bluuebunny/crossref_metadata_2025 huggingface dataset:
A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons.
Authors: Caponigro G , Muhlrad D , Parker R
Published: October 2015

Differences in decay rates of eukaryotic transcripts can be determined by discrete sequence elements within mRNAs. Through the analysis of chimeric transcripts and internal deletions, we have identified a 65-nucleotide segment of the MAT alpha 1 mRNA coding region, termed the MAT alpha 1 instability element, that is sufficient to confer instability to a stable PGK1 reporter transcript and that accelerates turnover of the unstable MAT alpha 1 mRNA. This 65-nucleotide element is composed of two parts, one located within the 5' 33 nucleotides and the second located in the 3' 32 nucleotides. The first part, which can be functionally replaced by sequences containing rare codons, is unable to promote rapid decay by itself but can enhance the action of the 3' 32 nucleotides (positions 234 to 266 in the MAT alpha 1 mRNA) in accelerating turnover. A second portion of the MAT alpha 1 mRNA (nucleotides 265 to 290) is also sufficient to destabilize the PGK1 reporter transcript when positioned 3' of rare codons, suggesting that the 3' half of the MAT alpha 1 instability element is functionally reiterated within the MAT alpha 1 mRNA. The observation that rare codons are part of the 65-nucleotide MAT alpha 1 instability element suggests possible mechanisms through which translation and mRNA decay may be linked.
Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites.
Authors: Hyman L E , Moore C L
Published: October 2015

Little is known about the transcriptional events which occur downstream of polyadenylation sites. Although the polyadenylation site of a gene can be easily identified, it has been difficult to determine the site of transcription termination in vivo because of the rapid processing of pre-mRNAs. Using an in vitro approach, we have shown that sequences from the 3' ends of two different Saccharomyces cerevisiae genes, ADH2 and GAL7, direct transcription termination and/or polymerase pausing in yeast nuclear extracts. In the case of the ADH2 sequence, the RNA synthesized in vitro ends approximately 50 to 150 nucleotides downstream of the poly(A) site. This RNA is not polyadenylated and may represent the primary transcript. A similarly sized nonpolyadenylated [poly(A)-] transcript can be detected in vivo from the same transcriptional template. A GAL7 template also directs the in vitro synthesis of an RNA which extends a short distance past the poly(A) site. However, a significant amount of the GAL7 RNA is polyadenylated at or close to the in vivo poly(A) site. Mutations of GAL7 or ADH2 poly(A) signals prevent polyadenylation but do not affect the in vitro synthesis of the extended poly(A)- transcript. Since transcription of the mutant template continues through this region in vivo, it is likely that a strong RNA polymerase II pause site lies within the 3'-end sequences. Our data support the hypothesis that the coupling of this pause site to a functional polyadenylation signal results in transcription termination.
Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression.
Authors: Unger T , Mietz J A , Scheffner M , Yee C L , Howley P M
Published: October 2015

The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of p53 was carried out. We show that different mutant p53 proteins found in cancers vary in the ability to inhibit the transcriptional transactivation and specific DNA binding activities of wt human p53. This transdominant effect was mediated through the carboxy-terminal oligomerization region. The role of the transactivation activity in transformation suppression by wt p53 was also examined by constructing an N-terminal deletion mutant lacking the transactivation domain. This mutant was unable to transactivate but could bind specifically to DNA. Although it was impaired in its ability to suppress transformation of primary rat embryo fibroblasts by adenovirus E1A plus activated ras, the N-terminal deletion mutant still had some suppression activity, suggesting that additional functions of p53 may contribute to transformation suppression.
Structure, mapping, and expression of erp, a growth factor-inducible gene encoding a nontransmembrane protein tyrosine phosphatase, and effect of ERP on cell growth.
Authors: Noguchi T , Metz R , Chen L , Mattéi M G , Carrasco D , Bravo R
Published: October 2015

