bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.
Authors: Shinomiya T , Ina S
Published: October 2015

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.
Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression.
Authors: Jhun B H , Rose D W , Seely B L , Rameh L , Cantley L , Saltiel A R , Olefsky J M
Published: October 2015

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.
ralGDS family members interact with the effector loop of ras p21.
Authors: Kikuchi A , Demo S D , Ye Z H , Chen Y W , Williams L T
Published: October 2015

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.
The GATA-4 transcription factor transactivates the cardiac muscle-specific troponin C promoter-enhancer in nonmuscle cells.
Authors: Ip H S , Wilson D B , Heikinheimo M , Tang Z , Ting C N , Simon M C , Leiden J M , Parmacek M S
Published: October 2015

The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac muscle-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Although previous reports have identified several cardiac muscle-specific transcriptional elements, relatively little is known about the lineage-specific transcription factors that regulate these elements. In this report, we demonstrate that the slow/cardiac muscle-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted zinc finger transcription factor GATA-4. This GATA-4-binding site is required for enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by overexpression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. In situ hybridization studies demonstrate that GATA-4 and cTnC have overlapping patterns of expression in the hearts of postimplantation mouse embryos and that GATA-4 gene expression precedes cTnC expression. Indirect immunofluorescence reveals GATA-4 expression in cultured cardiac myocytes from neonatal rats. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.
Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22.
Authors: Ono Y , Ohno M , Shimura Y
Published: October 2015

In the budding yeast Saccharomyces cerevisiae, a number of PRP genes known to be involved in pre-mRNA processing have been genetically identified and cloned. Three PRP genes (PRP2, PRP16, and PRP22) were shown to encode putative RNA helicases of the family of proteins with DEAH boxes. However, any such splicing factor containing the helicase motifs in vertebrates has not been identified. To identify human homologs of this family, we designed PCR primers corresponding to the highly conserved region of the DEAH box protein family and successfully amplified five cDNA fragments, using HeLa poly(A)+ RNA as a substrate. One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which was previously shown to be involved in the release of spliced mRNAs from the spliceosomes. Expression of HRH1 in a S. cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype. These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein. Interestingly, HRH1 but not Prp22 contains an arginine- and serine-rich domain (RS domain) which is characteristic of some splicing factors, such as members of the SR protein family. We could show that HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain. We speculate that HRH1 might be targeted to the spliceosome through this interaction.
Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function.
Authors: Theis J F , Newlon C S
Published: October 2015

ARS307 is highly active as a replication origin in its native location on chromosome III of Saccharomyces cerevisiae. Its ability to confer autonomous replication activity on plasmids requires the presence of an 11-bp autonomously replicating sequence (ARS) consensus sequence (ACS), which is also required for chromosomal origin function, as well as approximately 100 bp of sequence flanking the ACS called domain B. To further define the sequences required for ARS function, a linker substitution mutagenesis of domain B was carried out. The mutations defined two sequences, B1 and B2, that contribute to ARS activity. Therefore, like ARS1, domain B of ARS307 is composed of functional subdomains. Constructs carrying mutations in the B1 element were used to replace the chromosomal copy of ARS307. These mutations caused a reduction in chromosomal origin activity, demonstrating that the B1 element is required for efficient chromosomal origin function.
The basic helix-loop-helix-zipper domain of TFE3 mediates enhancer-promoter interaction.
Authors: Artandi S E , Cooper C , Shrivastava A , Calame K
Published: October 2015

Binding sites for three families of sequence-specific DNA-binding proteins, microE3, C/EBP, and OCT, are found in both the promoters and the intronic enhancer of the immunoglobulin heavy-chain gene. We have used a cotransfection system to investigate how proteins binding these sites may participate in enhancer-promoter interactions. Basic helix-loop-helix-zipper (BHLHZIP) proteins TFE3 and TFEB activate from a distance in this assay, but the basic zipper (BZIP) protein NF-IL6 and endogenous OCT-binding proteins do not. Our results suggest that remotely bound TFE3 is recruited to the initiation site by association with proximally bound TFE3; this interaction is mediated by the BHLHZIP domain and not by activation domains of TFE3. The BZIP domain of Ig/EBP lacks this activity, revealing an important functional difference between these structurally related dimerization domains. We also show that TFE3 can exist as a tetramer in solution and that tetramerization is determined by the HLHZIP domain. These data support a model in which protein-protein interactions between proximally and remotely bound TFE3 recruit TFE3 to the initiation site for activation. The IgH gene is the first example of a cellular gene in which proximal and distal binding sites are found for a protein capable of mediating enhancer-promoter interaction.
Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification.
Authors: Cohen S , Hassin D , Karby S , Lavi S
Published: October 2015

