bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of the Pla Protease Substrate α2-Antiplasmin on the Progression of Primary Pneumonic Plague
Authors: Eddy Justin L. , Schroeder Jay A. , Zimbler Daniel L. , Bellows Lauren E. , Lathem Wyndham W.
Published: October 2015

Many pathogens usurp the host hemostatic system during infection to promote pathogenesis.
Yersinia pestis
, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown
in vitro
to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor α2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP
in vivo
; the role of A2AP during respiratory
Y. pestis
infection is not known either. Here, we show that
Y. pestis
does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with
Y. pestis
, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection.
Airpnp: Auto- and Integrated Regulation of Polynucleotide Phosphorylase
Authors: Condon Ciarán
Published: October 2015

The properties and expression of polynucleotide phosphorylase (PNPase), capable of both RNA degradation and polymerization, have been studied for 60 years. In this issue of the
Journal of Bacteriology
,
Park et al.
(H. Park, H. Yakhnin, M. Connolly, T. Romeo, and P. Babitzke, J Bacteriol 197:3751–3759, 2015,
http://dx.doi.org/10.1128/JB.00721-15
) write the latest chapter on the complex regulation of
pnp
gene expression involving CsrA. I describe how this new piece of the puzzle fits into the global scheme of PNPase autoregulation and how this is influenced by central carbon metabolism at both the posttranscriptional level and that of enzyme activity.
Deletion or substitution within the alpha platelet-derived growth factor receptor kinase insert domain: effects on functional coupling with intracellular signaling pathways.
Authors: Heidaran M A , Pierce J H , Lombardi D , Ruggiero M , Gutkind J S , Matsui T , Aaronson S A
Published: October 2015

The tyrosine kinase domains of the platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1)/c-fms receptors are interrupted by kinase inserts (ki) which vary in length and amino acid sequence. To define the role of the ki in the human alpha PDGF receptor (alpha PDGFR), we generated deletion mutants, designated alpha R delta ki-1 and alpha R delta ki-2, which lacked 80 (710 to 789) and 95 (695 to 789) amino acids of the 104-amino-acid ki region, respectively. Their functional characteristics were compared with those of the wild-type alpha PDGFR following introduction into a naive hematopoietic cell line, 32D. Biochemical responses, including PDGF-stimulated PDGFR tyrosine phosphorylation, phosphatidylinositol (PI) turnover, and receptor-associated PI-3 kinase activity, were differentially impaired by the deletions. Despite a lack of any detectable receptor-associated PI-3 kinase activity, 32D cells expressing alpha R delta ki-1 showed only partially impaired chemotactic and mitogenic responses and were capable of sustained proliferation in vitro and in vivo under conditions of autocrine stimulation by the c-sis product. 32D transfectants expressing the larger ki deletion (alpha R delta ki-2) showed markedly decreased or abolished biochemical and biological responses. However, insertion of the highly unrelated smaller c-fms (685 to 750) ki domain into alpha R delta ki-2 restored each of these activities to wild-type alpha PDGFR levels. Since the CSF-1R does not normally induce PI turnover, the ability of the c-fms ki domain to reconstitute PI turnover in the alpha R delta ki-2 transfectant provides evidence that the ki domain of the alpha PDGFR does not directly couple with this pathway. Taken together, all od these bindings imply that their ki domains have evolved to play very similar roles in the known signaling functions PDGF and CSF-1 receptors.
Positive regulation of jun/AP-1 by E1A.
Authors: de Groot R , Foulkes N , Mulder M , Kruijer W , Sassone-Corsi P
Published: October 2015

