bluuebunny/crossref_metadata_2025 huggingface dataset:
urbs1, a gene regulating siderophore biosynthesis in Ustilago maydis, encodes a protein similar to the erythroid transcription factor GATA-1.
Authors: Voisard C , Wang J , McEvoy J L , Xu P , Leong S A
Published: October 2015

Ustilago maydis secretes ferrichrome-type siderophores, ferric-ion-binding compounds, in response to iron starvation. TA2701, a non-enterobactin-producing, non-ferrichrome-utilizing mutant of Salmonella typhimurium LT-2, was employed as a biological indicator in a novel screening method to isolate three N-methyl-N'-nitro-N-nitrosoguanidine-induced U. maydis mutants defective in the regulation of ferrichrome-type siderophore biosynthesis. These mutants displayed a constitutive phenotype; they produced siderophores in the presence of iron concentrations that would typically repress siderophore synthesis in wild-type strains. A 4.8-kb fragment of U. maydis genomic DNA capable of restoring normal regulation of siderophore biosynthesis in the constitutive mutants was identified. This segment of DNA contains an intronless open reading frame that specifies a protein of 950 amino acids containing two finger motifs similar to those found in the erythroid transcription factor GATA-1. Disruption of this open reading frame in a wild-type strain gave rise to cells that produced siderophores constitutively. Genetic studies indicated that the disruption mutation was allelic to the chemically induced mutations, confirming that the structural gene for a regulator rather than a suppressor gene had been cloned. Northern (RNA) analysis of the gene revealed a 4.2-kb transcript that is expressed constitutively at low levels in wild-type cells. The data support the hypothesis that this gene, which we designate urbs1 (Ustilago regulator of biosynthesis of siderophores), acts directly or indirectly to repress biosynthesis of siderophores in U. maydis.
Biphasic ordered induction of heme synthesis in differentiating murine erythroleukemia cells: role of erythroid 5-aminolevulinate synthase.
Authors: Lake-Bullock H , Dailey H A
Published: October 2015

During dimethyl sulfoxide (DMSO)-stimulated differentiation of murine erythroleukemia (MEL) cells, one of the early events is the induction of the heme biosynthetic pathway. While recent reports have clearly demonstrated that GATA-1 is involved in the induction of erythroid cell-specific forms of 5-aminolevulinate synthase (ALAS-2) and porphobilinogen (PBG) deaminase and that cellular iron status plays a regulatory role for ALAS-2, little is known about regulation of the remainder of the pathway. In the current study, we have made use of a stable MEL cell mutant (MEAN-1) in which ALAS-2 enzyme activity is not induced by DMSO, hexamethylene bisacetamide (HMBA), or butyric acid. In this cell line, addition of 2% DMSO to growing cultures results in the normal induction of PBG deaminase and coproporphyrinogen oxidase but not in the induction of the terminal two enzymes, protoporphyrinogen oxidase and ferrochelatase. These DMSO-treated cells did not produce mRNA for beta-globin and do not terminally differentiate. In addition, the cellular level of ALAS activity declines rapidly after addition of DMSO, indicating that ALAS-1 must turn over rapidly at this time. Addition of 75 microM hemin alone to the cultures did not induce cells to terminally differentiate or induce any of the pathway enzymes. However, the simultaneous addition of 2% DMSO and 75 microM hemin caused the cells to carry out a normal program of terminal erythroid differentiation, including the induction of ferrochelatase and beta-globin. These data suggest that induction of the entire heme biosynthetic pathway is biphasic in nature and that induction of the terminal enzymes may be mediated by the end product of the pathway, heme. We have introduced mouse ALAS-2 cDNA into the ALAS-2 mutant cell line (MEAN-1) under the control of the mouse metallothionein promoter (MEAN-RA). When Cd and Zn are added to cultures of MEAN-RA in the absence of DMSO, ALAS-2 is induced but erythroid differentiation does not occur and cells continue to grow normally. In the presence of metallothionein inducers and DMSO, the MEAN-RA cells induce in a fashion similar to that found with the wild-type 270 MEL cells. Induction of the activities of ALAS, PBG deaminase, coproporphyrinogen oxidase, and ferrochelatase occurs. In cultures of MEAN-RA where ALAS-2 had been induced with Cd plus Zn 24 h prior to DMSO addition, onset of heme synthesis occurs more rapidly than when DMSO and Cd plus Zn are added simultaneously. This study reveals that induction of ALAS-2 alone is not sufficient to induce terminal differentiation of the MEAN-RA cells, and it does not appear that ALAS-2 alone is the rate-limiting enzyme of the heme biosynthetic pathway during MEL cell differentiation.
Two novel transmembrane protein tyrosine kinases expressed during Caenorhabditis elegans hypodermal development.
Authors: Morgan W R , Greenwald I
Published: October 2015