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.
Replication factor A is required in vivo for DNA replication, repair, and recombination.
Authors: Longhese M P , Plevani P , Lucchini G
Published: October 2015

Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.
Regulated immunoglobulin (Ig) RNA processing does not require specific cis-acting sequences: non-Ig RNA can be alternatively processed in B cells and plasma cells.
Authors: Peterson M L
Published: October 2015

Alternative RNA processing of the heavy-chain immunoglobulin mu gene is regulated during B-cell maturation and requires competition between splice and cleavage-polyadenylation reactions that have balanced efficiencies. Studies with modified mu genes have failed to identify gene-specific sequences required for regulation. Thus, the only important feature for regulation may be the balanced competing splice and cleavage-polyadenylation reactions themselves. If this is so, then alternative RNA processing from any gene with similar competitive RNA processing pathways should also be regulated when expression is compared between B cells and plasma cells. To test this prediction, two nonimmunoglobulin genes engineered to have competing splice and cleavage-polyadenylation reactions were expressed in B cells and plasma cells. The ratios of alternative RNAs produced from both genes are different in the two cell types; like the mu gene, relatively more spliced RNA is produced in B cells than in plasma cells. Also, in a survey of mu gene expression in nine non-B-cell lines, only a T-cell line had an expression pattern similar to that of B cells; the expression patterns of all other lines resembled that of the plasma cells. Therefore, regulated mu RNA processing must be mediated by changes in general processing factors whose activity or abundance is regulated, most likely, in B cells.
Functional and conserved domains of the Drosophila transcription factor encoded by the segmentation gene knirps.
Authors: Gerwin N , La Rosée A , Sauer F , Halbritter H P , Neumann M , Jäckle H , Nauber U
Published: October 2015

The Drosophila gap gene knirps (kni) is required for abdominal segmentation. It encodes a steroid/thyroid orphan receptor-type transcription factor which is distributed in a broad band of nuclei in the posterior region of the blastoderm. To identify essential domains of the kni protein (KNI), we cloned and sequenced the DNA encompassing the coding region of nine kni mutant alleles of different strength and kni-homologous genes of related insect species. We also examined in vitro-modified versions of KNI in various assay systems both in vitro and in tissue culture. The results show that KNI contains several functional domains which are arranged in a modular fashion. The N-terminal 185-amino-acid region which includes the DNA-binding domain and a functional nuclear location signal fails to provide kni activity to the embryo. However, a truncated KNI protein that contains additional 47 amino acids exerts rather strong kni activity which is functionally defined by a weak kni mutant phenotype of the embryo. The additional 47-amino-acid stretch includes a transcriptional repressor domain which acts in the context of a heterologous DNA-binding domain of the yeast transcriptional activator GAL4. The different domains of KNI as defined by functional studies are conserved during insect evolution.
A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae.
Authors: Fox M E , Feldman B J , Chu G
Published: October 2015

DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.
Erythroid cell-specific determinants of alpha-globin mRNA stability.
Authors: Weiss I M , Liebhaber S A
Published: October 2015

