bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular involvement of the pvt-1 locus in a gamma/delta T-cell leukemia bearing a variant t(8;14)(q24;q11) translocation.
Authors: Kasai M , Maziarz R T , Aoki K , Macintyre E , Strominger J L
Published: October 2015

A highly malignant human T-cell receptor (TCR) gamma/delta+ T-cell leukemia was shown to have a productive rearrangement of the TCR delta locus on one chromosome 14 and a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on the other chromosome 14. Chromosome walking coupled with pulsed-field gel electrophoretic (PFGE) analysis determined that the TCR J delta 1 gene fragment of the involved chromosome was relocated approximately 280 kb downstream of the c-myc proto-oncogene locus found on chromosome band 8q24. This rearrangement was reminiscent of the Burkitt's lymphoma variants that translocate to a region identified as the pvt-1 locus. Sequence comparison of the breakpoint junctions of interchromosomal rearrangements in T-cell leukemias involving the TCR delta-chain locus revealed novel signal-like sequence motifs, GCAGA(A/T)C and CCCA(C/G)GAC. These sequences were found on chromosome 8 at the 5' flanking site of the breakpoint junction of chromosome 8 in the TCR gamma/delta leukemic cells reported here and also on chromosome 1 in T-cell acute lymphocytic leukemia patients carrying the t(1;14)(p32;q11) rearrangement. These results suggest that (i) during early stages of gamma delta T-cell ontogeny, the region 280 kb 3' of the c-myc proto-oncogene on chromosome 8 is fragile and accessible to the lymphoid recombination machinery and (ii) rearrangements to both 8q24 and 1p32 may be governed by novel sequence motifs and be subject to common enzymatic mechanisms.
cis-acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor mRNA.
Authors: Prats A C , Vagner S , Prats H , Amalric F
Published: October 2015

Four forms of basic fibroblast growth factor (bFGF) are synthesized from the same mRNA, resulting from alternative initiations of translation at three CUG start codons and one AUG start codon. The CUG- and AUG-initiated forms have distinct intracellular localizations and can modify cell phenotypes differently, indicating that control of the alternative expression of the different forms of bFGF has an important impact on the cell. In this study, we investigated the roles of the mRNA 5' untranslated region and the alternatively translated region located between the CUG and AUG codons in the regulation of alternative translation of the different forms of bFGF. Deletions and site-directed mutagenesis were carried out in bFGF mRNA leader, and translation was studied in vitro and in vivo. The results enabled us to identify five cis-acting RNA elements (two in the 5' untranslated region and three in the alternatively translated region) involved in the regulation of either global or alternative initiation of translation. Each of these elements had a specific effect on the level of synthesis of the different forms of bFGF. Furthermore, we showed that the 5' untranslated region regulatory elements had different effects on bFGF translation, depending on the translation system used. These results suggest that bFGF translation is modulated by cis-acting elements corresponding to secondary or tertiary RNA structures, which could be the targets of cell-specific trans-acting factors.
Identification of a novel dimer stabilization region in a plant bZIP transcription activator.
Authors: Katagiri F , Seipel K , Chua N H
Published: October 2015

We have carried out deletion analyses of a tobacco transcription activator, TGA1a, in order to define its functional domains. TGA1a belongs to the basic-region-leucine zipper (bZIP) class of DNA-binding proteins. Like other proteins of this class, it binds to its target DNA as a dimer, and its bZIP domain is necessary and sufficient for specific DNA binding. A mutant polypeptide containing the bZIP domain alone, however, shows a lower DNA-binding affinity than the full-length TGA1a. The C-terminal portion of TGA1a, which is essential for the higher DNA-binding affinity, contains a polypeptide region that can stabilize dimeric forms of the protein. This polypeptide region is designated the dimer stabilization (DS) region. Under our in vitro conditions, TGA1a derivatives with the DS region and those without the region do not form a detectable mixed dimer. This result indicates that in addition to the leucine zipper, the DS region can serve as another determinant of the dimerization specificity of TGA1a. In fact, the DS region, when fused to another bZIP protein, C/EBP, can inhibit dimer formation between the fusion protein and native C/EBP, whereas each of these can form homodimers. Such a portable determinant of dimerization specificity has potential application in studies of DNA-binding proteins as well as in biotechnology.
Indistinguishable nuclear factor binding to functional core sites of the T-cell receptor delta and murine leukemia virus enhancers.
Authors: Redondo J M , Pfohl J L , Hernandez-Munain C , Wang S , Speck N A , Krangel M S
Published: October 2015