We describe our characterization of kin-15 and kin-16, a tandem pair of homologous Caenorhabditis elegans genes encoding transmembrane protein tyrosine kinases (PTKs) with an unusual structure: the predicted extracellular domain of each putative gene product is only about 50 amino acids, and there are no potential autophosphorylation sites in the C-terminal domain. Using lacZ fusions, we found that kin-15 and kin-16 both appear to be expressed during postembryonic development in the large hypodermal syncytium (hyp7) around the time that specific hypodermal cells fuse with hyp7. kin-15 and kin-16 were positioned on the genetic and physical maps, but extrachromosomal arrays containing wild-type kin-15 and/or kin-16 genes were unable to complement candidate lethal mutations. The results suggest that kin-15 and kin-16 may be specifically involved in cell-cell interactions regulating cell fusions that generate the hypodermis during postembryonic development.
The murine alpha B-crystallin/small heat shock protein enhancer: identification of alpha BE-1, alpha BE-2, alpha BE-3, and MRF control elements.
Authors: Gopal-Srivastava R , Piatigorsky J
Published: October 2015

The murine alpha B-crystallin gene (a member of the small heat shock protein family) is expressed constitutively at high levels in the lens and at lower levels in many other tissues, including skeletal muscle. We have previously used the herpes simplex virus thymidine kinase promoter fused to the human growth hormone gene to identify an alpha B-crystallin enhancer at positions -427 to -259 that has high activity in muscle and low activity in lens cell lines. In the study reported here, we performed DNase I footprinting, transfection, mutagenesis, and electrophoretic mobility shift experiments using the murine C2C12 muscle and alpha TN4-1 lens cell lines and the rabbit N/N1003A lens cell line to identify sequences responsible for activity of this enhancer. Enhancer activity in both the muscle and lens cells was dependent on novel elements called alpha BE-1 (-407 to -397), alpha BE-2 (-360 to -327), and alpha BE-3 (-317 to -306). These elements were also weakly occupied by nuclear proteins in L929 cells, which appear to express the alpha B-crystallin gene at a very low level (detectable only by the polymerase chain reaction). A fourth element containing a consensus muscle regulatory factor-binding site called MRF (-300 to -288) was occupied and used only by the C2C12 muscle cells. Cotransfection in NIH 3T3 cells and antibody-gel shift experiments using C2C12 nuclear extracts indicated that MyoD, myogen, or a similar member of this family can activate the alpha B-crystallin enhancer by interaction with the MRF site. Taken together, we conclude that the alpha BE-1, alpha BE-2, and alpha BE-3 elements are shared by both lens and muscle cells, but the MRF element is used only in muscle cells, providing the first example of a muscle-specific control element in a crystallin gene.
Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.
Authors: Fabian J R , Daar I O , Morrison D K
Published: October 2015

The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that maturation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.
A common element involved in transcriptional regulation of two DNA alkylation repair genes (MAG and MGT1) of Saccharomyces cerevisiae.
Authors: Xiao W , Singh K K , Chen B , Samson L
Published: October 2015

The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents. MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA. We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression. Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS. One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S. cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase. Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements. We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S. cerevisiae DNA repair genes.
The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells.
Authors: Liu V F , Weaver D T
Published: October 2015