We have characterized a growth factor-inducible gene, erp, and demonstrated that it encodes a 367-amino-acid nontransmembrane tyrosine phosphatase protein with significant similarity to the vaccinia virus H1 protein. Immunoprecipitation analyses show that the erp protein, ERP, is rapidly induced following serum stimulation of quiescent fibroblasts. ERP has been expressed as a fusion protein with glutathione S-transferase and shown to have tyrosine as well as serine protein phosphatase activity. The enzymatic activity of ERP depends on the presence of reducing agents such as dithiothreitol, and its tyrosine phosphatase activity is inhibited by sodium vanadate, a potent inhibitor of protein tyrosine phosphatases. The number of stable NIH 3T3 clones obtained after transfection with a vector expressing the complete ERP protein is reduced more than 90% compared with that after transfection with a vector expressing a mutated inactive ERP protein. The remaining ERP-expressing clones present a significant increase in the proportion of bi- and multinucleated cells and a decrease in proliferation rate. Studies on the genomic structure reveal that the erp transcription unit is 2.8 kbp long and split into four exons. The erp gene maps to the 17A2-17C region of the murine genome. Our results demonstrate that the protein product of the immediate-early gene erp has a negative effect on cell proliferation.
Binding of myc proteins to canonical and noncanonical DNA sequences.
Authors: Blackwell T K , Huang J , Ma A , Kretzner L , Alt F W , Eisenman R N , Weintraub H
Published: October 2015

Using an in vitro binding-site selection assay, we have demonstrated that c-Myc-Max complexes bind not only to canonical CACGTG or CATGTG motifs that are flanked by variable sequences but also to noncanonical sites that consist of an internal CG or TG dinucleotide in the context of particular variations in the CA--TG consensus. None of the selected sites contain an internal TA dinucleotide, suggesting that Myc proteins necessarily bind asymmetrically in the context of a CAT half-site. The noncanonical sites can all be bound by proteins of the Myc-Max family but not necessarily by the related CACGTG- and CATGTG-binding proteins USF and TFE3. Substitution of an arginine that is conserved in these proteins into MyoD (MyoD-R) changes its binding specificity so that it recognizes CACGTG instead of the MyoD cognate sequence (CAGCTG). However, like USF and TFE3, MyoD-R does not bind to all of the noncanonical c-Myc-Max sites. Although this R substitution changes the internal dinucleotide specificity of MyoD, it does not significantly alter its wild-type binding sequence preferences at positions outside of the CA--TG motif, suggesting that it does not dramatically change other important amino acid-DNA contacts; this observation has important implications for models of basic-helix-loop-helix protein-DNA binding.
Interferons and interleukin-6 suppress the DNA-binding activity of E2F in growth-sensitive hematopoietic cells.
Authors: Melamed D , Tiefenbrun N , Yarden A , Kimchi A
Published: October 2015

Transcription factor E2F binds to cellular promoters of certain growth- and cell cycle-controlling genes and forms distinct heteromeric complexes with other nuclear proteins. We show here that alpha and beta interferons (alpha, beta) and interleukin-6 abolished the E2F-containing DNA-binding complexes in Daudi Burkitt lymphoma cells and in M1 myeloblastic cells, which responded to the cytokines by suppression of c-myc transcription. Time kinetics studies showed that the abolishment of E2F complexes coincided with reduction of c-myc expression and that both molecular events preceded the cell cycle block in G0/G1 phase. In contrast, the pattern of E2F complexes remained unchanged in an interferon-treated growth-resistant Daudi cell mutant that displayed relaxed regulation of c-myc. All of the DNA-binding E2F complexes, including those containing the retinoblastoma protein (pRB), cyclin A-p33cdk2, and the free forms of E2F, were reduced by interferons or interleukin-6. Their abolishment was unperturbed by pharmacological treatments that alleviated the cyclin A and pRB responses to interferon. Thus, changes in cyclin A expression and pRB phosphorylation are not primary events that influence the pattern of E2F responses to cytokines. Addition of EDTA to cell extracts of interferon-treated Daudi cells restored the DNA-binding activity of E2F, resulting in the appearance of a single E2F complex that exclusively contained pRB. It is suggested that the regulation of E2F by growth-inhibitory cytokines that induce cell cycle exit takes place at the level of the DNA-binding activity, and by that mean it differs basically from the phase-specific regulation of E2F in cycling cells.
Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain.
Authors: Wang Z E , Myles G M , Brandt C S , Lioubin M N , Rohrschneider L
Published: October 2015

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.
Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae.
Authors: Thorsness P E , White K H , Fox T D
Published: October 2015