Proteins encoded by the adenovirus E1A oncogene are capable of positive and negative transcriptional regulation of both viral and cellular genes. E1A regulatory function is commonly thought to involve modifications of specific cellular factors that interact with responsive promoters. In this report we present evidence that E1A induces the activity of the jun/AP-1 transcription factor in three different cell types: P19, JEG-3, and HeLa. AP-1 binds to 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs); therefore, E1A might modulate a specific signal transduction pathway normally induced by activation of the protein kinase C. Binding of jun/AP-1 to a TRE is induced in all cell types studied when E1A is expressed. We observe that the expression of endogenous c-jun and jun B genes is induced by E1A, which directly transactivates the promoters of c-fos, c-jun, and jun B. Similar inducibility is obtained by treatment with retinoic acid and differentiation of P19-embryonal carcinoma cells. The E1A 13S product transactivates TRE sequences and cooperates with c-jun in the transcriptional stimulation. The 12S E1A product does not activate a TRE sequence, but cotransfection with c-jun circumvents this lack of stimulation. Coexpression of c-fos and E1A 12S, however, blocks the transactivation by c-jun, suggesting an important role for fos in determining the dominance of the 12S or 13S protein.
The yeast rad18 mutator specifically increases G.C----T.A transversions without reducing correction of G-A or C-T mismatches to G.C pairs.
Authors: Kunz B A , Kang X L , Kohalmi L
Published: October 2015

Inactivation of the Saccharomyces cerevisiae RAD18 gene confers a mutator phenotype. To determine the specificity of this effect, a collection of 212 spontaneous SUP4-o mutants arising in a rad18 strain was characterized by DNA sequencing. Comparison of the resulting mutational spectrum with that for an isogenic wild-type (RAD18) strain revealed that the rad18 mutator specifically enhanced the frequency of single base pair substitutions. Further analysis indicated that an increase in the frequency of G.C----T.A transversions accounted for the elevated SUP4-o mutation frequency. Thus, rad18 is the first eucaryotic mutator found to generate only a particular base pair substitution. The majority of G.C pairs that were not mutated in the rad18 background were at sites where G.C----T.A events can be detected in SUP4-o, suggesting that DNA sequence context influences the rad18 mutator effect. Transformation of heteroduplex plasmid DNAs into the two strains demonstrated that the rad18 mutator did not reduce the efficiency of correcting G-A or C-T mismatches to G.C pairs or preferentially correct the mismatches to A.T pairs. We propose that the RAD18 gene product might contribute to the fidelity of DNA replication in S. cerevisiae by involvement in a process that serves to limit the formation of G-A and C-T mismatches at template guanine and cytosine sites during DNA synthesis.
A gene related to the proto-oncogene fps/fes is expressed at diverse times during the life cycle of Drosophila melanogaster.
Authors: Katzen A L , Montarras D , Jackson J , Paulson R F , Kornberg T , Bishop J M
Published: October 2015

The proto-oncogene fps/fes encodes a distinctive type of protein-tyrosine kinase. We identified a Drosophila gene (dfps85D) whose product resembles the proteins encoded by vertebrate fps/fes and the closely related gene fer. dfps85D is located at chromosomal position 85D10-13 and is unlikely to correspond to any previously defined genetic locus in Drosophila melanogaster. Expression of the gene is entirely zygotic in origin and occurs throughout the life cycle. But hybridization in situ revealed that the pattern of expression is specialized and evolves in a provocative manner. The most notable feature of expression is the diversity of developmental periods, tissues, and cells in which it occurs. In some tissues, expression is transient; in others, it is continuous. Expression occurs in both mitotic and terminally differentiated tissue and, at various times in development, is prominent in imaginal disks, gut, muscle, testes, ovaries, retina, and other neural tissues. It appears that the use of dfps85D is more diversified than that of other Drosophila protein-tyrosine kinases reported to date and contrasts sharply with the restricted expression of fps itself in vertebrates. The detailed description of expression provided here will help guide the search for mutants in dfps85D.
Structure and regulation of histone H2B mRNAs from Leishmania enriettii.
Authors: Genske J E , Cairns B R , Stack S P , Landfear S M
Published: October 2015

We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti. A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence. We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast. In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes. Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis. The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).
Maintenance of NF-kappa B activity is dependent on protein synthesis and the continuous presence of external stimuli.
Authors: Hohmann H P , Remy R , Scheidereit C , van Loon A P
Published: October 2015

