bluuebunny/crossref_metadata_2025 huggingface dataset:
Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73.
Authors: Smeal T , Binetruy B , Mercola D , Grover-Bardwick A , Heidecker G , Rapp U R , Karin M
Published: October 2015

In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun's activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun's activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.
Regulation and expression of a growth arrest-specific gene (gas5) during growth, differentiation, and development.
Authors: Coccia E M , Cicala C , Charlesworth A , Ciccarelli C , Rossi G B , Philipson L , Sorrentino V
Published: October 2015

The growth arrest-specific gas5 gene was isolated from mouse genomic DNA and structurally characterized. The transcriptional unit is divided into 12 exons that span around 7 kb. An alternative splicing mechanism gives rise to two mature mRNAs which contain either 11 or 12 exons, and both are found in the cytoplasm of growth-arrested cells. In vivo, the gas5 gene is ubiquitously expressed in mouse tissues during development and adult life. In Friend leukemia and NIH 3T3 cells, the levels of gas5 gene mRNA were high in saturation density-arrested cells and almost undetectable in actively growing cells. Run-on experiments indicated that the gas5 gene is transcribed at the same level in both growing and arrested cells. On the other hand, in dimethyl sulfoxide-induced differentiating cells a sharp decrease in the rate of transcription was observed shortly before the cells reached the postmitotic stage. These results indicate that in density-arrested cells accumulation of gas5 mRNA is controlled at the posttranscriptional level while in differentiating cells expression is regulated transcriptionally.
A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.
Authors: Paquette Y , Doyon L , Laperrière A , Hanna Z , Ball J , Sekaly R P , Jolicoeur P
Published: October 2015

The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to be derived from the progenitor double-positive T cells. These results suggest the existence of similar and common factors in CD4+ CD8- and CD4- CD8+ T cells and support a model of differentiation of CD4+ CD8+ T cells through common signal(s) involved in turning off the expression of the CD4 or CD8 gene.
A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome.
Authors: Schwer B , Guthrie C
Published: October 2015

PRP16 is an RNA-dependent ATPase required for the second catalytic step of splicing in vitro. A dominant suppressor of a branchpoint mutation in Saccharomyces cerevisiae, the prp16-1 allele, contains a Tyr to Asp change in the nucleotide-binding site consensus sequence. We now find that cells harboring the prp16-1 allele have a general growth defect that is exacerbated at cold temperatures. The mutant is dominant over the wild-type gene when overexpressed. Purified Prp16-1 protein binds to the spliceosome with apparently wild-type affinity; however, it only weakly complements the second-step block in a PRP16-depleted extract. Analysis of purified Prp16-1 revealed that the rate of ATP hydrolysis is greatly reduced. These results can account for the dominant negative growth phenotype and argue that the ATPase activity of PRP16 is essential for its role in splicing. Moreover, since PRP16 is a member of the DEAD/H box families, these findings have important implications for a large class of proteins.
Occupancy of upstream regulatory sites in vivo coincides with major histocompatibility complex class I gene expression in mouse tissues.
Authors: Dey A , Thornton A M , Lonergan M , Weissman S M , Chamberlain J W , Ozato K
Published: October 2015

The major histocompatibility complex (MHC) class I HLA-B7 transgene carrying a 660-bp upstream sequence is expressed in the mouse with tissue specificity that parallels that of the expression of endogenous mouse MHC class I (H-2) genes. We have performed in vivo genomic footprinting for the HLA-B7 transgene and the endogenous H-2Kb gene. We show that the upstream region of both the transgene and the endogenous gene was extensively occupied in spleen tissue, where these genes are expressed at high levels. In contrast, no occupancy was detected in brain tissue, where expression of these genes is virtually absent. Sites exhibiting in vivo protection correspond to cis elements previously shown to bind to nuclear factors in vitro, including the constitutive enhancer region I and the interferon response element. The strongest tissue-specific protection was detected at site alpha, located downstream from the interferon response element. Site alpha bound a constitutively expressed nuclear factor(s) in vitro that exhibited an overlapping specificity which may involve a nuclear hormone receptor, RXR, and an AP-1-related factor. Site alpha was functional in vivo, as it enhanced MHC class I transcription in lymphocytes. These results show that the tissue-specific occupancy of the MHC class I regulatory sequences in vivo correlates with their expression and suggest that in vivo occupancy is controlled by a mechanism other than the mere presence of factors capable of binding to these sites. Our results suggest that a sequence present in the 660-bp upstream region in a human leukocyte antigen gene directs tissue-specific occupancy of MHC class I genes in vivo, independently of their position and copy number, illustrating a potential advantage of using a transgene for delimitation of the sequence requirement for in vivo occupancy.
Effects of mutation position on frequency of marker rescue by homologous recombination.
Authors: Jiang L , Connor A , Shulman M J
Published: October 2015