Although globin mRNAs are considered prototypes of highly stable messages, the mechanisms responsible for their longevity remain largely undefined. As an initial step in identifying potential cis-acting elements or structures which contribute to their stability, we analyzed the defect in expression of a naturally occurring alpha 2-globin mutant, alpha Constant Spring (CS). The CS mutation is a single-base change in the translation termination codon (UAA-->CAA) that allows the ribosome to read through into the 3' nontranslated region (NTR). The presence of CS mRNA in transcriptionally active erythroid precursors and its absence (relative to normal alpha-globin mRNA) in the more differentiated transcriptionally silent erythrocytes suggest that this mutation disrupts some feature of the alpha-globin mRNA required for its stability. Using a transient transfection system, we demonstrate that in murine erythroleukemia cells the CS mRNA is unstable compared with the normal alpha 2-globin mRNA. The analyses of several other naturally occurring and site-directed mutant alpha-globin genes in murine erythroleukemia cells indicate that entry of a translating ribosome into the 3' NTR targets the message for accelerated degradation in erythroid cells. In contrast, both the CS and alpha 2-globin mRNAs are stable in several nonerythroid cell lines. These results suggest that translational readthrough disrupts a determinant associated with the alpha 2-globin 3' NTR which is required for mRNA stability in erythroid cells.
Direct binding to and tyrosine phosphorylation of the alpha subunit of the type I interferon receptor by p135tyk2 tyrosine kinase.
Authors: Colamonici O , Yan H , Domanski P , Handa R , Smalley D , Mullersman J , Witte M , Krishnan K , Krolewski J
Published: October 2015

Binding of type I interferons (IFNs) to their receptors induces rapid tyrosine phosphorylation of multiple proteins, including the alpha and beta subunits of the receptor, the polypeptides that form the transcriptional activator ISGF3 alpha (Stat113, Stat84, and Stat91), and the p135tyk2 and Jak-1 tyrosine kinases. In this report, we demonstrate that the alpha subunit of the type I IFN receptor (IFN-R) corresponds to the product of a previously cloned receptor subunit cDNA and, further, that the p135tyk2 tyrosine kinase directly binds and tyrosine phosphorylates this receptor subunit. Glutathione S-transferase (GST) fusion proteins encoding the different regions of the cytoplasmic domain of the alpha subunit can bind the p135tyk2 contained in human cell lysates. The association between the alpha subunit and Tyk2 was demonstrated by immunoblotting with anti-Tyk2 and antiphosphotyrosine antibodies and by using an in vitro kinase assay. Analogous experiments were then performed with recombinant baculoviruses encoding constitutively active Jak family tyrosine kinases. In this case, p135tyk2, but not Jak-1 or Jak-2 protein, binds to the GST-IFN-R proteins, suggesting that the interaction between these two proteins is both direct and specific. We also demonstrate that Tyk2, from extracts of either IFN alpha-treated human cells or insect cells infected with the recombinant baculoviruses, can catalyze in vitro phosphorylation of GST-IFN-R protein in a specific manner. Deletion mutants of the GST-IFN-R protein were used to localize both the binding and tyrosine phosphorylation site(s) to a 46-amino-acid juxtamembrane region of the alpha subunit, which shows sequence homology to functionally similar regions of other cytokine receptor proteins. These data support the hypothesis that the Tyk2 protein functions as part of a receptor complex to initiate intracellular signaling in response to type I IFNs.
Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements.
Authors: Chen C Y , Shyu A B
Published: October 2015

The metabolic lifetime of mRNA can be specified by specific cis-acting elements within mRNA. One type of element is an adenylate- and uridylate-rich element (ARE) found in the 3' untranslated region of many highly unstable mRNAs for mammalian early-response genes (ERGs). Among the better-characterized members of the ERG family are certain genes encoding nuclear transcription factors. Of particular significance was the finding that their mRNAs decay rapidly with kinetics similar to those of c-fos mRNA. Our previous studies of the c-fos ARE-directed mRNA decay have revealed the existence in this ARE of two structurally distinct and functionally interdependent domains, termed domain I and domain II. We proposed that the c-fos ARE-directed decay is a two-step mechanism in which rapid shortening of the poly(A) tail leads to the decay of the mRNA body and further hypothesized that this is a general mechanism by which the ERG AREs mediate rapid mRNA degradation. To test this hypothesis and to further address the generality of the critical structural characteristics within the c-fos ARE, the RNA-destabilizing functions of more than 10 different AU-rich sequences from various nuclear transcription factor mRNAs have been tested. Consistent with the above-mentioned hypothesis is the observation that mRNAs carrying the functional AREs display a biphasic decay, which is characteristic of the proposed two-step mechanism. Our results indicated that the presence of AUUUA pentanucleotides in an AU-rich region does not always guarantee an RNA-destabilizing function for this region. Our results also led to the identification of a novel class of AU-rich destabilizing elements which contains no AUUUA pentanucleotide. The results of sequence comparison and functional tests revealed that a continuous U-rich sequence is a unique feature among the functional AREs. Finally, our experiments further showed that the c-fos ARE domain II has an RNA decay-enhancing ability upon its fusion to heterologous AU-rich regions and defined for the first time an RNA decay-enhancing element, which we termed the RDE element.
Interactions among three proteins that specifically activate translation of the mitochondrial COX3 mRNA in Saccharomyces cerevisiae.
Authors: Brown N G , Costanzo M C , Fox T D
Published: October 2015

