bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro transcription of Drosophila rRNA genes shows stimulation by a phorbol ester and serum.
Authors: Chao Y , Pellegrini M
Published: October 2015

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum both stimulate rapid increases in the transcription of Drosophila rRNA genes in vivo. Here we report that this stimulation is observed in in vitro transcription assays using nuclear extracts from cells treated with TPA or serum. Experiments in which extracts from TPA- or serum-treated cells were mixed with extracts from cells grown in serum-restricted medium showed that there was an increased RNA polymerase I (Pol I) activity present in the cell extracts from treated cells. We used a series of plasmids that had been deleted in the region 5' to the start site of rRNA transcription to determine which sequences were necessary to support the increased transcription seen in extracts from stimulated cells. DNA templates that contain sequences between -150 and +32 (with +1 as the Pol I transcription start site) show dramatic increases in transcription with TPA- and serum-stimulated cell extracts; however, templates that contain 5' sequences to -60 or -43 show at most one-third of the stimulation level of transcription in nuclear extracts from treated cells in comparison with untreated cell extracts. The 5' deletion to -34 abolishes the stimulation effect and drops the basal-level transcription by 20-fold. These results indicate that the regulation of Pol I transcription in Drosophila cells by serum and TPA requires two DNA elements, sequences from -150 to -60 (upstream control element) and sequences from -43 to -34 (a portion of the core promoter.
Orientation and topography of RNA polymerase III in transcription complexes.
Authors: Bartholomew B , Durkovich D , Kassavetis G A , Geiduschek E P
Published: October 2015

A photo-cross-linking method has been used to map the subunits of Saccharomyces cerevisiae RNA polymerase (Pol) III with respect to DNA in binary (preinitiation) and ternary (RNA-elongating) transcription complexes. Transcription factor- and Pol III-containing complexes have been assembled on S. cerevisiae SUP4 tRNA(Tyr) gene probes containing the photoactive nucleotide 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP in different specified positions. Covalent DNA-protein linkages form upon irradiation of these complexes, and the Pol III subunits that are cross-linked to individual positions in the SUP4 tRNA gene have been identified. RNA Pol III cross-linking has been shown to require the box B downstream promoter element of the tRNA gene and the presence of transcription factor TFIIIB. Further proof of specificity has been provided by demonstrating that particular Pol III subunits move out of the range of upstream-placed photoactive nucleotides, and that others move into the range of downstream-placed photoactive nucleotides, as a consequence of initiating and elongating RNA chains. Binding and specific placement of Pol III have also been shown to require both the B' and the B" components of TFIIIB. Nine Pol III subunits are cross-linked from different positions of the SUP4 tRNA gene's nontranscribed strand. In binary transcription complexes, the two largest Pol III subunits are accessible to photo-cross-linking over the entire stretch of the DNase I footprint. The 27- and 34-kDa Pol III subunits are also relatively extended along DNA; its upstream projection makes the 34-kDa subunit a candidate for interaction with TFIIIB, while the 27-kDa subunit is accessible to photo-cross-linking from the leading edge of the Pol III binding site. Several subunits, including the 82- and 53-kDa subunits in binary transcription complexes, are relatively localized in their accessibility to cross-linking. Multiple Pol III subunits are accessible to specific cross-linking from a single photoactive nucleotide in the middle of the transcription bubble of an arrested ternary transcription complex. It is suggested that this precisely placed transcription complex comprises a dynamic ensemble of structural states rather than a single perfectly constrained entity.
Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein.
Authors: Huang P S , Patrick D R , Edwards G , Goodhart P J , Huber H E , Miles L , Garsky V M , Oliff A , Heimbrook D C
Published: October 2015

Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.
Combinatorial regulation by promoter and intron 1 regions of the metallothionein SpMTA gene in the sea urchin embryo.
Authors: Bai G , Stuebing E W , Parker H R , Harlow P , Nemer M
Published: October 2015

