +67. Both transcriptions were enhanced 10-fold by another UAS, whose 5' boundary is located between -195 and -185. Gal4p stimulated transcription, which depends on the TATA box located at -20, from all the downstream sites. By contrast, the constitutive transcription depended on a small region of less than 16 bp long encompassing the +1 site, which directed transcription even in the absence of both the TATA box and the UASs. When a fragment covering that region was inserted immediately upstream of the open reading frame of HIS3, the resulting gene fusion, if introduced into a his3 yeast strain, supported growth on histidine-lacking medium. We detected by gel retardation assay a protein specifically interacting with this fragment. All the transcriptions observed in the in vivo experiments were faithfully reproduced in a cell-free transcription system. From these results, we suggest that initiation of GAL80 transcription involves two alternative pathways; one is initiator dependent, and the other is Gal4p regulated and TATA dependent.
A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa.
Authors: Miao V P , Singer M J , Rountree M R , Selker E U
Published: October 2015

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.
Differential specificity for binding of retinoblastoma binding protein 2 to RB, p107, and TATA-binding protein.
Authors: Kim Y W , Otterson G A , Kratzke R A , Coxon A B , Kaye F J
Published: October 2015

The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2 can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.
Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 beta gene.
Authors: Tsukada J , Saito K , Waterman W R , Webb A C , Auron P E
Published: October 2015

A site located between -2782 and -2729 of the human prointerleukin-1 beta (IL1B) gene functions as a strong lipopolysaccharide (LPS)-responsive enhancer independent of the previously identified enhancer located between -2896 and -2846 (F. Shirakawa, K. Saito, C.A. Bonagura, D.L. Galson, M. J. Fenton, A. C. Webb, and P. E. Auron, Mol. Cell. Biol. 13:1332-1344, 1993). Although these two enhancers appear to function cooperatively in the native sequence context, they function independently as LPS-responsive elements upon removal of an interposed silencer sequence. The new enhancer is not induced by dibutyryl cyclic AMP (dbcAMP) alone but is superinduced by costimulation with LPS-dbcAMP. This pattern of induction depends upon the nature of the sequence, a composite NF-IL6-cAMP response element (CRE) binding site. This pseudosymmetrical sequence is shown to contrast with a classical symmetric CRE which responds to dbcAMP but not LPS. DNA binding studies using in vivo nuclear extract, recombinant proteins, and specific antibodies show that LPS induces the formation of two different complexes at the enhancer: (i) an NF-IL6-CREB heterodimer and (ii) a heterodimer consisting of NF-IL6 and a non-CREB, CRE-binding protein. Cotransfection studies using NF-IL6 and CREB expression vectors show that NF-IL6 transactivates the enhancer in the presence of LPS, whereas CREB acts either positively or negatively, depending upon its cAMP-regulated phosphorylation state. Our data demonstrate that the newly identified enhancer is a specialized LPS-responsive sequence which can be modulated by cAMP as a result of the involvement of NF-IL6-CRE-binding protein heterodimers.
A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins.
Authors: Cvekl A , Sax C M , Bresnick E H , Piatigorsky J
Published: October 2015

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.