The activation of NF-kappa B-like activities (called NF-kappa B) by tumor necrosis factor alpha (TNF alpha) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) were compared. High levels of NF-kappa B activity were found 2 to 4 min after TNF alpha addition to human HL60 cells and lasted for at least 3 h, although the half-life of active NF-kappa B was less than 30 min. Inactive NF-kappa B, however, was relatively stable. NF-kappa B activation by TNF alpha was initially cycloheximide insensitive, but maintenance of NF-kappa B activity required ongoing protein synthesis and continuous stimulation by TNF alpha. Thus, the cells did not remain in an activated state without stimulation. In HL60 cells, NF-kappa B induction by PMA required 30 to 45 min and was completely dependent on de novo protein synthesis, while PMA (and interleukin-1) induced NF-kappa B activity rapidly in mouse 70Z/3 cells via a protein synthesis-independent mechanism. The NF-kappa B-like activities obtained under each condition behaved identically in methylation interference and native proteolytic fingerprinting assays. The NF-kappa B-like factors induced are thus all very similar or identical. We suggest that cell-specific differences in the protein kinase C-dependent activation of NF-kappa B may exist and that TNF alpha and PMA may induce expression of the gene(s) encoding NF-kappa B.
Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors.
Authors: Lin H , Yutzey K E , Konieczny S F
Published: October 2015

The quail fast skeletal troponin I (TnI) gene is a member of the contractile protein gene set and is expressed exclusively in differentiated skeletal muscle cells. TnI gene transcription is controlled by an internal regulatory element (IRE), located within the first intron, that functions as a muscle-specific enhancer. Recent studies have shown that the TnI IRE may interact directly with the muscle regulatory factors MyoD, myogenin, and Myf-5 to produce a muscle-specific expression pattern, since these factors trans-activate cotransfected TnI gene constructs in C3H10T1/2 fibroblasts. In this study, we have examined the protein-IRE interactions that are responsible for transcriptionally activating the TnI gene during skeletal muscle development. We demonstrate that the helix-loop-helix muscle regulatory factors MyoD, myogenin, Myf-5, and MRF4, when complexed with the immunoglobulin enhancer-binding protein E12, interact with identical nucleotides within a muscle regulatory factor-binding site (MRF site) located in the TnI IRE. The nuclear proteins that bind to the MRF site are restricted to skeletal muscle cells, since protein extracts from HeLa, L, and C3H10T1/2 fibroblasts do not contain similar binding activities. Importantly, the TnI MRF site alone is not sufficient to elicit the full enhancer activity associated with the IRE. Instead, two additional regions (site I and site II) are required. The proteins that interact with site I and site II are expressed in both muscle and nonmuscle cell types and by themselves are ineffective in activating TnI gene expression. However, when the MRF site is positioned upstream or downstream of site I and site II, full enhancer activity is restored. We conclude that helix-loop-helix muscle regulatory factors must interact with ubiquitously expressed proteins to generate the active TnI transcription complex that is present in differentiated muscle fibers.
Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.
Authors: Marini N J , Benbow R M
Published: October 2015

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).
Molecular cloning and functional analysis of three subunits of yeast proteasome.
Authors: Emori Y , Tsukahara T , Kawasaki H , Ishiura S , Sugita H , Suzuki K
Published: October 2015

The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position.
Tissue-specific distribution of differentially phosphorylated forms of Cx43.
Authors: Kadle R , Zhang J T , Nicholson B J
Published: October 2015

Variants of the Cx43 gap junction protein have been detected on Western immunoblots by using an antipeptide antibody to the N-terminus of the protein. In heart ventricle, atrium, brain, retina, and uterus, different yet characteristic ratios of a broad 43-kDa band and a 39- to 40-kDa doublet were observed. These proteins (in lens epithelium, testes, and spleen) or their messages (in stomach, duodenum, kidney, and lung) were also detected in several nonexcitable systems but at consistently lower levels than found in electrically excitable tissues. The reproducible heterogeneity in electrophoretic mobility of Cx43 seen in different tissues does not appear to be due to proteolysis, since both the 43-kDa band and the 39- to 40-kDa doublet were recognized by an N-terminal as well as a C-terminal antibody. Furthermore, Northern (RNA) blots from different tissues show that both polypeptide profiles arise from indistinguishable transcripts. The conversion by alkaline phosphatase treatment of a predominantly 43-kDa profile (in heart) to a 39- to 40-kDa profile (characteristic of brain and protein translated in vitro from the RNA) suggests that the observed electrophoretic heterogeneity arises from tissue-wide differences in the phosphorylation state of Cx43.
Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase.
Authors: Minard K I , McAlister-Henn L
Published: October 2015