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.
Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.
Authors: Valsamakis A , Schek N , Alwine J C
Published: October 2015

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.
Meiotic induction of the yeast HOP1 gene is controlled by positive and negative regulatory sites.
Authors: Vershon A K , Hollingsworth N M , Johnson A D
Published: October 2015

The process of meiosis and sporulation in the yeast Saccharomyces cerevisiae is a highly regulated developmental pathway dependent on genetic as well as nutritional signals. The HOP1 gene, which encodes a component of meiotic chromosomes, is not expressed in mitotically growing cells, but its transcription is induced shortly after yeast cells enter the meiotic pathway. Through a series of deletions and mutations in the HOP1 promoter, we located two regulatory sites that are essential for proper regulation of HOP1. One site, called URS1H, brings about repression of HOP1 in mitotic cells and functions as an activator sequence in cells undergoing meiosis. The second site, which we designated UASH, acts as an activator sequence in meiotic cells and has similarity to the binding site of the mammalian CCAAT/enhancer binding protein (C/EBP). Both sites are required for full meiotic induction of the HOP1 promoter. We conclude that in mitotic yeast cells, the URS1H site maintains the repressed state of the HOP1 promoter, masking the effect of the UASH site. Upon entry into meiosis, repression is lifted, allowing the URS1H and UASH sites to activate high-level transcription.
Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid mimosine.
Authors: Dijkwel P A , Hamlin J L
Published: October 2015

In previous studies, we used two complementary two-dimensional gel electrophoretic methods to examine replication intermediates in the 240-kb amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells (J. P. Vaughn, P. A. Dijkwel, and J. L. Hamlin, Cell 61:1075-1087, 1990). Surprisingly, in both asynchronous and early-S-phase cultures, initiation bubbles were detected in several contiguous fragments from a previously defined 28-kb initiation locus. However, because of the low levels of bubblelike structures observed on gels, it has been suggested that these structures might represent artifacts, possibly unrelated to replication per se. In this study, we have achieved much more synchronous entry into S phase by using a novel inhibitor and have isolated replication intermediates by a new procedure that largely eliminates branch migration and shear. Under these conditions, we find that (i) the relative number of bubblelike structures detected in fragments from the initiation locus is markedly increased, (ii) bubbles are detected at multiple sites scattered throughout the region lying between the DHFR and 2BE2121 genes, and (iii) bubbles appear and disappear in this region with the kinetics expected of an early-firing origin. These data strengthen the proposal that in vivo, initiation can occur at any of a large number of sites scattered throughout a broad zone in the DHFR domain.
95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site.
Authors: Stephens R M , Sithanandam G , Copeland T D , Kaplan D R , Rapp U R , Morrison D K
Published: October 2015

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.
Multiple members of the retinoic acid receptor family are capable of mediating the granulocytic differentiation of HL-60 cells.
Authors: Robertson K A , Emami B , Mueller L , Collins S J
Published: October 2015

The complex and diverse biological effects of retinoic acid (RA) are mediated through specific receptors that are members of the steroid hormone family of nuclear transcription factors. The RA receptor family consists of multiple structurally distinct RA receptors, which diverge primarily at the NH2-terminal domain. The evolutionary conservation of this divergent region in individual RA receptors among different species together with their tissue-specific patterns of expression suggest that the biological function and activity of the individual RA receptors may be confined to specific tissues. To test this hypothesis in hematopoietic cells, we used retrovirus-mediated gene transduction to introduce the RA receptors RAR-alpha, RAR-beta, and RAR-gamma as well as RXR-alpha into a mutant subclone of the HL-60 promyelocytic leukemia cell line (designated HL-60R) that is relatively resistant to RA-induced granulocytic differentiation. We found that each of these structurally distinct RA receptors could restore sensitivity of the HL-60R cells to RA. A critical threshold number of transduced receptors per cell appears to be necessary to restore this functional activity. Thus, the capability to mediate granulocytic differentiation of HL-60 cells is shared among distinctly different RA receptors.
Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor.
Authors: Zhou P , Szczypka M S , Sosinowski T , Thiele D J
Published: October 2015