The yeast nuclear gene YME1 was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus. yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho- (cytoplasmic petite) cells. The cloned YME1 gene complements all of these phenotypes. The gene product, Yme1p, is immunologically detectable as an 82-kDa protein present in mitochondria. Yme1p is a member of a family of homologous putative ATPases, including Sec18p, Pas1p, Cdc48p, TBP-1, and the FtsH protein. Yme1p is most similar to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division. This observation suggests the hypothesis that Yme1p may play a role in mitochondrial fusion and/or division.
A novel POU domain protein which binds to the T-cell receptor beta enhancer.
Authors: Messier H , Brickner H , Gaikwad J , Fotedar A
Published: October 2015

POU domain proteins have been implicated in the regulation of a number of lineage-specific genes. Among the first POU domain proteins described were the immunoglobulin octamer-binding proteins Oct-1 and Oct-2. It was therefore of special interest when we identified a novel lymphoid POU domain protein in Southwestern (DNA-protein) screens of T-cell lambda gt11 libraries. This novel POU protein, TCF beta 1, binds in a sequence-specific manner to a critical motif in the T-cell receptor (TCR) beta enhancer. Sequence analysis revealed that TCF beta 1 represents a new class of POU domain proteins which are distantly related to other POU proteins. TCF beta 1 is encoded by multiple exons whose organization is distinct from that of other POU domain proteins. The expression of TCF beta 1 in a tissue-restricted manner and its ability to bind to multiple motifs in the TCR beta enhancer support a role in regulating TCR beta gene expression. The expression of TCF beta 1 in both B and T cells and the ability of recombinant TCF beta 1 to bind octamer and octamer-related motifs suggest that TCF beta 1 has additional roles in lymphoid cell function. The ability of TCF beta 1 to transactivate in a sequence-specific manner is consistent with a role for regulating lymphoid gene expression.
Genetic transfer and expression of reconstructed yeast artificial chromosomes containing normal and translocated BCL2 proto-oncogenes.
Authors: Silverman G A , Yang E , Proffitt J H , Zutter M , Korsmeyer S J
Published: October 2015

The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection. The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus. Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles. Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line. Fifty-eight percent of the clones contained a copy of the entire YAC insert. Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein. These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S. cerevisiae.
Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.
Authors: Brazas R M , Stillman D J
Published: October 2015

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.
Multiple proteins interact with the fushi tarazu proximal enhancer.
Authors: Han W , Yu Y , Altan N , Pick L
Published: October 2015

The expression of the Drosophila segmentation gene fushi tarazu (ftz) is controlled at the level of transcription. The proximal enhancer, located approximately 3.4 kb upstream of the transcription start site, directs lacZ fusion gene expression in a ftz-like seven-stripe pattern in transgenic fly embryos. We have taken a biochemical approach to identify DNA-binding proteins that regulate ftz gene expression through the proximal enhancer. DNase I footprinting and methylation interference experiments with staged Drosophila embryo nuclear extracts identified nine protein binding sites in the proximal enhancer. Ten different sequence-specific DNA-binding complexes that interact with eight of these sites were identified. Some interact with multiple sites, while others bind to single sites in the enhancer. Two of the complexes that interact with multiple sites appear to contain the previously described ftz regulators, FTZ-F1 and TTK/FTZ-F2. These in vitro studies allowed us to narrow down the proximal enhancer to a 323-bp DNA fragment that contains all of the protein binding sites. Expression directed by this minimal enhancer element in seven ftz-like stripes in transgenic embryos is identical to that directed by the full-length enhancer. Internal deletions of several sites abolish reporter gene expression in vivo. Thus, the ftz proximal enhancer, like other cell-type-specific eukaryotic enhancers, interacts with an array of proteins that are expected to mediate the establishment, maintenance, and repression of transcription of the ftz gene in seven stripes in the developing embryo.
Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A.
Authors: Blacketer M J , Koehler C M , Coats S G , Myers A M , Madaule P
Published: October 2015

The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.
Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53.
Authors: Nibbs R J , Itoh K , Ostertag W , Harrison P R
Published: October 2015

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.
An mRNA-type intron is present in the Rhodotorula hasegawae U2 small nuclear RNA gene.
Authors: Takahashi Y , Urushiyama S , Tani T , Ohshima Y
Published: October 2015

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.
A heterodimer of HEB and an E12-related protein interacts with the CD4 enhancer and regulates its activity in T-cell lines.
Authors: Sawada S , Littman D R
Published: October 2015