The PET54, PET122, and PET494 proteins, which are associated with the yeast inner mitochondrial membrane, specifically activate translation of the mitochondrially encoded COX3 mRNA. We used the two-hybrid system to test whether pairs of these proteins, when fused to either the GAL4 DNA-binding or transcriptional activating domain, can physically associate as measured by the expression of the GAL4-dependent reporter, lacZ. PET54 and PET122 interacted in this system, and an amino-terminally truncated PET494 fragment showed an interaction with PET54. We also detected functional interactions between PET54 and PET122 genetically: a pet54 missense substitution (Phe to Gly at position 244) that caused a severe respiratory defect was suppressed both by a missense substitution affecting PET122 (Gly to Val at position 211) and by overproduction of wild-type PET122. Both Gly and Ala, substituted at PET54 position 244, disrupted the two-hybrid interactions with PET122 and PET494. While Ala at PET54 position 244 caused only a modest respiratory phenotype alone, it caused a severe respiratory defect when combined with a cold-sensitive mitochondrial mutation affecting the COX3 mRNA 5' leader. This synthetic defect was suppressed by a missense substitution in PET122 and by overproduction of wild-type PET122, indicating functional interactions among PET54, PET122, and the mRNA. Taken together with previous work, these data suggest that a complex containing PET54, PET122, and PET494 mediates the interaction of the COX3 mRNA with mitochondrial ribosomes at the surface of the inner membrane.
A factor induced by differentiation signals in cells of the macrophage lineage binds to the gamma interferon activation site.
Authors: Eilers A , Baccarini M , Horn F , Hipskind R A , Schindler C , Decker T
Published: October 2015

Rapid transcriptional induction of genes in response to gamma interferon (IFN-gamma) is mediated by the IFN-gamma activation site (GAS) and its cognate protein, the IFN-gamma activation factor (GAF). We describe a GAS-associated, differentiation-induced factor (DIF) as a potential molecular link between the activities of IFN-gamma and of growth and differentiation factors. DIF DNA binding was activated by colony-stimulating factor 1 in murine macrophages and also during tetradecanoyl phorbol acetate-induced differentiation or IFN-gamma treatment in myeloid U937 cells. IFN-gamma activation of DIF decreased significantly upon monocytic differentiation. DIF binding to DNA was inhibited by antiphosphotyrosine antibodies and could be induced by treatment of U937 cells with vanadate. Unlike GAF, DIF-DNA complexes did not contain the 91-kDa protein (p91) from ISGF-3. DIF bound with high affinity to GAS from the promoters of the IFP 53/tryptophanyl-tRNA synthetase and Fc gamma RI genes, intermediate affinity to the Ly6A/E GAS, and low affinity to the guanylate-binding protein GAS. DIF may belong to a family of cytokine- or growth factor-induced factors binding with variable affinities to GAS-related elements: the interleukin-6-responsive acute-phase response factor associated with GAS from different IFN-inducible promoters but with a different preference of binding compared with DIF. The sis-inducible element of the c-fos promoter bound GAF but not DIF. However, the sis-inducible element could be changed by point mutation to compete for GAF and DIF binding. Our data show DIF to be a novel DNA-binding protein which is activated in response to differentiating signals. Moreover, they suggest that a family of cytokine- or growth factor-regulated proteins integrates and coordinates the responses to cytokines and to growth and differentiation factors by binding to GAS-related elements.
Structure and regulation of the human interferon regulatory factor 1 (IRF-1) and IRF-2 genes: implications for a gene network in the interferon system.
Authors: Harada H , Takahashi E , Itoh S , Harada K , Hori T A , Taniguchi T
Published: October 2015