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.
Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways.
Authors: Burgering B M , de Vries-Smits A M , Medema R H , van Weeren P C , Tertoolen L G , Bos J L
Published: October 2015

Expression of p21rasAsn-17, a dominant negative mutant of p21ras that blocks p21ras activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived growth factor in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21rasAsn-17 does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p21ras in these cells, this indicates that EGF induces a p21ras-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21ras-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21ras and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21ras activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.
Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones.
Authors: Altiok S , Groner B
Published: October 2015

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.
Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.
Authors: Stone J C , Colleton M , Bottorff D
Published: October 2015

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.
E2A expression, nuclear localization, and in vivo formation of DNA- and non-DNA-binding species during B-cell development.
Authors: Jacobs Y , Vierra C , Nelson C
Published: October 2015

A monoclonal antibody (Yae) was characterized and shown to specifically recognize E2A proteins in vivo, including the E2A-Pbx1 fusion gene products, p77E2A-Pbx1 and p85E2A-Pbx1. E2A proteins of a predominant molecular mass of 72 kDa, which comigrated with in vitro-produced rat E12 and and rat E47, were detected in human pro-B, pre-B, mature B, and plasma cell lines. The Yae antibody detected an E2A-containing microE2 enhancer element-binding complex (BCF-1) in pre-B- and mature B-cell lines in electrophoretic mobility shift assays which displayed a migration rate similar to that of in vitro-produced rat E12 and rat E47. A new E2A-containing microE2-binding species (P-E2A) was identified in plasma cells by using electrophoretic mobility shift assays. E2A proteins were detected in pro-B cells but were unable to bind the microE2 site. These observations suggest that the microE2 site is the target of stage-specific E2A regulatory complexes during B-cell development. Immunostaining analyses demonstrated the predominant nuclear localization of E2A proteins. Finally, we have identified an E2A form, designated I-E2A, which is unable to bind DNA. Our observations demonstrate novel in vivo mechanisms for the regulation of transcription by E2A proteins during B-cell development.
Elevated levels of cyclin D1 protein in response to increased expression of eukaryotic initiation factor 4E.
Authors: Rosenwald I B , Lazaris-Karatzas A , Sonenberg N , Schmidt E V
Published: October 2015

Cyclin D1 is a G1-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF-4E]) is a potential regulatory of several posttranscriptional events, and it can itself induce neoplastic transformation. Consequently, we examined eIF-4E as a potential regulator of cyclin D1. Overexpression of cyclin D1 mRNA in NIH 3T3 cells did not increase cyclin D1 protein. In contrast, overexpression of eIF-4E markedly increased the amount of cyclin D1 protein in NIH 3T3 cells. This increase was specific to cyclin D1 in comparison with the retinoblastoma gene product, c-Myc, actin, and eukaryotic initiation factor 2 alpha. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.
The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT.
Authors: Masuda E S , Tokumitsu H , Tsuboi A , Shlomai J , Hung P , Arai K , Arai N
Published: October 2015

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation.
Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements.
Authors: Trepicchio W L , Dyer M A , Baron M H
Published: October 2015

The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene.
Yin-yang 1 activates the c-myc promoter.
Authors: Riggs K J , Saleque S , Wong K K , Merrell K T , Lee J S , Shi Y , Calame K
Published: October 2015

Previous studies on the murine c-myc promoter demonstrated that a ubiquitously present protein, common factor 1 (CF1), bound at two sites located -260 and -390 bp from the P1 transcription start site. CF1 has been purified to near homogeneity and shown to be identical to the zinc finger protein Yin-yang 1 (YY1) as judged by similarity of molecular weight and other biochemical properties, immunological cross-reactivity, and the ability of recombinant YY1 to bind to CF1 sites. In cotransfection experiments, YY1 is a strong activator of transcription from c-myc promoter-based reporters. Furthermore, in murine erythroleukemia cells, overexpressed YY1 causes increased levels of c-myc mRNA initiated from both major transcription initiation sites of the endogenous c-myc gene.
Signals in chicken beta-globin DNA influence chromatin assembly in vitro.
Authors: Liu K , Lauderdale J D , Stein A
Published: October 2015