The SpMTA metallothionein gene of the sea urchin Strongylocentrotus purpuratus is regulated developmentally, histospecifically, and by heavy-metal induction. The sequenced 5' flank of the gene can be divided into proximal, middle, and distal regions, each containing a pair of metal response elements (MREs). Canonical 7-bp core sequences are present in all except the middle-region MREs c and d, which contain 1-bp mismatches. Metal-induced expression in transgenic blastulae was increased with each consecutive addition of the middle and distal regions to a chimeric reporter gene construct containing the proximal SpMTA promoter region. Reduced metal induction through point mutation of the distal MREs e and f indicated that the MREs themselves were largely responsible for the transcriptional increase. These activities were further enhanced by SpMTA intron 1, but not when a specific interior region of the intron had been deleted. The atypical MREs c and d did not support induction by themselves, i.e., when present alone with mutated proximal MREs a and b. However, in the presence of intron 1, they were able to substitute for the nullified MREs a and b in the promotion of metal-induced expression. This capability suggests, furthermore, that these atypical MREs, in addition to responding to an intron 1 region, participate cooperatively with the canonical proximal MREs.
Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3).
Authors: Duckett C S , Perkins N D , Kowalik T F , Schmid R M , Huang E S , Baldwin A S , Nabel G J
Published: October 2015

Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.
Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins.
Authors: Lai C C , Boguski M , Broek D , Powers S
Published: October 2015

The Saccharomyces cerevisiae CDC25 gene and closely homologous genes in other eukaryotes encode guanine nucleotide exchange factors for Ras proteins. We have determined the minimal region of the budding yeast CDC25 gene capable of activity in vivo. The region required for full biological activity is approximately 450 residues and contains two segments homologous to other proteins: one found in both Ras-specific exchange factors and the more distant Bud5 and Lte1 proteins, and a smaller segment of 48 amino acids found only in the Ras-specific exchange factors. When expressed in Escherichia coli as a fusion protein, this region of CDC25 was found to be a potent catalyst of GDP-GTP exchange on yeast Ras2 as well as human p21H-ras but inactive in promoting exchange on the Ras-related proteins Ypt1 and Rsr1. The CDC25 fusion protein catalyzed replacement of GDP-bound to Ras2 with GTP (activation) more efficiently than that of the reverse reaction of replacement of GTP for GDP (deactivation), consistent with prior genetic analysis of CDC25 which indicated a positive role in the activation of Ras. To more directly study the physical interaction of CDC25 and Ras proteins, we developed a protein-protein binding assay. We determined that CDC25 binds tightly to Ras2 protein only in the absence of guanine nucleotides. This higher affinity of CDC25 for the nucleotide-free form than for either the GDP- or GTP-bound form suggests that CDC25 catalyzes exchange of guanine nucleotides bound to Ras proteins by stabilization of the transitory nucleotide-free state.
cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs.
Authors: Watanabe H , Sawada J , Yano K , Yamaguchi K , Goto M , Handa H
Published: October 2015

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.
SH2 domains exhibit high-affinity binding to tyrosine-phosphorylated peptides yet also exhibit rapid dissociation and exchange.
Authors: Felder S , Zhou M , Hu P , Ureña J , Ullrich A , Chaudhuri M , White M , Shoelson S E , Schlessinger J
Published: October 2015

src homology 2 (SH2) domains of intracellular signaling molecules such as phospholipase C-gamma and phosphatidylinositol 3'-kinase-associated protein p85 represent recognition motifs for specific phosphotyrosine-containing regions on activated growth factor receptors. The binding of SH2 domains to activated growth factor receptors controls the interaction with signaling molecules and the regulation of their activities. In this report, we describe the kinetic parameters and binding affinities of SH2 domains of p85 toward short phosphotyrosine-containing peptides with the amino acid sequence motif YMXM, derived from a major insulin receptor substrate, IRS-1, by using real time biospecific interaction analysis (BIAcore). Associations were specific and of very high affinity, with dissociation constants of 0.3 to 3 nM, between phosphopeptides and the two separate SH2 domains contained within p85. Nonphosphorylated peptides showed no measurable binding, and the interactions were specific for the primary sequence very close to the phosphotyrosine residue. Moreover, the interactions between phosphopeptides and SH2 domains of other signaling molecules were of much lower affinity. Interestingly, the binding of the SH2 domains to the tyrosine-phosphorylated peptides was of high affinity as a result of a very high on rate, of 3 x 10(7) to 40 x 10(7)/M/s; at the same time, the rate of dissociation, of 0.11 to 0.19/s, was rapid, allowing for rapid exchange of associating proteins with the tyrosine phosphorylation sites.
The immunoglobulin heavy chain locus contains another B-cell-specific 3' enhancer close to the alpha constant region.
Authors: Matthias P , Baltimore D
Published: October 2015