The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.
Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome.
Authors: Johnson R E , Henderson S T , Petes T D , Prakash S , Bankmann M , Prakash L
Published: October 2015

rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.
A delay in the Saccharomyces cerevisiae cell cycle that is induced by a dicentric chromosome and dependent upon mitotic checkpoints.
Authors: Neff M W , Burke D J
Published: October 2015

Dicentric chromosomes are genetically unstable and depress the rate of cell division in Saccharomyces cerevisiae. We have characterized the effects of a conditionally dicentric chromosome on the cell division cycle by using microscopy, flow cytometry, and an assay for histone H1 kinase activity. Activating the dicentric chromosome induced a delay in the cell cycle after DNA replication and before anaphase. The delay occurred in the absence of RAD9, a gene required to arrest cell division in response to DNA damage. The rate of dicentric chromosome loss, however, was elevated in the rad9 mutant. A mutation in BUB2, a gene required for arrest of cell division in response to loss of microtubule function, diminished the delay. Both RAD9 and BUB2 appear to be involved in the cellular response to a dicentric chromosome, since the conditionally dicentric rad9 bub2 double mutant was highly inviable. We conclude that a dicentric chromosome results in chromosome breakage and spindle aberrations prior to nuclear division that normally activate mitotic checkpoints, thereby delaying the onset of anaphase.
Alternative splicing of a human alpha-tropomyosin muscle-specific exon: identification of determining sequences.
Authors: Graham I R , Hamshere M , Eperon I C
Published: October 2015

The human alpha-tropomyosin gene hTMnm has two mutually exclusive versions of exon 5 (NM and SK), one of which is expressed specifically in skeletal muscle (exon SK). A minigene construct expresses only the nonmuscle (NM) isoform when transfected into COS-1 cells and both forms when transfected into myoblasts. Twenty-four mutants were produced to determine why the SK exon is not expressed in COS cells. The results showed that exons NM and SK are not in competition for splicing to the flanking exons and that there is no intrinsic barrier to splicing between the exons. Instead, exon SK is skipped whenever there are flanking introns. Splicing of exon SK was induced when the branch site sequence 70 nucleotides upstream of the exon was mutated to resemble the consensus and when the extremities of the exon itself were changed to the corresponding NM sequence. Precise swaps of the NM and SK exon sequences showed that the exon sequence effect was dominant to that of intron sequences. The mechanism of regulation appears to be unlike that of other tropomyosin genes. We propose that exclusion of exon SK arises because its 3' splicing signals are weak and are prevented by an exon-specific repressor from competing for splice site recognition.
HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster.
Authors: Biessmann H , Valgeirsdottir K , Lofsky A , Chin C , Ginther B , Levis R W , Pardue M L
Published: October 2015

Eight terminally deleted Drosophila melanogaster chromosomes have now been found to be "healed." In each case, the healed chromosome end had acquired sequence from the HeT DNA family, a complex family of repeated sequences found only in telomeric and pericentric heterochromatin. The sequences were apparently added by transposition events involving no sequence homology. We now report that the sequences transposed in healing these chromosomes identify a novel transposable element, HeT-A, which makes up a subset of the HeT DNA family. Addition of HeT-A elements to broken chromosome ends appears to be polar. The proximal junction between each element and the broken chromosome end is an oligo(A) tract beginning 54 nucleotides downstream from a conserved AATAAA sequence on the strand running 5' to 3' from the chromosome end. The distal (telomeric) ends of HeT-A elements are variably truncated; however, we have not yet been able to determine the extreme distal sequence of a complete element. Our analysis covers approximately 2,600 nucleotides of the HeT-A element, beginning with the oligo(A) tract at one end. Sequence homology is strong (greater than 75% between all elements studied). Sequence may be conserved for DNA structure rather than for protein coding; even the most recently transposed HeT-A elements lack significant open reading frames in the region studied. Instead, the elements exhibit conserved short-range sequence repeats and periodic long-range variation in base composition. These conserved features suggest that HeT-A elements, although transposable elements, may have a structural role in telomere organization or maintenance.
Early meiotic transcripts are highly unstable in Saccharomyces cerevisiae.
Authors: Surosky R T , Esposito R E
Published: October 2015