We have confirmed the result that chicken beta-globin gene chromatin, which possesses the characteristics of active chromatin in erythroid cells, has shortened internucleosome spacings compared with bulk chromatin or that of the ovalbumin gene, which is inactive. To understand how the short (approximately 180-bp) nucleosome repeat arises specifically on beta-globin DNA, we have studied chromatin assembly of cloned chicken beta-globin DNA in a defined in vitro system. With chicken erythrocyte core histones and linker histone H5 as the only cellular components, a cloned 6.2-kb chicken beta-globin DNA fragment assembled into chromatin possessing a regular 180 +/- 5-bp repeat, very similar to what is observed in erythroid cells. A 2-kb DNA subfragment containing the beta A gene and promoter region, but lacking the downstream intergenic region between the beta A and epsilon genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic region facilitates linker histone-induced nucleosome alignment. When the beta A gene was placed on a plasmid that contained a known chromatin-organizing signal, nucleosome alignment with a 180-bp periodicity was restored, whereas nucleosomes on flanking plasmid sequences possessed a 210-bp spacing periodicity. Our results suggest that the shortened 180-bp nucleosome spacing periodicity observed in erythroid cells is encoded in the beta-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the beta A-epsilon intergenic region.
A regulatory element in the CHA1 promoter which confers inducibility by serine and threonine on Saccharomyces cerevisiae genes.
Authors: Bornaes C , Ignjatovic M W , Schjerling P , Kielland-Brandt M C , Holmberg S
Published: October 2015

CHA1 of Saccharomyces cerevisiae is the gene for the catabolic L-serine (L-threonine) dehydratase, which is responsible for biodegradation of serine and threonine. We have previously shown that expression of the CHA1 gene is transcriptionally induced by serine and threonine. Northern (RNA) analysis showed that the additional presence of good nitrogen sources affects induction. This may well be due to inducer exclusion. To identify interactions of cis-acting elements with trans activators of the CHA1 promoter, we performed band shift assays of nuclear protein extracts with CHA1 promoter fragments. By this approach, we identified a protein-binding site of the CHA1 promoter. The footprint of this protein contains the ABF1-binding site consensus sequence. This in vitro binding activity is present irrespectively of CHA1 induction. By deletion analysis, two other elements of the CHA1 promoter, UAS1CHA and UAS2CHA, which are needed for induction of the CHA1 gene were identified. Each of the two sequence elements is sufficient to confer serine and threonine induction upon the CYC1 promoter when substituting its upstream activating sequence. Further, in a cha4 mutant strain which is unable to grow with serine or threonine as the sole nitrogen source, the function of UAS1CHA, as well as that of UAS2CHA, is obstructed.
A C/EBP-binding site in the transferrin promoter is essential for expression in the liver but not the brain of transgenic mice.
Authors: Theisen M , Behringer R R , Cadd G G , Brinster R L , McKnight G S
Published: October 2015

The gene for the iron-binding protein transferrin is transcribed at a high level in liver hepatocytes but is also active in several other cell types, including oligodendrocytes in the brain. Enhancer elements between bp -560 and -44 of the transferrin gene promoter specifically activated transcription from a heterologous promoter in transgenic mouse liver and brain. Within this region, a potent cis-acting element between bp -98 and -83 was found to be essential for gene activity in both cultured hepatocytes and transgenic mouse liver. The -98 to -83 element contains a CCAAT sequence and is specifically bound by a nuclear factor from mouse liver that is homologous to rat liver C/EBP (CAAT enhancer-binding protein). Point mutations within this binding site inhibit factor binding and abolish transcription in transfected hepatoma cells. When placed in the context of the 3,000-bp transferrin promoter, the C/EBP binding site mutation causes a complete loss of transcription in transgenic mouse liver; however, transgene expression in the brain of the same animals was unaffected. These results suggest a modular structure for the transferrin promoter and demonstrate that deletions or specific point mutations can be used to generate transgene promoters with an activity more restricted than that of their endogenous counterparts.
Formation of retinoid X receptor homodimers leads to repression of T3 response: hormonal cross talk by ligand-induced squelching.
Authors: Lehmann J M , Zhang X K , Graupner G , Lee M O , Hermann T , Hoffmann B , Pfahl M
Published: October 2015