The transcription of immunoglobulin genes is controlled by variable region promoters and by enhancers, both of which are lymphoid specific. Because immunoglobulin genes are subject to an extremely complex regulation, we anticipated that there might be additional control elements for these genes. We therefore sought additional enhancers and demonstrate here that there is indeed another weak transcriptional enhancer just 3' to the mouse alpha constant region. This novel immunoglobulin enhancer is lymphoid specific and at two positions can bind members of the Oct family of transcription factors.
Complex telomere-associated repeat units in members of the genus Chironomus evolve from sequences similar to simple telomeric repeats.
Authors: Nielsen L , Edström J E
Published: October 2015

The dipteran Chironomus tentans has complex tandemly repeated 350-bp DNA sequences at or near the chromosome ends. As in Drosophila melanogaster, short simple repeats with cytosines and guanines in different strands have never been observed. We were therefore interested in learning whether the Chironomus repeats could have evolved from simple sequence telomeric DNA, which might suggest that they constitute a functional equivalent. We screened for repeat units with evolutionarily ancient features within the tandem arrays and recovered two clones with a less-evolved structure. Sequence analysis reveals that the present-day 350-bp unit probably evolved from a simpler 165-bp unit through the acquisition of transposed sequences. The 165-bp unit contains DNA with a highly biased distribution of cytosine and guanine between the two strands, although with the ratios inverted in two minor parts of the repeat. It is largely built up of short degenerate subrepeats for which most of the sequence can be reconstructed. The consensus for the subrepeat sequence is similar to the simple telomeric repeat sequences of several kinds of eukaryotes. We propose that the present-day unit has evolved from telomeric, simple sequence, asymmetric DNA from which it has retained some original sequence features and possibly functions.
Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein.
Authors: Ananthan J , Baler R , Morrissey D , Zuo J , Lan Y , Weir M , Voellmy R
Published: October 2015

Synergistic activation of transcription by Drosophila segmentation genes in tissue culture cells provides a model with which to study combinatorial regulation. We examined the synergistic activation of an engrailed-derived promoter by the pair-rule proteins paired (PRD) and fushi tarazu (FTZ). Synergistic activation by PRD requires regions of the homeodomain or adjacent sequences, and that by FTZ requires the first 171 residues. Surprisingly, deletion of the FTZ homeodomain does not reduce the capacity of the protein for synergistic activation, although this mutation abolishes any detectable DNA-binding activity. This finding suggests that FTZ can function through protein-protein interactions with PRD or other components of the homeoprotein transcription complex, adding a new layer of mechanisms that could underlie the functional specificities and combinatorial regulation of homeoproteins.
In vitro activation of the transcription factor gamma interferon activation factor by gamma interferon: evidence for a tyrosine phosphatase/kinase signaling cascade.
Authors: Igarashi K , David M , Finbloom D S , Larner A C
Published: October 2015