Meiosis in Saccharomyces cerevisiae requires the induction of a large number of genes whose mRNAs accumulate at specific times during meiotic development. This study addresses the role of mRNA stability in the regulation of meiosis-specific gene expression. Evidence is provided below demonstrating that the levels of meiotic mRNAs are exquisitely regulated by both transcriptional control and RNA turnover. The data show that (i) early meiotic transcripts are extremely unstable when expressed during either vegetative growth or sporulation, and (ii) transcriptional induction, rather than RNA turnover, is the predominant mechanism responsible for meiosis-specific transcript accumulation. When genes encoding the early meiotic mRNAs are fused to other promoters and expressed during vegetative growth, their mRNA half-lives, of under 3 min, are among the shortest known in S. cerevisiae. Since these mRNAs are only twofold more stable when expressed during sporulation, we conclude that developmental regulation of mRNA turnover can be eliminated as a major contributor to meiosis-specific mRNA accumulation. The rapid degradation of the early mRNAs at all stages of the yeast life cycle, however, suggests that a specific RNA degradation system operates to maintain very low basal levels of these transcripts during vegetative growth and after their transient transcriptional induction in meiosis. Studies to identify specific cis-acting elements required for the rapid degradation of early meiotic transcripts support this idea. A series of deletion derivatives of one early meiosis-specific gene, SPO13, indicate that its mRNA contains determinants, located within the coding region, which contribute to the high instability of this transcript. Translation is another component of the degradation mechanism since frameshift and nonsense mutations within the SPO13 mRNA stabilize the transcript.
Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.
Authors: Dingermann T , Werner H , Schütz A , Zündorf I , Nerke K , Knecht D , Marschalek R
Published: October 2015

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.
Housekeeping Na,K-ATPase alpha 1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind.
Authors: Suzuki-Yagawa Y , Kawakami K , Nagano K
Published: October 2015

Na,K-ATPase alpha 1 subunit gene (ATP1A1) is one of the housekeeping genes involved in homeostasis of Na+ and K+ in all animal cells. We identified and characterized the cis-acting elements that regulate the expression of ATP1A1. The region between -155 and -49 was determined as a positive regulatory region in five cultured cell lines of different tissue origins (MDCK, B103, L6, 3Y1, and HepG2). The region was divided into three subregions: from -120 to -106 (including the Sp1 binding site), from -102 to -61, and from -58 to -49 (including an Sp1 consensus sequence). Cell type-specific factors binding to the middle subregion (from -102 to -61) were detected by gel retardation analysis, using nuclear extracts prepared from MDCK and B103 cells. Two gel retardation complexes were formed in the B103 nuclear extract, and three were formed in the MDCK nuclear extract. DNA binding regions of these factors were located at -88 to -69 and differed from each other in DNase I footprinting experiments. These factors also showed different binding characteristics in gel retardation competition and methylation interference experiments. The identified cis element was named the ATP1A1 regulatory element. The core sequence of this element is found in several other genes involved in cellular energy metabolism, suggesting that the sequence is a common regulatory element responsive to the state of energy metabolism.
Replication forks pause at yeast centromeres.
Authors: Greenfeder S A , Newlon C S
Published: October 2015

The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used two-dimensional agarose gel electrophoresis to analyze the replication of the chromosomal copies of yeast CEN1, CEN3, and CEN4 and determine the fate of replication forks that encounter the protein-DNA complex at the centromere. We have shown that replication fork pause sites are coincident with each of these centromeres and therefore probably with all yeast centromeres. We have analyzed the replication of plasmids containing mutant derivatives of CEN3 to determine whether the replication fork pause site is a result of an unusual structure adopted by centromere DNA or a result of the protein-DNA complex formed at the centromere. The mutant centromere derivatives varied in function as well as the ability to form the nuclease-resistant core structure. The data obtained from analysis of these derivatives indicate that the ability to cause replication forks to pause correlates with the ability to form the nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. Our findings further suggest that the centromere protein-DNA complex is present during S phase when replication forks encounter the centromere and therefore may be present throughout the cell cycle.
The DNA-binding defect observed in major histocompatibility complex class II regulatory mutants concerns only one member of a family of complexes binding to the X boxes of class II promoters.
Authors: Herrero Sanchez C , Reith W , Silacci P , Mach B
Published: October 2015