A T-lymphocyte-specific enhancer located 13 kb upstream of the murine CD4 gene was recently shown to be required for the developmentally regulated expression of CD4. We have previously identified three nuclear protein binding sites in this enhancer; one of these sites, CD4-3, is essential for expression and contains two E-box core motifs (CANNTG) adjacent to each other in the sequence TAACAGGTGTCAGCTGGT. In electrophoretic mobility shift assays using the CD4-3 oligonucleotide as a probe, three nuclear protein complexes, termed CD4-3A, -B, and -C, were detected with nuclear extracts from T-cell lines. CD4-3A, which involves nuclear protein binding to the 5' E-box, was detected only with nuclear extracts from lymphoid cells. Specific antisera were used to show that the CD4-3A complex contains a heterodimer or heterooligomer of basic helix-loop-helix transcriptional factors, E12 or a related factor and HEB, which is expressed predominantly in thymus. Consistent with this finding, in vitro-translated E12 and HEB proteins, as homodimers or heterodimers, bound preferentially to the 5' E-box. Point mutations in the 5' E-box, but not in the 3' E-box, abolished CD4 enhancer activity. Furthermore, overexpression of Id, a protein that forms inactive heterodimers with E12/E47, blocked CD4 enhancer activity in T cells. These results suggest that a heterodimer composed of HEB and E12 or a closely related protein plays a critical role in CD4 enhancer function by interacting with the 5' E-box motif of the CD4-3 site in vivo.
Elements in the immunoglobulin heavy-chain enhancer directly regulate simian virus 40 ori-dependent DNA replication.
Authors: Ariizumi K , Ghosh M R , Tucker P W
Published: October 2015

In a previous study, we showed that the immunoglobulin heavy-chain (IgH) enhancer (IgHe) is near or in an initiation zone of chromosomal DNA replication, which is preferentially active in B cells (K. Ariizumi, Z. Wang, and P. W. Tucker, Proc. Natl. Acad. Sci. USA 90:3695-3699, 1993). This suggests the existence of a functional relationship between IgHe-mediated transcription and DNA replication. To test this theory, we utilized simian virus 40 (SV40) DNA replication as a model of chromosomal replication. IgHe or its operationally divisible domains (5'-En, core, and 3'-En) were introduced into SV40 minichromosomes (IgHe-SV40). Results of replication assays with IgHe-SV40 replicons indicated that the 5'-En and 3'-En activated or suppressed SV40 DNA replication regardless of the presence of SV40 enhancers or promoters in these replicons. The activity did not reside in IgHe core sequences. The results suggested that the 5'- and 3'-En regulated SV40 replication through direct interaction with the origin, not through suppression at the SV40 enhancer and/or promoter. In an effort to identify elements within the 5'-En motif that contributed to this effect, we found that the E site, but not microE5 and microE2 boxes, upregulated DNA replication. Our results provide another possible regulatory function for the 5'-En and 3'-En domains besides transcriptional suppression of IgHe.
Saccharomyces cerevisiae HSP70 heat shock elements are functionally distinct.
Authors: Young M R , Craig E A
Published: October 2015

The Saccharomyces cerevisiae HSP70 gene SSA1 has multiple heat shock elements (HSEs). To determine the significance of each of these sequences for expression of SSA1, we analyzed expression from a set of promoters containing point mutations in each of the HSEs, individually and in pairwise combinations. Of the three HSE-like sequences, two (HSE2 and HSE3) were active promoter elements; only one, HSE2, was active under basal growth conditions. Either HSE2 or HSE3 alone was able to drive SSA1 transcription at near-normal rates after heat shock. Both HSE2 and HSE3 were capable of driving basal transcription when placed in the context of the CYC1 promoter. Previous analysis had identified an upstream repressing sequence overlapping HSE2 that repressed basal transcription driven by HSE2. Our analysis showed that basal transcription driven by HSE3 was repressed both by the distant upstream repressing sequence and by closer flanking sequences. The ability to drive basal transcription is not inherent in all natural HSEs, since the HSEs from the heat-inducible SSA3 and SSA4 genes showed no basal activity when placed in the CYC1 vector. Gel mobility shift experiments showed that the same population of heat shock transcription factor molecules bound to HSEs capable of driving basal activity and to HSEs having very low or undetectable basal activity.
Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters.
Authors: DesJardins E , Hay N
Published: October 2015