Although it has been well documented that the biological activities of gamma interferon (IFN-gamma) are initiated through interaction with its cell surface receptor, the signal transduction mechanisms which mediate the effects of this cytokine have remained unclear. In order to facilitate a better understanding of IFN-gamma signaling, we have designed an assay using human fibroblast cell homogenates in which IFN-gamma activates the formation of the IFN-gamma activation factor (GAF) transcription complex. GAF mediates the rapid transcriptional activation of the guanylate-binding protein gene by IFN-gamma. Activation of GAF in homogenates required ATP, but not Ca2+ or GTP. Fractionation of homogenates indicated that both the pellet (18,000 x g) and the remaining cytoplasmic fraction were required for GAF activation by IFN-gamma. In intact cells and cell homogenates, the activation of GAF was prevented by the specific tyrosine kinase inhibitor genistein. Treatment of GAF-containing nuclear extracts with either monoclonal antiphosphotyrosine antibody or protein tyrosine phosphatase prevented the assembly of the transcription complex, indicating that its formation required phosphorylation of tyrosine residues. Furthermore, the tyrosine phosphatase inhibitors phenylarsine oxide and zinc chloride also inhibited GAF formation in vitro, but only if these agents were added to cell homogenates before IFN-gamma was added. The addition of either agent 5 min after IFN-gamma had no effect. These results provide the first evidence for an IFN-gamma-regulated tyrosine phosphatase/kinase signaling cascade that permits this cytokine to activate the transcription of an early-response gene.
A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.
Authors: Carpenter C L , Auger K R , Duckworth B C , Hou W M , Schaffhausen B , Cantley L C
Published: October 2015

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.
Mechanisms of p34cdc2 regulation.
Authors: Atherton-Fessler S , Parker L L , Geahlen R L , Piwnica-Worms H
Published: October 2015

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.
Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets.
Authors: Clark E A , Brugge J S
Published: October 2015

Thrombin stimulation of platelets induces a transient increase in the specific activity of pp60c-src followed by a redistribution of pp60c-src to the Triton X-100-insoluble, cytoskeleton-rich fraction. Concomitant with the observed increase in pp60c-src activity was a rapid dephosphorylation of tyrosine 527 in 10 to 15% of pp60c-src molecules. In addition, we found that pp60c-src from the Triton-insoluble fraction was phosphorylated on tyrosine 416, the autophosphorylation site which is phosphorylated in activated oncogenic variants of pp60src. Furthermore, in platelets from patients with Glanzmann's thrombasthenia (which are deficient in the integrin receptor GPIIb-IIIa), pp60c-src was not translocated to the Triton-insoluble fraction, and there was a sustained increase in pp60c-src activity following thrombin treatment. These results suggest that pp60c-src is rapidly activated in thrombin-stimulated platelets, potentially by a protein tyrosine phosphatase, before it translocates to a cytoskeletal fraction, where many of its potential substrates are found. The evidence that the cytoskeletal association of pp60c-src is dependent upon engagement of the integrin receptor GPIIb-IIIa suggests that integrin-cytoskeletal complexes may serve to compartmentalize and anchor activated enzymes involved in signal transduction.
Near-zero linking difference upon transcription factor IID binding to promoter DNA.
Authors: Lorch Y , Kornberg R D
Published: October 2015

Binding of yeast transcription factor IID (TFIID) to the adenoviral major late promoter in circular DNA molecules caused a linking number change of less than 0.1. TFIID on its own therefore fails to unwind DNA appreciably, or else it causes both unwinding and compensatory writhing. Highly purified, recombinant yeast TFIID relaxed supercoiled DNA, because of a contaminant of bacterial topoisomerase I. Relaxing activity of topoisomerase I was enhanced by the adenoviral major late promoter, suggesting an instability of the TATA sequence or a destabilizing effect on flanking DNA.
The NFAT-1 DNA binding complex in activated T cells contains Fra-1 and JunB.
Authors: Boise L H , Petryniak B , Mao X , June C H , Wang C Y , Lindsten T , Bravo R , Kovary K , Leiden J M , Thompson C B
Published: October 2015