The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000. Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2. Transformation and complementation of the acetate- growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library. The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins. Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source. Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDH1 gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source. The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.
Two different mutants blocked in synthesis of dolichol-phosphoryl-mannose do not add glycophospholipid anchors to membrane proteins: quantitative correction of the phenotype of a CHO cell mutant with tunicamycin.
Authors: Singh N , Tartakoff A M
Published: October 2015

The addition of glycophospholipid (GPL) anchors to certain membrane proteins occurs in the rough endoplasmic reticulum and is essential for transport of the proteins to the plasma membrane. Limited circumstantial evidence suggests that dolichol-phosphoryl-mannose (DPM) is a donor of mannose residues of these anchors. We here report studies of a CHO cell mutant (B421) transfected to express the GPL-anchored protein, placental alkaline phosphatase (AP). Only a few transfectants were found to express GPL-anchored AP on their surface, and these clones synthesized DPM. Moreover, and most strikingly, when surface AP-negative transfectants were treated with tunicamycin to cause accumulation of DPM, these cells expressed lipid-anchored AP. Fusion of a cloned surface AP-negative transfectant of B421 with the Thy-1-class E mutant thymoma, which is also deficient in DPM synthesis, produced hybrids that synthesized DPM and expressed AP and Thy-1. Thus, two mutations can interrupt DPM synthesis, and three sets of observations point to an essential role of DPM for addition of GPL anchors.
Aspergillus nidulans wetA activates spore-specific gene expression.
Authors: Marshall M A , Timberlake W E
Published: October 2015

The Aspergillus nidulans wetA gene is required for synthesis of cell wall layers that make asexual spores (conidia) impermeable. In wetA mutant strains, conidia take up water and autolyze rather than undergoing the final stages of maturation. wetA is activated during conidiogenesis by sequential expression of the brlA and abaA regulatory genes. To determine whether wetA regulates expression of other sporulation-specific genes, its coding region was fused to a nutritionally regulated promoter that permits gene activation in vegetative cells (hyphae) under conditions that suppress conidiation. Expression of wetA in hyphae inhibited growth and caused excessive branching. It did not lead to activation of brlA or abaA but did cause accumulation of transcripts from genes that are normally expressed specifically during the late stages of conidiation and whose mRNAs are stored in mature spores. Thus, wetA directly or indirectly regulates expression of some spore-specific genes. At least one gene (wA), whose mRNA does not occur in spores but rather accumulates in the sporogenous phialide cells, was activated by wetA, suggesting that wetA may have a regulatory function in these cells as well as in spores. We propose that wetA is responsible for activating a set of genes whose products make up the final two conidial wall layers or direct their assembly and through this activity is responsible for acquisition of spore dormancy.
The oncogenic potential of an activated Hox-2.4 homeobox gene in mouse fibroblasts.
Authors: Aberdam D , Negreanu V , Sachs L , Blatt C
Published: October 2015

The homeobox gene Hox-2.4 is transcriptionally activated in cells of the mouse myeloid leukemia WEHI-3B. The constitutive Hox-2.4 expression in WEHI-3B cells is due to insertion of a transposable element belonging to the family of intracisternal A particles. In this study, we demonstrated the oncogenic potential of this activated homeobox gene. NIH 3T3 fibroblast clones bearing the activated Hox-2.4 gene produced fibrosarcomas in nude mice.
STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases.
Authors: Howell B W , Afar D E , Lew J , Douville E M , Icely P L , Gray D A , Bell J C
Published: October 2015

We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.
Liver-specific gene expression: A-activator-binding site, a promoter module present in vitellogenin and acute-phase genes.
Authors: Kaling M , Kugler W , Ross K , Zoidl C , Ryffel G U
Published: October 2015