We have previously shown that the delta E3 site is an essential element for transcriptional activation by the human T-cell receptor (TCR) delta enhancer and identified two factors, NF-delta E3A and NF-delta E3C, that bound to overlapping core (TGTGGTTT) and E-box motifs within delta E3. In this study, we show that protein binding to the core motif is necessary but not sufficient for transcriptional activation by the delta E3 element. In contrast, protein binding to the E-box motif does not contribute significantly to enhancer activity. A similar core motif present within the enhancers of T-cell-tropic murine retroviruses has been shown to contribute to transcriptional activity of the viral long terminal repeat in T lymphocytes and to viral T-cell tropism. We therefore determined the relationship between the nuclear factors that bind to the TCR delta and Moloney murine leukemia virus core motifs. On the basis of electrophoretic mobility shift binding and competition studies, biochemical analysis of affinity-labeled DNA-binding proteins, and the binding of a purified core binding factor, the proteins that bound to the TCR delta core site were indistinguishable from those that bound to the murine leukemia virus core site. These data argue that DNA-binding proteins that interact with the core site of murine leukemia virus long terminal repeats and contribute to viral T-cell tropism also play an essential role in the T-cell-specific expression of cellular genes.
Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements.
Authors: Aneskievich B J , Fuchs E
Published: October 2015

Terminal differentiation of epidermal keratinocytes is inhibited by 1 microM retinoic acid, a concentration which induces differentiation in a number of cell types, including F9 teratocarcinoma cells. The molecular basis for these opposing retinoid responses is unknown, although retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have been detected in both cell types. When F9 cells are stably transfected with a truncated RAR alpha lacking the E/F domain necessary for ligand binding and RAR/RXR dimerization, action at retinoid response elements is suppressed and cells produce a retinoic acid-resistant phenotype; i.e., they are blocked in differentiation (A. S. Espeseth, S. P. Murphy, and E. Linney, Genes Dev. 3:1647-1656, 1989). If retinoid receptors influence epidermal differentiation only in a negative fashion, then suppression of transactivation at retinoid response elements would be expected to enhance, rather than block, keratinocyte differentiation. In this study, we show that surprisingly, even though constitutive expression of an analogous truncated RAR gamma in keratinocytes specifically suppressed transactivation at retinoid response elements, keratinocytes were blocked, rather than enhanced, in their ability to undergo morphological and biochemical features of differentiation. These findings demonstrate a direct and hitherto unrecognized role for RARs and RXRs in positively as well as negatively regulating epidermal differentiation. Additionally, our studies extend those of Espeseth et al. (Genes Dev. 3:1647-1656, 1989), indicating a novel RAR function independent of the E/F domain.
Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.
Authors: Ruediger R , Roeckel D , Fait J , Bergqvist A , Magnusson G , Walter G
Published: October 2015

Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.
The EBNA2-related resistance towards alpha interferon (IFN-alpha) in Burkitt's lymphoma cells effects induction of IFN-induced genes but not the activation of transcription factor ISGF-3.
Authors: Kanda K , Decker T , Aman P , Wahlström M , von Gabain A , Kallin B
Published: October 2015

Transfection of a plasmid encoding the Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) gene confers resistance to the antiproliferative effect of alpha interferon (IFN-alpha) in EBV-negative U968 cells (P. Aman and A. von Gabain, EMBO J. 9:147-152, 1990). We studied the expression of IFN-stimulated genes (ISGs) in two pairs of Burkitt's lymphoma cell lines, differing in the expression of the putative immortalizing gene of EBV, EBNA2. In EBNA2-expressing cells, the induction of four ISGs by IFN-alpha was strongly reduced or, in some cases, abolished. Chloramphenicol acetyltransferase reporter gene constructs containing different IFN-stimulated response elements were transfected into EBNA2-negative and EBNA2-positive cells. Induction of chloramphenicol acetyltransferase activity by IFN was impaired in EBNA2-positive cells. Also, a reporter gene construct driven by an IFN-gamma-sensitive promoter element was affected. However, as revealed by gel shift assays, EBNA2-positive and EBNA2-negative cells exhibited a nearly identical pattern of IFN-stimulated response element-binding proteins. Most important, activation of the factor ISGF-3, which previously has been shown to be required and sufficient for transcriptional activation of IFN-induced genes, was not inhibited in IFN-resistant cells expressing EBNA2. The mechanism of the EBNA2-related IFN resistance seems to be distinct both from the resistance mediated by hepatitis virus and adenovirus gene products and from the IFN resistance in Daudi cell variants. In these three cases, the transcriptional block of IFN-induced genes is due to inhibition of ISGF-3 activation and binding. Our data suggest that the EBNA2-related IFN resistance in Burkitt's lymphoma cells acts downstream of the activation of ISGF-3.
A novel myoblast enhancer element mediates MyoD transcription.
Authors: Tapscott S J , Lassar A B , Weintraub H
Published: October 2015