Activation of T cells induces transcription of the interleukin-2 (IL-2) gene. IL-2 expression is regulated through the binding of transcription factors to multiple sites within the IL-2 enhancer. One such cis-acting element within the IL-2 enhancer is the NFAT-1 (nuclear factor of activated T cells) binding site. NFAT-1 binding activity is absent in resting cells but is induced upon T-cell activation. The induction of NFAT-1 binding activity can be inhibited by cyclosporin A, potentially accounting for the ability of cyclosporin A to inhibit IL-2 production by T cells. We have previously reported that the NFAT-1 binding complex is composed of at least two proteins and that the 5' portion of the NFAT-1 sequence acts as a binding site for one or more proteins from the Ets family of transcription factors. We now report that the 3' portion of the NFAT-1 sequence contains a variant AP-1 binding site. NFAT-1 binding can be specifically inhibited by oligonucleotides containing a consensus AP-1 site. Moreover, mutation of the AP-1 site at the 3' end of the NFAT-1 sequence inhibits both NFAT-1 binding and the ability of the NFAT-1 binding site to activate expression from a reporter plasmid upon T-cell activation. Since AP-1 sites bind dimeric protein complexes composed of individual members of the Fos and Jun families of transcription factors, we used antibodies specific for individual Fos and Jun family members to determine whether they are present in the NFAT-1 binding complex. These experiments demonstrated that the NFAT-1 binding complex contains JunB and Fra-1 proteins. Northern (RNA) blot analyses demonstrate that both fra-1 and junB mRNAs are induced upon T-cell activation, although fra-1 mRNA is present even in quiescent T cells. Of interest, junB is not expressed in quiescent T cells, and it is induced with kinetics that are similar to those for the induction of IL-2 mRNA expression. Taken together, these results suggested that the JunB-Fra-1 heterodimer is the inducible nuclear component of the NFAT-1 binding activity and that JunB expression regulates the formation of the heterodimer. In addition, these data indicated that specific heterodimers of Fos and Jun family members may have selective roles in the induction of transcription during cellular activation.
A retrovirus carrying the K-fgf oncogene induces diffuse meningeal tumors and soft-tissue fibrosarcomas.
Authors: Talarico D , Ittmann M M , Bronson R , Basilico C
Published: October 2015

The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF.
Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways.
Authors: Yu C L , Prochownik E V , Imperiale M J , Jove R
Published: October 2015

Immediate early genes involved in controlling cell proliferation are rapidly and transiently induced following stimulation of susceptible cells with serum. To study how oncoproteins regulate immediate early genes, we examined serum inducibility of these genes in cells transformed by various oncoproteins. We found that induction of the immediate early gene, c-fos, by serum stimulation was markedly attenuated in four independent cell lines stably transformed by the v-Src tyrosine kinase. Cells chronically transformed by other oncoproteins implicated in tyrosine kinase signaling pathways, including v-Sis, v-Ras, and v-Raf, showed the same pattern of attenuation. In contrast, serum inducibility of c-fos was not attenuated in cells transformed by simian virus 40, which is thought to transform cells through a different pathway. Cell cycle analyses showed that proliferation of these transformed cell lines could be arrested effectively in 0.1% serum, demonstrating that the attenuation was not simply due to continuous cycling of transformed cells after serum deprivation. Moreover, serum inducibility of other immediate early genes, including c-jun, junB, egr-1, and NGFI-B, also was strikingly attenuated by these same oncoproteins. Nuclear run-on transcription assays established that this attenuation of serum inducibility occurred at the transcriptional level. Finally, flow cytometric analysis demonstrated that serum-starved v-Src-transformed cells were viable and able to progress into S phase of the cell cycle after serum stimulation, even though the induction of immediate early genes was greatly attenuated in these cells. Our results suggest that activation of immediate early genes is repressed by chronic stimulation of tyrosine kinase signaling pathways in transformed cells.
The product of a novel growth factor-activated gene, fic, is a biologically active "C-C"-type cytokine.
Authors: Heinrich J N , Ryseck R P , Macdonald-Bravo H , Bravo R
Published: October 2015