Interferon regulatory factor 1 (IRF-1) and IRF-2 are structurally similar DNA-binding factors which were originally identified as regulators of the type I interferon (IFN) system; the former functions as a transcriptional activator, and the latter represses IRF-1 function by competing for the same cis elements. More recent studies have revealed new roles of the two factors in the regulation of cell growth; IRF-1 and IRF-2 manifest antioncogenic and oncogenic activities, respectively. In this study, we determined the structures and chromosomal locations of the human IRF-1 and IRF-2 genes and further characterized the promoters of the respective genes. Comparison of exon-intron organization of the two genes revealed a common evolutionary structure, notably within the exons encoding the N-terminal portions of the two factors. We confirmed the chromosomal mapping of the human IRF-1 gene to 5q31.1 and newly assigned the IRF-2 gene to 4q35.1, using fluorescence in situ hybridization. The 5' regulatory regions of both genes contain highly GC-rich sequences and consensus binding sequences for several known transcription factors, including NF-kappa B. Interestingly, one IRF binding site was found within the IRF-2 promoter, and expression of the IRF-2 gene was affected by both transient and stable IRF-1 expression. In addition, one potential IFN-gamma-activated sequence was found within the IRF-1 promoter. Thus, these results may shed light on the complex gene network involved in regulation of the IFN system.
Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells.
Authors: Klingelhutz A J , Barber S A , Smith P P , Dyer K , McDougall J K
Published: October 2015

Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.
Identification of the functional components of the Ras signaling pathway regulating pituitary cell-specific gene expression.
Authors: Conrad K E , Oberwetter J M , Vaillancourt R , Johnson G L , Gutierrez-Hartmann A
Published: October 2015

Ras, a small GTP-binding protein, is required for functional receptor tyrosine kinase signaling. Ultimately, Ras alters the activity of specific nuclear transcription factors and regulates novel patterns of gene expression. Using a rat prolactin promoter construct in transient transfection experiments, we show that both oncogenic Ras and activated forms of Raf-1 kinase selectively stimulated the cellular rat prolactin promoter in GH4 rat pituitary cells. We also show that the Ras signal is completely blocked by an expression vector encoding a dominant-negative Raf kinase. Additionally, using a molecular genetic approach, we determined that inhibitory forms of p42 mitogen-activated protein kinase and an Ets-2 transcription factor interfere with both the Ras and the Raf activation of the rat prolactin promoter. These findings define a functional requirement for these signaling constituents in the activation of the prolactin gene, a cell-specific gene which marks the lactotroph pituitary cell type. Further, this analysis allowed us to order the components in the Ras signaling pathway as it impinges on regulation of prolactin gene transcription as Ras-->Raf kinase-->mitogen-activated protein kinase-->Ets. In contrast, we show that intact c-Jun expression inhibited the Ras-induced activation of the prolactin promoter, defining it as a negative regulator of this pathway, whereas c-Jun was able to enhance the Ras activation of an AP-1-driven promoter in GH4 cells. These data show that c-Jun is not the nuclear mediator of the Ras signal for the highly specialized, pituitary cell-specific prolactin cellular promoter. Thus, we have defined a model system which provides an ideal paradigm for studying Ras/Raf signaling pathways and their effects on neuroendocrine cell-specific gene regulation.
Normal peripheral T-cell function in c-Fos-deficient mice.
Authors: Jain J , Nalefski E A , McCaffrey P G , Johnson R S , Spiegelman B M , Papaioannou V , Rao A
Published: October 2015