Transcription of the human proto-oncogene c-myc is governed by two tandem principal promoters, termed P1 and P2. In general, the downstream promoter, P2, is predominant, which is in contrast to the promoter occlusion phenomenon usually observed in genes containing tandem promoters. A shift in human c-myc promoter usage has been observed in some tumor cells and in certain physiological conditions. However, the mechanisms that regulate promoter usage are not well understood. The present studies identify regulators which are required to promote transcription from both human c-myc promoters, P1 and P2, and have a role in determining their relative activities in vivo. A novel regulatory region located 101 bp upstream of P1 was characterized and contains five tandem repeats of the consensus sequence CCCTCCCC (CT element). The integrity of the region containing all five elements is required to promote transcription from P1 and for maximal activity from P2 in vivo. A single copy of this same element, designated CT-I2, also appears in an inverted orientation 53 bp upstream of the P2 transcription start site. This element has an inhibitory effect on P1 transcription and is required for P2 transcription. The transcription factor Sp1 was identified as the factor that binds specifically to the tandem CT elements upstream of P1 and to the CT-I2 element upstream of P2. In addition, the recently cloned zinc finger protein ZF87, or MAZ, was also able to bind these same elements in vitro. The five tandem CT elements can be functionally replaced by a heterologous enhancer that only in the absence of CT-I2 reverses the promoter usage, similar to what is observed in the translocated c-myc allele of Burkitt's lymphoma cells.
Saccharomyces cerevisiae BUF protein binds to sequences participating in DNA replication in addition to those mediating transcriptional repression (URS1) and activation.
Authors: Luche R M , Smart W C , Marion T , Tillman M , Sumrada R A , Cooper T G
Published: October 2015

The heteromeric BUF protein was originally shown to bind to URS1 elements which are situated upstream of many genes in Saccharomyces cerevisiae and mediate negative control of their transcription. Among the genes regulated through the URS1 site and the proteins interacting with it are those participating in carbon, nitrogen, and inositol metabolism; electron transport; meiosis; sporulation; and mating-type switching. We show here that pure BUF protein, in addition to binding to the negatively acting URS1 site, also binds to CAR1 sequences supporting transcriptional activation (upstream activation sequences). To determine the BUF protein structure, we cloned and sequenced the BUF1 and BUF2 genes and found them to be identical to the RF-A (RP-A) gene whose products participate in yeast DNA replication as single-stranded DNA binding proteins. These data argue that BUF protein-binding sites serve multiple roles in transcription and replication.
Cell cycle-regulated phosphorylation of the pre-mRNA-binding (heterogeneous nuclear ribonucleoprotein) C proteins.
Authors: Piñol-Roma S , Dreyfuss G
Published: October 2015

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, the structures that contain heterogeneous nuclear RNA and its associated proteins, constitute one of the most abundant components of the eukaryotic nucleus. hnRNPs appear to play important roles in the processing, and possibly also in the transport, of mRNA. hnRNP C proteins (C1, M(r) of 41,000; C2, M(r) of 43,000 [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis]) are among the most abundant pre-mRNA-binding proteins, and they bind tenaciously to sequences relevant to pre-mRNA processing, including the polypyrimidine stretch of introns (when it is uridine rich). C proteins are found in the nucleus during the interphase, but during mitosis they disperse throughout the cell. They have been shown previously to be phosphorylated in vivo, and they can be phosphorylated in vitro by a casein kinase type II. We have identified and partially purified at least two additional C protein kinases. One of these, termed Cs kinase, caused a distinct mobility shift of C proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These phosphorylated C proteins, the Cs proteins, were the prevalent forms of C proteins during mitosis, and Cs kinase activity was also increased in extracts prepared from mitotic cells. Thus, hnRNP C proteins undergo cell cycle-dependent phosphorylation by a cell cycle-regulated protein kinase. Cs kinase activity appears to be distinct from the well-characterized mitosis-specific histone H1 kinase activity. Several additional hnRNP proteins are also phosphorylated during mitosis and are thus also potential substrates for Cs kinase. These novel phosphorylations may be important in regulating the assembly and disassembly of hnRNP complexes and in the function or cellular localization of RNA-binding proteins.
Retention of fibroblast growth factor 3 in the Golgi complex may regulate its export from cells.
Authors: Kiefer P , Peters G , Dickson C
Published: October 2015