Thyroid hormone receptors (TRs) form heterodimers with retinoid X receptors (RXRs). Heterodimerization is required for efficient TR DNA binding to most response elements and transcriptional activation by thyroid hormone. RXRs also function as auxiliary proteins for several other receptors. In addition, RXR alpha can be induced by specific ligands to form homodimers. Here we report that RXR-specific retinoids that induce RXR homodimers are effective repressors of the T3 response. We provide evidence that this repression by RXR-specific ligands occurs by sequestering of RXR from TR-RXR heterodimers into RXR homodimers. This ligand-induced squelching may represent an important mechanism by which RXR-specific retinoids and 9-cis retinoic acid mediate hormonal cross talk among a subfamily of nuclear receptors activated by structurally unrelated ligands.
Sex-lethal autoregulation requires multiple cis-acting elements upstream and downstream of the male exon and appears to depend largely on controlling the use of the male exon 5' splice site.
Authors: Horabin J I , Schedl P
Published: October 2015

The on/off state of the binary switch gene Sex-lethal (Sxl), which controls somatic sexual development in Drosophila melanogaster, is regulated at the level of alternative splicing. In males, in which the gene is off, the default splicing machinery produces nonfunctional mRNAs; in females, in which the gene is on, the autoregulatory activity of the Sxl proteins directs the splicing machinery to produce functional mRNAs. We have used germ line transformation to analyze the mechanism of default and regulated splicing. Our results demonstrate that a blockage mechanism is employed in Sxl autoregulation. However, in contrast to transformer, in which Sxl appears to function by preventing the interaction of splicing factors with the default 3' splice site, a different strategy is used in autoregulation. (i) Multiple cis-acting elements, both upstream and downstream of the male exon, are required. (ii) These cis-acting elements are distant from the splice sites they regulate, suggesting that the Sxl protein cannot function in autoregulation by directly competing with splicing factors for interaction with the regulated splice sites. (iii) The 5' splice site of the male exon appears to be dominant in regulation while the 3' splice site plays a subordinate role.
The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function.
Authors: Watkins J F , Sung P , Prakash L , Prakash S
Published: October 2015

In eukaryotes, the posttranslational conjugation of ubiquitin to various cellular proteins marks them for degradation. Interestingly, several proteins have been reported to contain ubiquitin-like (ub-like) domains that are in fact specified by the DNA coding sequences of the proteins. The biological role of the ub-like domain in these proteins is not known; however, it has been proposed that this domain functions as a degradation signal rendering the proteins unstable. Here, we report that the product of the Saccharomyces cerevisiae RAD23 gene, which is involved in excision repair of UV-damaged DNA, bears a ub-like domain at its amino terminus. This finding has presented an opportunity to define the functional significance of this domain. We show that deletion of the ub-like domain impairs the DNA repair function of RAD23 and that this domain can be functionally substituted by the authentic ubiquitin sequence. Surprisingly, RAD23 is highly stable, and the studies reported herein indicate that its ub-like domain does not mediate protein degradation. Thus, in RAD23 at least, the ub-like domain affects protein function in a nonproteolytic manner.
Mutational analysis of yeast profilin.
Authors: Haarer B K , Petzold A S , Brown S S
Published: October 2015

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.
Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate.
Authors: Flynn D C , Leu T H , Reynolds A B , Parsons J T
Published: October 2015

Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.
DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.
Authors: Ripmaster T L , Vaughn G P , Woolford J L
Published: October 2015