The ubiquitous transcription factors Fos and Jun are rapidly induced in T cells stimulated through the T-cell antigen receptor and regulate transcription of cytokines, including interleukin 2, in activated T cells. Since positive and negative selection of thymocytes during T-cell development also depends on activation through the T-cell receptor, Fos and Jun may play a role in thymocyte development as well. Fos and Jun act at several regulatory elements in the interleukin 2 promoter, including the AP-1 and NFAT sites. Using antisera specific to individual Fos and Jun family members, we show that c-Fos as well as other Fos family members are present in the inducible AP-1 and NFAT complexes of activated murine T cells. Nevertheless, c-Fos is not absolutely required for the development or function of peripheral T cells, as shown by using mice in which both copies of the c-fos gene were disrupted by targeted mutagenesis. c-Fos-deficient mice were comparable to wild-type mice in their patterns of thymocyte development and in the ability of their peripheral T cells to proliferate and produce several cytokines in response to T-cell receptor stimulation. Our results suggest that other Fos family members may be capable of substituting functionally for c-Fos during T-cell development and cytokine gene transcription in activated T cells.
Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system.
Authors: Resnitzky D , Gossen M , Bujard H , Reed S I
Published: October 2015

Conditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor. After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition. We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts. We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity. Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence. However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition. Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E. These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence.
Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.
Authors: Schaller M D , Hildebrand J D , Shannon J D , Fox J W , Vines R R , Parsons J T
Published: October 2015

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.
Retroviral insertions in the murine His-1 locus activate the expression of a novel RNA that lacks an extensive open reading frame.
Authors: Askew D S , Li J , Ihle J N
Published: October 2015

The His-1 locus is a common site of viral insertion in murine myeloid leukemias induced by the wild mouse ecotropic retrovirus, CasBrM. In this report, we describe the cloning of a novel gene at the His-1 locus and show that His-1 expression is associated with the transformed phenotype. Northern (RNA) blot analysis identified His-1 transcripts in four transformed myeloid cell lines but in no normal tissues examined. Two of these cell lines were derived from retrovirus-induced myeloid leukemias that harbor integrated proviruses which drive His-1 gene expression by promoter insertion. The two other cell lines expressed a discrete 3-kb His-1 RNA that is derived from a novel gene consisting of three exons that span 6 kb on mouse chromosome 2. The His-1 gene is conserved as a single-copy sequence in multiple vertebrate species and is expressed as a spliced and polyadenylated RNA. A protein-coding region is not evident from analysis of the His-1 sequence because of the presence of multiple small open reading frames, none of which are greater than 219 bp. This lack of an extensive open reading frame is an unusual feature that is shared by other RNA molecules believed to function in the absence of translation.
A single element of the elastase I enhancer is sufficient to direct transcription selectively to the pancreas and gut.
Authors: Rose S D , Kruse F , Swift G H , MacDonald R J , Hammer R E
Published: October 2015

The elastase I (EI) gene is expressed at high levels in the exocrine pancreas and at lower levels in other regions of the gut. The transcriptional enhancer of the EI gene, from nucleotides -205 to -72, recapitulates the expression of the endogenous gene in transgenic mice; it directs not only pancreatic acinar cell expression of a human growth hormone (hGH) transgene but also expression to the stomach, duodenum, and colon. This pattern of selective expression limited to the gastroenteropancreatic organ system is specified by the A element, one of three functional elements in the EI enhancer. When multimerized, the A element directed expression of a hGH reporter gene selectively to the pancreas, stomach, and intestine in transgenic mice. Immunofluorescent localization of hGH indicated that the A element multimer transgenes were expressed in the acinar cells of the pancreas as well as in Brunner's gland cells of the duodenum. The A element binds a pancreatic acinar cell-specific factor, PTF1. Our results show that while the A element is responsible for directing tissue and cell type specificity, other elements of the enhancer must be involved in the regulation of the level of gene expression.
Sap1, a protein that binds to sequences required for mating-type switching, is essential for viability in Schizosaccharomyces pombe.
Authors: Arcangioli B , Copeland T D , Klar A J
Published: October 2015