The MyoD gene can orchestrate the expression of the skeletal muscle differentiation program. We have identified the regions of the gene necessary to reproduce transcription specific to skeletal myoblasts and myotubes. A proximal regulatory region (PRR) contains a conserved TATA box, a CCAAT box, and a GC-rich region that includes a consensus SP1 binding site. The PRR is sufficient for high levels of skeletal muscle-specific activity in avian muscle cells. In murine cells the PRR alone has only low levels of activity and requires an additional distal regulatory region to achieve high levels of muscle-specific activity. The distal regulatory region differs from a conventional enhancer in that chromosomal integration appears necessary for productive interactions with the PRR. While the Moloney leukemia virus long terminal repeat can enhance transcription from the MyoD PRR in both transient and stable assays, the simian virus 40 enhancer cannot, suggesting that specific enhancer-promoter interactions are necessary for PRR function.
Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins.
Authors: Matviw H , Yu G , Young D
Published: October 2015

The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: the N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expression of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals.
The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor.
Authors: Porcu P , Ferber A , Pietrzkowski Z , Roberts C T , Adamo M , LeRoith D , Baserga R
Published: October 2015

We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).
A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction.
Authors: Muller A J , Pendergast A M , Havlik M H , Puil L , Pawson T , Witte O N
Published: October 2015

SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.
Temporal order of DNA replication in the H-2 major histocompatibility complex of the mouse.
Authors: Spack E G , Lewis E D , Paradowski B , Schimke R T , Jones P P
Published: October 2015

As an approach to mapping replicons in an extended chromosomal region, the temporal order of DNA replication was analyzed in the murine major histocompatibility gene complex (MHC). Replicating DNA from T-lymphoma and myelomonocyte cell lines was density labeled with bromodeoxyuridine and extracted from cells which had been fractionated into different stages of S phase by centrifugal elutriation. The replicating DNA from each fraction of S phase was separated from nonreplicating DNA on density gradients, blotted, and hybridized with 34 specific MHC probes. The earliest replication occurred in the vicinity of transcribed genes K, HAM1 and HAM2, RD, B144, D, L, T18, and T3. The temporal order of replication of groups of DNA segments suggests the location of five or six replicons within the H-2 complex, some of which appear to be either unidirectional or markedly asymmetric. The rates of replication through each of these apparent replicons appear to be similar. The TL region of the S49.1 T-lymphoma cells, which contains at least three transcribed genes, replicates earlier than the inactive TL region of WEHI-3 myelomonocytic cells. These results provide further evidence of a relationship between transcription and the initiation of DNA replication in mammalian cells. The mouse MHC examined in this study is the largest chromosomal region (> 2,000 kb) measured for timing of replication to date.
Characterization of the human immunoglobulin kappa gene 3' enhancer: functional importance of three motifs that demonstrate B-cell-specific in vivo footprints.
Authors: Judde J G , Max E E
Published: October 2015

Using a combination of in vivo footprinting and site-directed mutagenesis, we have functionally characterized an enhancer located 12 kb downstream of the human immunoglobulin kappa constant-region gene. The core enhancer region is highly homologous to the murine 3' kappa enhancer. However, in addition to two regulatory elements homologous to the functional motifs of the murine enhancer, we find a third positive regulatory element in the human enhancer. This element is associated with an 11/12-bp direct repeat (DR) that is well conserved in the murine locus but was not recognized as functionally important in the murine enhancer. Mutation of any of the three motifs of the human enhancer decreases its activity to 3 to 20% of the wild-type level, indicating cooperative interaction between these elements. The DR motif does not resemble any known enhancer element and does not appear to function as a transcriptional activator on its own when present in multiple copies. Interestingly, nuclear extracts from both B- and T-cell lines contain factors binding to DR in vitro, but in vivo footprinting shows no evidence of protein-DNA binding in the T-cell line. This finding suggests that an additional regulatory mechanism, such as the effect of chromatin configuration on accessibility, may be involved in the B-cell-restricted activity of the human 3' kappa enhancer.
Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.
Authors: Su T Z , Logsdon C D , Oxender D L
Published: October 2015