We have characterized a new member of the superfamily of proinflammatory peptides encoded by a growth factor-inducible gene, fic, previously isolated by differential screening of a cDNA library of mRNA from serum-stimulated NIH 3T3 cells. Immunoprecipitation analyses showed that the protein was rapidly induced following serum stimulation and secreted unglycosylated into the medium. The fic protein, FIC, shows highest sequence homology (57%) to human and rabbit monocyte chemoattractant protein 1 (MCP-1), an established monocyte activator. To determine the biological activity of FIC and to compare it with that of mouse MCP-1 (muMCP-1), both proteins were expressed in the baculovirus system. FIC and muMCP-1 were purified to near homogeneity by a two-step chromatography protocol. Both proteins elicited changes in intracellular calcium concentration in human monocytes. The effect was dependent on external Ca2+ and was inhibited by pretreatment of cells with pertussis toxin. FIC did not desensitize human monocytes to the three related cytokines muMCP-1, human MCP-1 (huMCP-1), and huMCP-2. However, pretreatment with muMCP-1 or huMCP-1, but not with huMCP-2, desensitized human monocytes to FIC. Specific binding of [125I]FIC was found in human monocytes, mouse monocytic cultured cells, and human endothelial cells but not in lymphocytes, neutrophils, or primary mouse fibroblasts. Scatchard analysis of the binding of [125I]FIC to human monocytes showed the presence of two classes of receptors, with apparent KdS of 1.2 and 7.7 nM and receptor numbers per cell of 2,400 and 6,300, respectively. FIC, muMCP-1, and huMCP-1 competed to the same extent for the binding of [125I]FIC to human monocytes, contrary to huMCP-2, which competed very ineffectively, if at all.
Function of the ste signal transduction pathway for mating pheromones sustains MAT alpha 1 transcription in Saccharomyces cerevisiae.
Authors: Mukai Y , Harashima S , Oshima Y
Published: October 2015

Sterile mutants of Saccharomyces cerevisiae were isolated from alpha * cells having the a/alpha aar1-6 genotype (exhibiting alpha mating ability and weak a mating ability as a result of a defect in a1-alpha 2 repression). Among these sterile mutants, we found two ste5 mutants together with putative ste7, ste11, and ste12 mutants of the signal transduction pathway of mating pheromones. The amino acid sequence of the Ste5p protein predicted from the nucleotide sequence of a cloned STE5 DNA has a domain rich in acidic amino acids close to its C terminus, a cysteine-rich sequence, resembling part of a zinc finger structure, in its N-terminal half, and a possible target site of cyclic AMP-dependent protein kinase at its C terminus. Northern (RNA) blot analysis revealed that STE5 transcription is under a1-alpha 2-Aar1p repression. The MAT alpha 1 cistron has a single copy of the pheromone response element in its 5' upstream region, and its basal level of transcription was reduced in these ste mutant cells. However, expression of the MAT alpha 1 cistron was not enhanced appreciably by pheromone signals. One of the ste5 mutant alleles conferred a sterile phenotype to a/alpha aar1-6 cells but a mating ability to MATa cells.
Bipartite structure of an early meiotic upstream activation sequence from Saccharomyces cerevisiae.
Authors: Bowdish K S , Mitchell A P
Published: October 2015

Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation. Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells. We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene. Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS). Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis. Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes. Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes. In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1. However, the URS1 mutations prevent any further stimulation of UAS activity by IME1. Repression through URS1 has been shown to require the UME6 gene product. We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product. Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS. We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor.
Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen.
Authors: Gatignol A , Buckler C , Jeang K T
Published: October 2015

TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. Here, we show that the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) of this sequence bound TAR RNA with affinities similar to that of the entire TRBP, thus suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, we determined that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovered TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment revealed that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. We suggest that these proteins are related by virtue of sharing a common RNA-binding moiety.
Mouse GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart.
Authors: Arceci R J , King A A , Simon M C , Orkin S H , Wilson D B
Published: October 2015