The A2 vitellogenin gene of Xenopus laevis, which is expressed liver specifically, contains an A-activator-binding site (AABS) that mediates high in vitro transcriptional activity in rat liver nuclear extracts. Footprint experiments with DNase I and gel retardation assays revealed the binding of several proteins to AABS. Using binding sites of known DNA-binding proteins as competitors in the gel retardation assay, we found that the transcription factor C/EBP and/or one of its "iso-binders" as well as LFB1/HNF1 bound AABS. These interactions were confirmed by in vitro transcription experiments using various oligonucleotides as competitors. However, saturating amounts of C/EBP- and LFB1/HNF1-binding sites as competitors only partially blocked AABS-mediated transcriptional activity. This finding implies that at least a third distinct transcription factor interacts with AABS. In vitro transcription experiments revealed that AABS was present not only in the closely related Xenopus A1 vitellogenin gene but also in acute-phase genes as a liver-specific regulatory element known to confer the interleukin-6 response. Both AABS and the interleukin-6 response element are promoter modules interacting with at least three distinct transcription factors, including C/EBP and LFB1/HNF1.
A mutation in the putative Mg(2+)-binding site of Gs alpha prevents its activation by receptors.
Authors: Hildebrandt J D , Day R , Farnsworth C L , Feig L A
Published: October 2015

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.
Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis.
Authors: Ronne H , Carlberg M , Hu G Z , Nehlin J O
Published: October 2015

We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.
Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation.
Authors: Miller C L , Feldhaus A L , Rooney J W , Rhodes L D , Sibley C H , Singh H
Published: October 2015

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.
v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.
Authors: Song J G , Pfeffer L M , Foster D A
Published: October 2015

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.
Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknüllt protein.
Authors: Yamaguchi M , Hirose F , Nishida Y , Matsukage A
Published: October 2015

A 631-bp fragment containing the 5'-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5'-deletion derivatives revealed that the promoter function resided within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknüllt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even-skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region (-119 to -86) of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region (-607 to +137 or -168 to +137) fused with lacZ were established. When these flies were crossed with the zen mutant, ectopic expression of lacZ was observed in the dorsal region of gastrulating embryos carrying the transgene with either construct. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.
Exocrine pancreas transcription factor 1 binds to a bipartite enhancer element and activates transcription of acinar genes.
Authors: Weinrich S L , Meister A , Rutter W J
Published: October 2015

Exocrine pancreas (XP) enhancers, which contain a conserved core sequence, are active only in XP cells. A core enhancer-binding activity also appears to be restricted to XP nuclei. Here we describe the properties of a factor, purified approximately 100,000-fold from pancreas nuclei, which displays core enhancer-binding activity. It is not identical to previously characterized factors and is termed exocrine pancreas transcription factor 1 (XPF-1). In the highly purified preparation, only a single major protein of 60 kDa was detected by silver staining on sodium dodecyl sulfate-gels and by UV cross-linking. XPF-1 binds to the core enhancer of all tested XP genes and not to a mutant sequence which is inactive in vivo. High-affinity binding sites are bipartite. The results of competition binding and UV-cross-linking assays suggest that XPF-1 interacts with both motifs. XPF-1 selectively stimulates transcription of core enhancer templates in an in vitro transcription system. We hypothesize that XPF-1 plays a role in activation of the transcription of XP-specific genes.
Progestins both stimulate and inhibit breast cancer cell cycle progression while increasing expression of transforming growth factor alpha, epidermal growth factor receptor, c-fos, and c-myc genes.
Authors: Musgrove E A , Lee C S , Sutherland R L
Published: October 2015

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.
A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.
Authors: Mohammadi M , Honegger A M , Rotin D , Fischer R , Bellot F , Li W , Dionne C A , Jaye M , Rubinstein M , Schlessinger J
Published: October 2015

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.
Identification and characterization of a novel cytoskeleton-associated pp60src substrate.
Authors: Wu H , Reynolds A B , Kanner S B , Vines R R , Parsons J T
Published: October 2015

Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies revealed a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 revealed an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure.
Two distinct alpha-interferon-dependent signal transduction pathways may contribute to activation of transcription of the guanylate-binding protein gene.
Authors: Decker T , Lew D J , Darnell J E
Published: October 2015