The X box of major histocompatibility complex class II promoters is essential for proper expression of class II genes. Here we show that two distinct protein-DNA complexes (A and B), which exhibit similar binding characteristics and identical contact points on the X box, can be formed. This suggests the existence of a family of related X box-binding factors. Complex B (and not complex A) is specifically affected in primary combined immunodeficiency, a congenital defect in class II gene regulation. RFX1, the first X box-binding protein cloned, encodes a functionally relevant factor present in complex A and not in complex B as originally suspected. This report also illustrates the need for caution in correlating specific cloned proteins with nuclear factors identified by DNA-binding assays, particularly when dealing with families of related proteins.
Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.
Authors: McCabe P C , Haubruck H , Polakis P , McCormick F , Innis M A
Published: October 2015

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.
Formation of the 3' end of sea urchin U1 small nuclear RNA occurs independently of the conserved 3' box and on transcripts initiated from a histone promoter.
Authors: Wendelburg B J , Marzluff W F
Published: October 2015

The formation of the 3' end of vertebrate small nuclear RNAs (snRNAs) requires that transcription initiate from an snRNA promoter. There is a loosely conserved required box 5 to 20 nucleotides (nt) 3' of the gene. The sea urchin snRNA genes contain promoter elements different from those of the vertebrate snRNAs. They also contain a characteristic 3' 15-nt sequence which is conserved among different sea urchin snRNA genes. We used microinjection of sea urchin U1 snRNA genes into sea urchin zygotes to define the sequence requirements for U1 snRNA 3'-end formation. Surprisingly, the conserved 3' box is not required for efficient 3'-end formation in vivo. Deletion analysis reveals that the 6 nt immediately 3' of the U1 snRNA are involved in 3'-end formation. Substitution analysis revealed that either these 6 nt 3' of the U1 RNA or the conserved 3' box could direct 3'-end formation. Transcripts initiated from a histone H4 promoter formed U1 3' ends about 50% as efficiently as transcripts initiated from the U1 promoter, even when the U1 end was placed in tandem with a histone 3'-processing signal, suggesting that transcription from an snRNA promoter is not necessary for formation of the 3' end of sea urchin U1 snRNA.
Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.
Authors: Archambault J , Lacroute F , Ruet A , Friesen J D
Published: October 2015

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.
Functional analysis of the human adenosine deaminase gene thymic regulatory region and its ability to generate position-independent transgene expression.
Authors: Aronow B J , Silbiger R N , Dusing M R , Stock J L , Yager K L , Potter S S , Hutton J J , Wiginton D A
Published: October 2015

We previously observed that human ADA gene expression, required for the intrathymic maturation of T cells, is controlled by first-intron sequences. Used as a cis activator, the intron generates copy-dependent reporter expression in transgenic thymocytes, and we here dissect its critical determinants. Of six DNase I-hypersensitive sites (HS sites) in the intron, only HS III was a transfection-active classic enhancer in T cells. The enhancer contains a critical core region, ACATGGCAGTTGGTGGTGGAGGGGAACA, that interacts with at least two factors, ADA-NF1 and ADA-NF2. Activity of the core is strongly augmented by adjacent elements contained within a 200-bp domain corresponding to the limits of HS III hypersensitivity. These core-adjacent sequences include consensus matches for recognition by the AP-1, TCF-1 alpha, mu E, and Ets transcription factor families. In contrast, considerably more extensive sequences flanking the enhancer domain were required for position-independent and copy-proportional expression in transgenic mouse thymocytes. The additionally required upstream segment encompassed the nonenhancer HS II site. The required downstream segment, composed largely of Alu-repetitive DNA, was non-DNase I hypersensitive. Transgenes that lacked either segment were subject to strong positional effects. Among these variably expressing lines, the expression level correlated with the degree of hypersensitivity at HS III. This finding suggests that formation of hypersensitivity is normally facilitated by the flanking segments. These results delineate a complex thymic regulatory region within the intron and indicate that a series of interactions is necessary for the enhancer domain to function consistently within chromatin.
Thymic overexpression of Ttg-1 in transgenic mice results in T-cell acute lymphoblastic leukemia/lymphoma.
Authors: McGuire E A , Rintoul C E , Sclar G M , Korsmeyer S J
Published: October 2015