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.
Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe.
Authors: Speiser D M , Ortiz D F , Kreppel L , Scheel G , McDonald G , Ow D W
Published: October 2015

Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems.
Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation.
Authors: Hornstra I K , Yang T P
Published: October 2015

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.
Biological function of the retinoblastoma protein requires distinct domains for hyperphosphorylation and transcription factor binding.
Authors: Qian Y , Luckey C , Horton L , Esser M , Templeton D J
Published: October 2015

Despite the importance of the retinoblastoma susceptibility gene to tumor growth control, the structural features of its encoded protein (pRb) and their relationship to protein function have not been well explored. We constructed a panel of deletion mutants of pRb expression vectors and used a biological assay for pRb that measures growth inhibition and morphologic changes in pRb-transfected Saos-2 cells to correlate structural alterations of the pRb coding region with function. We tested the deleted proteins for the ability to bind to viral oncoprotein E1A and to the transcription factor E2F. We also measured the ability of the mutant proteins to become hyperphosphorylated in vivo and to be recognized as substrates in vitro by a cell cycle-regulatory kinase associated with cyclin A. We identified two regions of pRb that are required for E2F binding and for hyperphosphorylation. E1A binding domains partially overlap but are distinct from both of these other two regions. Biological function of pRb is dependent on retention of the integrity of both of these biochemically defined domains. These data support the model that pRb is a transducer of afferent signals (via the kinase that phosphorylates it) and efferent signals (through transcription factor binding), using distinct structural elements. Preservation of both of these features is essential for the ability of pRb to induce growth inhibition and morphologic changes upon reintroduction into transfected cells.
p59fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain.
Authors: Timson Gauen L K , Kong A N , Samelson L E , Shaw A S
Published: October 2015

Several lines of evidence link the protein tyrosine kinase p59fyn to the T-cell receptor. The molecular basis of this interaction has not been established. Here we show that the tyrosine kinase p59fyn can associate with chimeric proteins that contain the cytoplasmic domains of CD3 epsilon, gamma, zeta (zeta), and eta. Mutational analysis of the zeta cytoplasmic domain demonstrated that the membrane-proximal 41 residues of zeta are sufficient for p59fyn binding and that at least two p59fyn binding domains are present. The association of p59fyn with the zeta chain was specific, as two closely related Src family protein tyrosine kinases, p60src and p56lck, did not associate with a chimeric protein that contained the cytoplasmic domain of zeta. Mutational analysis of p59fyn revealed that a 10-amino-acid sequence in the unique amino-terminal domain of p59fyn was responsible for the association with zeta. These findings support evidence that p59fyn is functionally and structurally linked to the T-cell receptor. More importantly, these studies support a critical role for the unique amino-terminal domains of Src family kinases in the coupling of tyrosine kinases to the signalling pathways of cell surface receptors.
Fission yeast pap1-dependent transcription is negatively regulated by an essential nuclear protein, crm1.
Authors: Toda T , Shimanuki M , Saka Y , Yamano H , Adachi Y , Shirakawa M , Kyogoku Y , Yanagida M
Published: October 2015

The fission yeast pap1+ gene encodes an AP-1-like transcription factor that contains a leucine zipper motif. We identified a target gene of pap1, the p25 gene. The 5' upstream region of the p25 gene contains an AP-1 site, and by DNase I footprint analysis, we showed that the pap1 protein binds to the AP-1 site as well as to a 14-bp palindrome sequence. p25 is overproduced when the pap1+ gene is overexpressed, whereas p25 is not produced at all in the pap1 deletion mutant. p25 was previously found to be overproduced in strains carrying cold-sensitive crm1 mutations whose gene product is essential for viability and is thought to play an important role in maintenance of a proper chromosomal architecture. Deletion and site-directed mutagenesis of sequences upstream of the p25 gene demonstrated that the AP-1 site as well as the palindrome sequence are crucial for transcriptional activation either by pap1 overproduction or by the cold-sensitive crm1 mutation; pap1+ is apparently negatively regulated by crm1+. Moreover, we found that cold-sensitive crm1 mutations are suppressed by the deletion of pap1+, further indicating a close relationship between crm1+ and pap1+. The crm1 protein is highly conserved; the budding yeast homolog, CRM1, which complements the fission yeast cold-sensitive crm1 mutation, was isolated and found to also be essential for viability. These results suggest the functional importance of chromosome structure on the regulation of gene expression through the pap1 transcription factor.
A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.
Authors: Boorstein R J , Chiu L N , Teebor G W
Published: October 2015