The pattern of mating-type switching in cell pedigrees of the fission yeast Schizosaccharomyces pombe is dictated by the inheritance of specific DNA chains at the mating-type locus (mat1). The recombination event essential for switching is initiated by a site-specific double-strand break at mat1. The switch-activating protein, Sap1, binds in vitro to a mat1 cis-acting site that was shown earlier to be essential for efficient mating-type switching. We isolated the sap1 gene by using oligonucleotides corresponding to the amino acid sequence of purified Sap1 protein. The sequence of that gene predicted a 30-kDa protein with no significant homology to other canonical DNA-binding protein motifs. To facilitate its biochemical characterization, Sap1 was expressed in Escherichia coli. The protein expressed in bacteria displayed the same DNA-binding specificities as the protein purified from S. pombe. Interestingly, analysis of a sap1 null mutation showed that the gene is essential for growth even in a strain in which mating-type switching is prohibited because of a defect in generation of the double-strand break. Thus, the sap1 gene product implicated in mating-type switching is shown to be essential for cell viability.
Large exon size does not limit splicing in vivo.
Authors: Chen I T , Chasin L A
Published: October 2015

Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.
Biological activity of the growth factor-induced cytokine N51: structure-function analysis using N51/Interleukin-8 chimeric molecules.
Authors: Heinrich J N , O'Rourke E C , Chen L , Gray H , Dorfman K S , Bravo R
Published: October 2015

The immediate-early gene N51/KC encodes a protein which following expression in the baculovirus system and purification to apparent homogeneity is able to induce chemotaxis and intracellular Ca2+ flux, to compete for 125I-labeled interleukin-8 (IL-8) binding, and upon iodination, to bind specifically to human neutrophils. The activity of N51/KC can be distinguished from that of IL-8 by a number of criteria. First, at equivalent concentrations, the specific binding of [125I]N51/KC to human neutrophils is about 10 times less than that of [125I]IL-8. Second, the competition studies of [125I]IL-8 with IL-8 define a single class of high-affinity receptors, while the presence of both a high- and a low-affinity class of receptors is defined by N51/KC. Third, although the changes in intracellular Ca2+ of fura-2/AM-preloaded human neutrophils elicited by N51/KC and IL-8 are similar, pretreatment of the cells with N51/KC did not result in a loss of response to a subsequent treatment with IL-8; in contrast, treatment with IL-8 did result in the subsequent desensitization to N51/KC. To further characterize N51/KC, mutants and hybrids of N51/KC and IL-8 were produced and analyzed for the ability to compete for [125I]IL-8 binding and elicit intracellular Ca2+ changes in human neutrophils. Two important observations came from these studies. First, the N51/IL-8I hybrid in which the N51/KC sequence between cysteines 2 and 3 (or first disulfide bond) is replaced by the corresponding sequence in IL-8 shows IL-8-like properties, indicating that this region is important for specific receptor recognition. Second, the N51 delta III and IL-8 delta III C-terminus deletion mutants were biologically inactive, but the hybrid molecules N51/IL-8III and IL-8/N51III, in which the C termini were exchanged, had biological activities similar to that of the wild-type molecules, demonstrating that the presence of the C terminus is essential for the biological activity of these chemokines but does not confer receptor specificity.
Poly(A) polymerase contains multiple functional domains.
Authors: Raabe T , Murthy K G , Manley J L
Published: October 2015

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.
MOT2 encodes a negative regulator of gene expression that affects basal expression of pheromone-responsive genes in Saccharomyces cerevisiae.
Authors: Cade R M , Errede B
Published: October 2015

Pheromones induce haploid cells of Saccharomyces cerevisiae to differentiate into a mating-competent state. Ste11p is one of several protein kinases required to transmit the pheromone-induced signal and to maintain basal expression of certain mating-specific genes in the absence of pheromone stimulation. To identify potential regulators of Ste11p, we screened for suppressors that restored mating and basal transcriptional competence to a strain with a conditionally functional Ste11p. This screen uncovered a novel gene we call MOT2, for modulator of transcription. A mot2 deletion mutation leads to modest increases in the basal amounts of mRNA for several pheromone-responsive genes. Yet mot2 deletion does not affect the signal transmission activity of the pathway in either the presence or absence of pheromone stimulation. Therefore, we propose that Mot2p, directly or indirectly, represses basal transcription of certain mating-specific genes. Because mot2 deletion mutants also have a conditional cell lysis phenotype, we expect that Mot2p regulatory effects may be more global than for mating-specific gene expression.