T-cell translocation gene 1 (Ttg-1), also called rhombotin, is deregulated upon translocation into the alpha/delta T-cell receptor loci in acute lymphoblastic leukemias bearing the t(11;14)(p15;q11). Ttg-1 encodes a nuclear protein, expressed predominantly in neuronal cells, which belongs to a novel family of transcription factors possessing LIM domains. We utilized the lck proximal promoter to overexpress this candidate oncogene in immature thymocytes of transgenic mice. lckPr Ttg-1 mice develop immature, aggressive T-cell leukemia/lymphomas. Tumor incidence is proportional to the level of Ttg-1 expression. Most tumors contain CD4+8+ cells as well as CD4-8+ cells, which have an immature rather than a mature peripheral phenotype. Ttg-1-induced tumorigenesis preferentially affects a minority population of thymocytes representing an immature CD4-8+ intermediate stage between double-negative CD4-8- cells and double-positive CD4+8+ cells. This model indicates that the aberrant expression of putative transcription factors plays a primary role in the genesis of T-cell acute lymphoblastic leukemias.
RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.
Authors: Hartshorne T , Agabian N
Published: October 2015

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.
Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras.
Authors: Medema R H , de Vries-Smits A M , van der Zon G C , Maassen J A , Bos J L
Published: October 2015

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.
An abundant high-mobility-group-like protein is targeted to micronuclei in a cell cycle-dependent and developmentally regulated fashion in Tetrahymena thermophila.
Authors: Wang T , Allis C D
Published: October 2015

In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei. Biochemical data document the fact that HMG B is extremely labile in micronuclei. Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei. Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication. This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair. Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin. However, micronuclei are transcriptionally active during early stages of conjugation. Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented. Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration. We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.
Sperm-specific expression of angiotensin-converting enzyme (ACE) is mediated by a 91-base-pair promoter containing a CRE-like element.
Authors: Howard T , Balogh R , Overbeek P , Bernstein K E
Published: October 2015

The gene encoding the testis isozyme of angiotensin-converting enzyme (testis ACE) is one example of the many genes expressed uniquely during spermatogenesis. This protein is expressed by developing germ cells late in their development and results from the activation of a sperm-specific promoter that is located within intron 12 of the gene encoding the somatic isozyme of ACE. In vitro transcription, DNase footprinting, gel shift assays, and transgenic mouse studies have been used to define the minimal testes ACE promoter and to characterize DNA-protein interactions mediating germ cell-specific expression. These studies show that proper cell- and stage-specific expression of testis ACE requires only a small portion of the immediate upstream sequence extending to -91. A critical motif within this core promoter is a cyclic AMP-responsive element sequence that interacts with a testis-specific transactivating factor. Since this putative cyclic AMP-responsive element has been conserved within the testis ACE promoters of different species and is found at the same site in other genes that are expressed specifically in the testis, it may provide a common mechanism for the recognition of sperm-specific promoters.
The mdm-2 oncogene can overcome wild-type p53 suppression of transformed cell growth.
Authors: Finlay C A
Published: October 2015

Expression of a p53-associated protein, Mdm-2 (murine double minute-2), can inhibit p53-mediated transactivation. In this study, overexpression of the Mdm-2 protein was found to result in the immortalization of primary rat embryo fibroblasts (REFs) and, in conjunction with an activated ras gene, in the transformation of REFs. The effect of wild-type p53 on the transforming properties of mdm-2 was determined by transfecting REFs with ras, mdm-2, and normal p53 genes. Transfection with ras plus mdm-2 plus wild-type p53 resulted in a 50% reduction in the number of transformed foci (relative to the level for ras plus mdm-2); however, more than half (9 of 17) of the cell lines derived from these foci expressed low levels of a murine p53 protein with the characteristics of a wild-type p53. These results are in contrast to previous studies which demonstrated that even minimal levels of wild-type p53 are not tolerated in cells transformed by ras plus myc, E1A, or mutant p53. The mdm-2 oncogene can overcome the previously demonstrated growth-suppressive properties of p53.