To identify Saccharomyces cerevisiae mutants defective in assembly or function of ribosomes, a collection of cold-sensitive strains generated by treatment with ethyl methanesulfonate was screened by sucrose gradient analysis for altered ratios of free 40S to 60S ribosomal subunits or qualitative changes in polyribosome profiles. Mutations defining seven complementation groups deficient in ribosomal subunits, drs1 to drs7, were identified. We have previously shown that DRS1 encodes a putative ATP-dependent RNA helicase necessary for assembly of 60S ribosomal subunits (T. L. Ripmaster, G. P. Vaughn, and J. L. Woolford, Jr., Proc. Natl. Acad. Sci. USA 89:11131-11135, 1992). Strains bearing the drs2 mutation process the 20S precursor of the mature 18S rRNA slowly and are deficient in 40S ribosomal subunits. Cloning and sequencing of the DRS2 gene revealed that it encodes a protein similar to membrane-spanning Ca2+ ATPases. The predicted amino acid sequence encoded by DRS2 contains seven transmembrane domains, a phosphate-binding loop found in ATP- or GTP-binding proteins, and a seven-amino-acid sequence detected in all classes of P-type ATPases. The cold-sensitive phenotype of drs2 is suppressed by extra copies of the TEF3 gene, which encodes a yeast homolog of eukaryotic translation elongation factor EF-1 gamma. Identification of gene products affecting ribosome assembly and function among the DNAs complementing the drs mutations validates the feasibility of this approach.
Defective replication of psoralen adducts detected at the gene-specific level in xeroderma pigmentosum variant cells.
Authors: Misra R R , Vos J M
Published: October 2015

Replication of damaged DNA is suspected to play an important role in cell cycle, genetic stability, and survival pathways. Using psoralen photoaddition as prototype DNA damage and the renaturing agarose gel electrophoresis technique to measure DNA cross-linking in individual genes, Vos and Hanawalt previously observed efficient bypass replication of psoralen monoadducts in human genes (J.-M. H. Vos and P. C. Hanawalt, Cell 50:789-799, 1987). To understand the mechanism of bypass replication in human cells, mutants affected in such a process would be useful. We now report that cells from individuals suffering from the hereditary recessive syndrome xeroderma pigmentosum variant (XPV) are hypersensitive to killing induced by photoactivated psoralen. In addition, analysis of psoralen-mediated DNA cross-linking in the rRNA genes indicated that although repair of psoralen adducts was similar to that of normal individuals, XPV cells were markedly deficient in the ability to bypass psoralen adducts during replication; in comparison with normal cells, approximately half as many monoadducts were bypassed during replication in XPV cells. Furthermore, in contrast to normal cells, replication of interstrand cross-links was not detected in XPV. This is the first demonstration of a deficiency in bypass replication detected at the gene-specific level in vivo. A model involving a strand-specific defect in recombinational bypass in XPV is proposed.
A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae.
Authors: Muscarella D E , Vogt V M
Published: October 2015

Pp LSU3 is a mobile group I intron in the extrachromosomal nuclear ribosomal DNA (rDNA) of Physarum polycephalum. As found for other mobile introns, Pp LSU3 encodes a site-specific endonuclease, I-Ppo, which mediates "homing" to unoccupied target sites in Physarum rDNA. The recognition sequence for this enzyme is conserved in all eucaryotic nuclear rDNAs. We have introduced this intron into a heterologous species, Saccharomyces cerevisiae, in which nuclear group I introns have not been detected. The expression of Pp LSU3, under control of the inducible GAL10 promoter, was found to be lethal as a consequence of double-strand breaks in the rDNA. However, surviving colonies that are resistant to the lethal effects of I-Ppo because of alterations in the rDNA at the cleavage site were recovered readily. These survivors are of two classes. The first comprises cells that acquired one of three types of point mutations. The second comprises cells in which Pp LSU3 became inserted into the rDNA. In both cases, each resistant survivor appears to carry the same alterations in all approximately 150 rDNA repeats. When it is embedded in yeast rDNA, Pp LSU3 leads to the synthesis of I-Ppo and appears to be mobile in appropriate genetic crosses. The existence of yeast cells carrying a mobile intron should allow dissection of the steps that allow expression of the highly unusual I-Ppo gene.
A nucleosome assembly factor is a constituent of simian virus 40 minichromosomes.
Authors: Krude T , de Maddalena C , Knippers R
Published: October 2015