We report the cDNA cloning and characterization of mouse GATA-4, a new member of the family of zinc finger transcription factors that bind a core GATA motif. GATA-4 cDNA was identified by screening a 6.5-day mouse embryo library with oligonucleotide probes corresponding to a highly conserved region of the finger domains. Like other proteins of the family, GATA-4 is approximately 50 kDa in size and contains two zinc finger domains of the form C-X-N-C-(X17)-C-N-X-C. Cotransfection assays in heterologous cells demonstrate that GATA-4 trans activates reporter constructs containing GATA promoter elements. Northern (RNA) analysis and in situ hybridization show that GATA-4 mRNA is expressed in the heart, intestinal epithelium, primitive endoderm, and gonads. Retinoic acid-induced differentiation of mouse F9 cells into visceral or parietal endoderm is accompanied by increased expression of GATA-4 mRNA and protein. In vitro differentiation of embryonic stem cells into embryoid bodies is also associated with increased GATA-4 expression. We conclude that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally regulated transcription factor. On the basis of its tissue distribution, we speculate that GATA-4 plays a role in gene expression in the heart, intestinal epithelium, primitive endoderm, and gonads.
Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.
Authors: Segars J H , Marks M S , Hirschfeld S , Driggers P H , Martinez E , Grippo J F , Brown M , Wahli W , Ozato K
Published: October 2015

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.
v-myb blocks granulocyte colony-stimulating factor-induced myeloid cell differentiation but not proliferation.
Authors: Patel G , Kreider B , Rovera G , Reddy E P
Published: October 2015

To understand the effects of v-myb expression on mammalian hematopoietic cell differentiation, we have constructed a retroviral vector which can efficiently express v-myb gene product in mammalian cells. Infection of interleukin-3-dependent murine progenitor cell line 32D Cl3, which undergoes terminal differentiation to mature granulocytes in the presence of granulocyte colony-stimulating factor (GCSF), with this recombinant retrovirus does not abrogate its requirement of interleukin-3 for growth. However, expression of v-myb in these cells blocks their ability to differentiate in response to GCSF. Instead, the v-myb-infected cells proliferate indefinitely in the presence of GCSF. 32D Cl3 cells infected with empty vector carrying only the neomycin resistance gene responded to the addition of GCSF in a manner identical to that of the uninfected cells and underwent terminal differentiation into granulocytes. These results suggest that oncogenic forms of myb gene bring about transformation by blocking the differentiation signal derived by cytokines while promoting the proliferative signal transduction pathways.
Linear mitochondrial DNAs of yeasts: frequency of occurrence and general features.
Authors: Fukuhara H , Sor F , Drissi R , Dinouël N , Miyakawa I , Rousset S , Viola A M
Published: October 2015

In most yeast species, the mitochondrial DNA (mtDNA) has been reported to be a circular molecule. However, two cases of linear mtDNA with specific termini have previously been described. We examined the frequency of occurrence of linear forms of mtDNA among yeasts by pulsed-field gel electrophoresis. Among the 58 species from the genera Pichia and Williopsis that we examined, linear mtDNA was found with unexpectedly high frequency. Thirteen species contained a linear mtDNA, as confirmed by restriction mapping, and labeling, and electron microscopy. The mtDNAs from Pichia pijperi, Williopsis mrakii, and P. jadinii were studied in detail. In each case, the left and right terminal fragments shared homologous sequences. Between the terminal repeats, the order of mitochondrial genes was the same in all of the linear mtDNAs examined, despite a large variation of the genome size. This constancy of gene order is in contrast with the great variation of gene arrangement in circular mitochondrial genomes of yeasts. The coding sequences determined on several genes were highly homologous to those of the circular mtDNAs, suggesting that these two forms of mtDNA are not of distant origins.
Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein.
Authors: Li X , Coffino P
Published: October 2015

Polyamine-mediated degradation of vertebrate ornithine decarboxylase (ODC) is associated with the production of antizyme, a reversible tightly binding protein inhibitor of ODC activity. The interaction of antizyme with a binding element near the N terminus of ODC is essential but not sufficient for regulation of the enzyme by polyamines (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-2562, 1992). We now show that a second element present at the C terminus is required for the degradation process. Antizyme caused a conformational change in ODC, which made the C terminus of ODC more accessible. Blocking the C terminus with antibody prevented degradation. Tethering the C terminus by creating a circularly permuted, enzymatically active form of ODC prevented antizyme-mediated degradation. These data elucidate a form of feedback regulation whereby excess polyamines induce destruction of ODC, the enzyme that initiates their biosynthesis.
Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.
Authors: Huang D C , Marshall C J , Hancock J F
Published: October 2015

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.