The fibroblast growth factors (FGFs) fall into two distinct groups with respect to their mode of release from cells. Whereas FGF1 and FGF2 lack conventional signal peptides, the remaining members have typical features of secreted proteins. However, the behavior of mouse FGF3 is anomalous, since, despite entering the secretory pathway and undergoing primary glycosylation, its release from transfected COS-1 cells is very inefficient compared with that of FGF4 and FGF5. To investigate the unusual properties of FGF3, we analyzed the processing, secretion, and intracellular localization of a series of site-directed mutants as well as chimeras produced by fusing parts of FGF3, FGF4, and FGF5. Wild-type FGF3 was shown to accumulate in an immature form in the Golgi complex, from where it is slowly released into the extracellular matrix. Removing or relocating the Asn-linked glycosylation site further impaired its release, and exchanging the signal peptide or carboxy terminus had little effect. In contrast, a chimeric protein with an amino terminus from FGF5 was efficiently secreted and biologically active in cell transformation assays. The data suggest that a structural feature of FGF3 involving the amino-terminal region and glycosylation site has a significant bearing on its passage through the Golgi complex and may regulate the secretion of the ligand.
Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.
Authors: Wang M M , Tsai R Y , Schrader K A , Reed R R
Published: October 2015

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.
SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase.
Authors: Zheng P , Fay D S , Burton J , Xiao H , Pinkham J L , Stern D F
Published: October 2015

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.
A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1-mediated pathway.
Authors: Lee K S , Hines L K , Levin D E
Published: October 2015

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth. Loss of PKC1 function results in cell lysis due to an inability to remodel the cell wall properly during growth. The PKC1 gene has been proposed to regulate a bifurcated pathway, on one branch of which function four putative protein kinases that catalyze a linear cascade of protein phosphorylation culminating in the activation of the mitogen-activated protein kinase homolog, Mpk1p. Here we describe two genes whose overexpression suppress both an mpk1 delta mutation and a pkc1 delta mutation. One of these genes is identical to the previously identified PPZ2 gene. The PPZ2 gene is predicted to encode a type 1-related protein phosphatase and is functionally redundant with a closely related gene, designated PPZ1. Deletion of both PPZ1 and PPZ2 resulted in a temperature-dependent cell lysis defect similar to that observed for bck1 delta, mkk1,2 delta, or mpk1 delta mutants. However, ppz1,2 delta mpk1 delta triple mutants displayed a cell lysis defect at all temperatures. The additivity of the ppz1,2 delta defect with the mpk1 delta defect, combined with the results of genetic epistasis experiments, suggested either that the PPZ1- and PPZ2-encoded protein phosphatases function on a branch of the PKC1-mediated pathway different from that defined by the protein kinases or that they play an auxiliary role in the pathway. The other suppressor gene, designated BCK2 (for bypass of C kinase), is predicted to encode a 92-kDa protein that is rich in serine and threonine residues. Genetic interactions between BCK2 and other pathway components suggested that BCK2 functions on a common pathway branch with PPZ1 and PPZ2.
Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.
Authors: Pleiman C M , Clark M R , Gauen L K , Winitz S , Coggeshall K M , Johnson G L , Shaw A S , Cambier J C
Published: October 2015

Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.
Elements controlling follicular expression of the s36 chorion gene during Drosophila oogenesis.
Authors: Tolias P P , Konsolaki M , Halfon M S , Stroumbakis N D , Kafatos F C
Published: October 2015

An 84-bp proximal regulatory protein (PRR) of the Drosophila melanogaster s36 chorion gene is sufficient for directing proper temporal and spatial expression of a reporter gene in three domains of the follicle: anterior, posterior, and main body. Here we show that the fidelity of PRR-directed s36 expression is dependent on the proper dorsal-ventral differentiation of the follicular epithelium, which requires the Drosophila epidermal growth factor receptor homolog. Transgenic analysis of site-directed mutants of the PRR suggests that s36 expression is regulated by the concerted action of multiple positive activators. Several cis-acting transcriptional elements have been identified: some appear to function in a quantitative manner, while others either are essential or appear to regulate expression in particular spatial domains. The approximate locations of these regulatory elements have been defined; some map within sequences that are strongly conserved in widely divergent dipteran species. In fact, the PRR analog of the medfly Ceratitis capitata Ccs36 gene directs expression in a manner similar to the D. melanogaster s36 PRR. We propose a model for transcriptional regulation of s36 based on the prechoriogenic polarization of the follicular epithelium that surrounds the developing egg chamber.
Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding.
Authors: Sap J , Jiang Y P , Friedlander D , Grumet M , Schlessinger J
Published: October 2015

Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation.
Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.
Authors: Cobb B S , Schaller M D , Leu T H , Parsons J T
Published: October 2015

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.