Using in vitro replication assays, we compared native with salt-treated simian virus 40 minichromosomes isolated from infected cell nuclei. Minichromosomes from both preparations contain the full complement of nucleosomes, but salt treatment removes histone H1 and a fraction of nonhistone chromatin proteins. Both types of minichromosomes served well as templates for in vitro replication, but the structures of the replication products were strikingly different. Replicated salt-treated minichromosomes contained, on average, about half the normal number of nucleosomes as previously shown (T. Krude and R. Knippers, Mol. Cell. Biol. 11:6257-6267, 1991). In contrast, the replicated untreated minichromosomes were found to be densely packed with nucleosomes, indicating that an assembly of new nucleosomes occurred during in vitro replication. Biochemical and immunological data showed that the fraction of nonhistone chromatin proteins associated with native minichromosomes includes a nucleosome assembly activity that appears to be closely related to chromatin assembly factor I (S. Smith and B. W. Stillman, Cell 58:15-25, 1989). Furthermore, this minichromosome-bound nucleosome assembly factor is able to exert its activity in trans to replicating protein-free competitor DNA. Thus, native chromatin itself contains the activities required for an ordered assembly of nucleosomes during the replication process.
P nucleotides in V(D)J recombination: a fine-structure analysis.
Authors: Meier J T , Lewis S M
Published: October 2015

Antigen receptor genes acquire junctional inserts upon assembly from their component, germ line-encoded V, D, and J segments. Inserts are generally of random sequence, but a small number of V-D, D-J, or V-J junctions are exceptional. In such junctions, one or two added base pairs inversely repeat the sequence of the abutting germ line DNA. (For example, a gene segment ending AG might acquire an insert beginning with the residues CT upon joining). It has been proposed that the nonrandom residues, termed "P nucleotides," are a consequence of an obligatory end-modification step in V(D)J recombination. P insertion in normal, unselected V(D)J joining products, however, has not been rigorously established. Here, we use an experimentally manipulable system, isolated from immune selection of any kind, to examine the fine structure of V(D)J junctions formed in wild-type lymphoid cells. Our results, according to statistical tests, show the following, (i) The frequency of P insertion is influenced by the DNA sequence of the joined ends. (ii) P inserts may be longer than two residues in length. (iii) P inserts are associated with coding ends only. Additionally, a systematic survey of published P nucleotide data shows no evidence for variation in P insertion as a function of genetic locus and ontogeny. Together, these analyses establish the generality of the P nucleotide pattern within inserts but do not fully support previous conjectures as to their origin and centrality in the joining reaction.
Erythroid differentiation of mouse erythroleukemia cells results in reorganization of protein-DNA complexes in the mouse beta maj globin promoter but not its distal enhancer.
Authors: Reddy P M , Shen C K
Published: October 2015

Dimethyl sulfoxide (DMSO) induction of mouse erythroleukemia (MEL) cells represents a well-defined in vitro system of terminal erythroid differentiation. We have studied the molecular mechanisms of transcriptional activation of the mouse beta maj globin gene during MEL cell differentiation by analyzing nuclear factor-DNA interactions in vivo at the gene's upstream promoter and a distal enhancer, 5'HS-2. Genomic footprinting data indicate that three motifs, CAC, NF-E2/AP1, and GATA-1, of the 5'HS-2 enhancer are bound with nuclear factors in MEL cells both prior to and after DMSO induction. No obvious conformational change of these nuclear factor-DNA complexes could be detected upon terminal differentiation of MEL cells. On the other hand, DMSO induction of MEL cells leads to the formation of specific nuclear factor-DNA complexes at several transcriptional regulatory elements of the mouse beta maj globin upstream promoter. Our genomic footprinting data have interesting implications with respect to the molecular mechanisms of transcriptional regulation and chromatin change of the mouse beta maj globin gene during erythroid differentiation.