The promoter of the gene encoding a cytoplasmic guanylate-binding protein (GBP) contains two overlapping elements: the interferon stimulation response element (ISRE), which mediates alpha interferon (IFN-alpha)-dependent transcription, and the IFN-gamma activation site (GAS), which is required for IFN-gamma-mediated stimulation. The ISRE binds a factor called ISGF-3 that is activated by IFN-alpha but not by IFN-gamma. The GAS binds a protein that is activated by IFN-gamma, which we have termed GAF (IFN-gamma activation factor; T. Decker, D. J. Lew, J. Mirkovitch, and J. E. Darnell, Jr., EMBO J., in press; D. J. Lew, T. Decker, I. Strehlow, and J. E. Darnell, Jr., Mol. Cell. Biol. 11:182-191, 1991). We now find that the GAS is also an IFN-alpha-responsive element in vivo and that IFN-alpha (in addition to activating ISGF-3) rapidly activates a GAS-binding factor, the IFN-alpha activation factor (AAF). The AAF has characteristics very similar to those of the previously described GAF. Through the use of inhibitors of protein synthesis and inhibitors of protein kinases, the activation conditions of AAF, GAF, and ISGF-3 could be distinguished. Therefore, not only do IFN-alpha and IFN-gamma stimulate transcription of GBP through different receptors linked to different signaling molecules, but occupation of the IFN-alpha receptor apparently leads to the rapid activation of two different DNA-binding proteins through the use of different intracellular pathways.
Convergent transcription initiates from oppositely oriented promoters within the 5' end regions of Drosophila melanogaster F elements.
Authors: Minchiotti G , Di Nocera P P
Published: October 2015

Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that likely propagate by the retrotranscription of RNA intermediates. Plasmids bearing DNA segments from the left-hand region of a full-length F element fused to the CAT gene were used as templates for transient expression assays in Drosophila Schneider II cultured cells. Protein and RNA analyses led to the identification of two promoters, Fin and Fout, that transcribe in opposite orientations. The Fin promoter drives the synthesis of transcripts that initiate around residue +6 and are directed toward the element. Fin, that probably controls the formation of F transposition RNA intermediates and gene products, is internal to the transcribed region. Sequences important for accumulation of Fin transcripts are included within the +1 to +30 interval; an additional regulatory element may coincide with a heptamer located downstream of this region also found in the 5' end regions of F-like Drosophila retrotransposons. Analysis of the template activity of 3' deletion derivatives indicates that the level of accumulation of Fin RNA is also dependent upon the presence of sequences located within the +175 to +218 interval. The Fout promoter drives transcription in the opposite orientation with respect to Fin. Fout transcripts initiate at nearby sites within the +92 to +102 interval. Sequences downstream of these multiple RNA start sites are not required for the activity of the Fout promoter. Deletions knocking out the Fin promoter do not impair Fout transcription; conversely, initiation at the Fin promoter still takes place in templates that lack the Fout promoter. At a low level, both promoters are active in cultured cells.
Two components of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically located upstream of a tRNA gene and interact with the second-largest subunit of TFIIIC.
Authors: Bartholomew B , Kassavetis G A , Geiduschek E P
Published: October 2015

A novel photocrosslinking method has been used to identify the components of transcription factor IIIB (TFIIIB) and TFIIIC that associate with DNA upstream of the Saccharomyces cerevisiae SUP4 tRNATyr gene and to map these components to specific positions in DNA. When TFIIIC binds to the tRNA gene, only its second-largest subunit (135 kDa) is accessible for reaction with a photoactive nucleotide, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP, inserted into DNA upstream of the transcriptional start. Formation of TFIII(C + B)-tRNA gene complexes specifically brings two additional polypeptides (90 and 70 kDa) within reach of upstream photoprobes. A collection of 13 probes has been used to map the locations of these three proteins along a 45-bp segment of DNA upstream of the transcriptional start site. Evidence is presented that the 90- and 70-kDa polypeptides are separate and distinct components of yeast TFIIIB, that they are accessible to crosslinking on opposite sides of the DNA helix in a 6-bp segment centered 35 bp upstream of the tRNATyr gene transcriptional start, and that they interact with the second-largest subunit of TFIIIC.
c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site.
Authors: Pal S K , Zinkel S S , Kiessling A A , Cooper G M
Published: October 2015

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.