We isolated a mutant mammalian cell line lacking activity for the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). The mutant was isolated through its resistance to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (HmdUrd). The mutant incorporates HmdUrd into DNA to the same extent as the parent line but, lacking the repair enzyme, does not remove it. The phenotype of the mutant demonstrates that the toxicity of HmdUrd does not result from substitution of thymine in DNA by HmUra but rather from the removal via base excision of large numbers of HmUra residues in DNA. This finding elucidates a novel mechanism of toxicity for a xenobiotic nucleoside. Furthermore, the isolation of this line supports our hypothesis that the enzymatic repairability of HmUra derives not from its formation opposite adenine via the oxidation of thymine, but rather from its formation opposite guanine as a product of the oxidation and subsequent deamination of 5-methylcytosine.
Association of Src-like protein tyrosine kinases with the CD2 cell surface molecule in rat T lymphocytes and natural killer cells.
Authors: Bell G M , Bolen J B , Imboden J B
Published: October 2015

The cell surface molecule CD2 has a signaling role in the activation of T lymphocytes and natural killer cells. Because perturbation of CD2 leads to the appearance of tyrosine-phosphorylated proteins, we investigated the possibility that CD2 associates with cytoplasmic protein tyrosine kinases. As determined by in vitro kinase assays and phosphoamino acid analysis, protein tyrosine kinase activity coprecipitated with CD2 from rat T lymphocytes, T lymphoblasts, thymocytes, interleukin-2-activated natural killer cells, and RNK-16 cells (a rat natural killer cell line). In each case, both p56lck and p59fyn were identified in the CD2 immunoprecipitate. In the thymus, the association between CD2 and these kinases occurred predominately in a small subset of thymocytes that had the cell surface phenotype of mature T cells, indicating that the association is a regulated event and occurs late in T-cell ontogeny. The finding that CD2 is associated with p56lck and p59fyn in detergent lysates suggests that interactions with these Src-like protein kinases play a critical role in CD2-mediated signal transduction.
The fission yeast genes pyp1+ and pyp2+ encode protein tyrosine phosphatases that negatively regulate mitosis.
Authors: Ottilie S , Chernoff J , Hannig G , Hoffman C S , Erikson R L
Published: October 2015

We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PTPases) to identify two Schizosaccharomyces pombe genes encoding PTPases. We previously described the cloning of pyp1+ (S. Ottilie, J. Chernoff, G. Hannig, C. S. Hoffman, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 88:3455-3459, 1991), and here we describe a second gene, called pyp2+. The C terminus of each protein contains sequences conserved in the apparent catalytic domains of all known PTPases. Disruption of pyp2+ results in viable cells, as was the case for pyp1+, whereas disruption of pyp2+ and pyp1+ results in synthetic lethality. Overexpression of either pyp1+ or pyp2+ in wild-type strains leads to a delay in mitosis but is suppressed by a wee1-50 mutation at 35 degrees C or a cdc2-1w mutation. A pyp1 disruption suppresses the temperature-sensitive lethality of a cdc25-22 mutation. Our data suggest that pyp1+ and pyp2+ act as negative regulators of mitosis upstream of the wee1+/mik1+ pathway.
Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding.
Authors: Shaulian E , Zauberman A , Ginsberg D , Oren M
Published: October 2015

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.
Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.
Authors: Singh K K , Small G M , Lewin A S
Published: October 2015

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.
Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.
Authors: Shan B , Zhu X , Chen P L , Durfee T , Yang Y , Sharp D , Lee W H
Published: October 2015

The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Ap12, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Ap12 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Ap12 encodes a protein with properties known to be characteristic of transcription factor E2F.
Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae.
Authors: Yano R , Oakes M , Yamaghishi M , Dodd J A , Nomura M
Published: October 2015

The SRP1-1 mutation is an allele-specific dominant suppressor of temperature-sensitive mutations in the zinc-binding domain of the A190 subunit of Saccharomyces cerevisiae RNA polymerase I (Pol I). We found that it also suppresses temperature-sensitive mutations in the zinc-binding domain of the Pol I A135 subunit. This domain had been suggested to be in physical proximity to the A190 zinc-binding domain. We have cloned the SRP1 gene and determined its nucleotide sequence. The gene encodes a protein of 542 amino acids consisting of three domains: the central domain, which is composed of eight (degenerate) 42-amino-acid contiguous tandem repeats, and the surrounding N-terminal and C-terminal domains, both of which contain clusters of acidic and basic amino acids and are very hydrophilic. The mutational alteration (P219Q) responsible for the suppression was found to be in the central domain. Using antibody against the SRP1 protein, we have found that SRP1 is mainly localized at the periphery of the nucleus, apparently more concentrated in certain regions, as suggested by a punctate pattern in immunofluorescence microscopy. We suggest that SRP1 is a component of a larger macromolecular complex associated with the nuclear envelope and interacts with Pol I either directly or indirectly through other components in the structure containing SRP1.
Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.
Authors: Rose A M , Joyce P B , Hopper A K , Martin N C
Published: October 2015

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.
Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2.
Authors: Wek R C , Cannon J F , Dever T E , Hinnebusch A G
Published: October 2015

GCN2 is a protein kinase in Saccharomyces cerevisiae that is required for increased expression of the transcriptional activator GCN4 in amino acid-starved cells. GCN2 stimulates GCN4 synthesis at the translational level by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2). We identified a truncated form of the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by its ability to restore derepression of GCN4 expression in a strain containing the partially defective gcn2-507 allele. Genetic analysis suggests that the truncated GLC7 allele has a dominant negative phenotype, reducing the level of native type 1 protein phosphatase activity in the cell. The truncated form of GLC7 does not suppress the regulatory defect associated with a gcn2 deletion or a mutation in the phosphorylation site of eIF-2 alpha (Ser-51). In addition, the presence of multiple copies of wild-type GLC7 impairs the derepression of GCN4 that occurs in response to amino acid starvation or dominant-activating mutations in GCN2. These findings suggest that the phosphatase activity of GLC7 acts in opposition to the kinase activity of GCN2 in modulating the level of eIF-2 alpha phosphorylation and the translational efficiency of GCN4 mRNA. This conclusion is supported by biochemical studies showing that the truncated GLC7 allele increases the level of eIF-2 alpha phosphorylation in the gcn2-507 mutant to a level approaching that seen in wild-type cells under starvation conditions. The truncated GLC7 allele also leads to reduced glycogen accumulation, indicating that this protein phosphatase is involved in regulating diverse metabolic pathways in yeast cells.
Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP.
Authors: Park D , Rhee S G
Published: October 2015

The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.
Segments of the POU domain influence one another's DNA-binding specificity.
Authors: Aurora R , Herr W
Published: October 2015

The ubiquitously expressed mammalian POU-domain protein Oct-1 specifically recognizes two classes of cis-acting regulatory elements that bear little sequence similarity, the octamer motif ATGCAAAT and the TAATGARAT motif. The related pituitary-specific POU protein Pit-1 also recognizes these two motifs but, unlike Oct-1, binds preferentially to the TAATGARAT motif. Yet in our assay, Pit-1 still binds octamer elements better than does the octamer motif-binding protein Oct-3. The POU domain is responsible for recognizing these diverse regulatory sequences through multiple DNA contacts that include the two POU subdomains, the POU-specific region, and the POU homeodomain. The DNA-binding properties of 10 chimeric POU domains, in which different POU-domain segments are derived from either Oct-1 or Pit-1, reveal a high degree of structural plasticity; these hybrid proteins all bind DNA well and frequently bind particular sites better than does either of the parental POU domains. In these chimeric POU domains, the POU-specific A and B boxes and the hypervariable POU linker can influence DNA-binding specificity. The surprising result is that the influence a particular segment has on DNA-binding specificity can be greatly affected by the origin of other segments of the POU domain and the sequence of the binding site. Thus, the broad but selective DNA-binding specificity of Oct-1 is conferred both by multiple DNA contacts and by dynamic interactions within the DNA-bound POU domain.