bluuebunny/crossref_metadata_2025 huggingface dataset:
The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.
Authors: Buermeyer A B , Thompson N E , Strasheim L A , Burgess R R , Farnham P J
Published: October 2015

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.
Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases.
Authors: Campbell M A , Sefton B M
Published: October 2015

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.
Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants.
Authors: Irniger S , Sanfaçon H , Egli C M , Braus G H
Published: October 2015

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.
The -6.1-kilobase chicken lysozyme enhancer is a multifactorial complex containing several cell-type-specific elements.
Authors: Grewal T , Theisen M , Borgmeyer U , Grussenmeyer T , Rupp R A , Stief A , Qian F , Hecht A , Sippel A E
Published: October 2015

In the chromatin domain of the chicken lysozyme gene of myeloid and oviduct cells, which both have the potential to activate the gene, a developmentally stable DNase I-hypersensitive site is formed around 6.1 kb upstream of the gene. This implies that this DNA region, which has previously been demonstrated to function as a transcriptional enhancer element in myeloid cells, is intimately involved in the cell-type-specific activation of the lysozyme gene locus. Deletion analysis identifies a 157-bp minimal fragment that confers the same promacrophage-specific enhancer activity as the originally described 562-bp -6.1-kb enhancer fragment. By introducing specific point mutations, we demonstrate in transient gene transfer experiments that the minimal fragment consists of at least six adjacent elements, each substantially contributing to enhancer function. The compact multifactorial enhancer complex includes a nuclear factor I (NF-I)/TGGCA binding site, homologies to AP1, and octanucleotide or enhancer core consensus motifs. Point mutation of the NF-I binding site results in the loss of NF-I binding in vitro and enhancer activity in vivo after gene transfer. Surprisingly, four overlapping oligonucleotides, each consisting of at least two elements of the -6.1-kb enhancer, confer myeloid-cell-specific enhancer activity. We found several myeloid-cell-specific DNA-binding proteins interacting with the -6.1-kb enhancer, a result consistent with that described above. Therefore, we suggest that more than a single trans-acting factor mediates the cell type specificity of the -6.1-kb enhancer.
Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro.
Authors: Arndt K M , Ricupero S L , Eisenmann D M , Winston F
Published: October 2015

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.
The retinoblastoma gene product regulates Sp1-mediated transcription.
Authors: Kim S J , Onwuta U S , Lee Y I , Li R , Botchan M R , Robbins P D
Published: October 2015

We have demonstrated that the retinoblastoma gene product (Rb) can positively regulate transcription from the fourth promoter of the insulinlike growth factor II gene. Two copies of a motif (the retinoblastoma control element) similar to that found in the human c-fos, transforming growth factor beta 1, and c-myc promoters are responsible for conferring Rb regulation to the fourth promoter of the insulinlike growth factor II gene. We have shown that the transcription factor Sp1 can bind to and stimulate transcription from the retinoblastoma control element motif. Moreover, by using a GAL4-Sp1 fusion protein, we have directly demonstrated that Rb positively regulates Sp1 transcriptional activity in vivo. These results indicate that Rb can function as a positive regulator of transcription and that Sp1 is one potential target, either directly or indirectly, for transcriptional regulation by Rb.
Point mutations in the Drosophila hairy gene demonstrate in vivo requirements for basic, helix-loop-helix, and WRPW domains.
Authors: Wainwright S M , Ish-Horowicz D
Published: October 2015

The Drosophila pair-rule gene, hairy (h), encodes a nuclear basic helix-loop-helix (bHLH) protein that regulates embryonic segmentation and adult bristle patterning. In both cases, the h protein behaves as a transcriptional repressor. In this study, we determined the molecular nature of 12 h alleles. One mutation maps within the HLH domain, consistent with h function requiring homodimerization or heterodimerization with other HLH proteins. A second mutation lies in the basic domain, suggesting that DNA binding is required for h activity. Several mutations show that the h C terminus, in particular the WRPW domain, is also required for h activity, perhaps by interacting with other proteins to mediate transcriptional repression. We show that the h protein in Drosophila virilis closely resembles that in D. melanogaster and includes completely conserved bHLH and WRPW domains.
Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication.
Authors: Guo Z S , DePamphilis M L
Published: October 2015

The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.
Functional dissection of the lck proximal promoter.
Authors: Allen J M , Forbush K A , Perlmutter R M
Published: October 2015

The lck gene encodes a protein tyrosine kinase that participates in lymphocyte-specific signal transduction pathways. Previous studies have established that lck transcription is regulated by two distinct promoter elements termed proximal (or 3') and distal (or 5'). The proximal promoter is active almost exclusively in thymocytes and becomes inactive later during T-cell maturation. To dissect the mechanisms responsible for lck gene regulation, we generated transgenic animals bearing 5' truncations in the proximal promoter element. Sequences between -584 and +37 with respect to the proximal promoter transcription start site act to direct tissue-specific and temporally correct transcription of either a tagged version of the lck gene itself or a heterologous reporter sequence (lacZ). This region contains binding sites for at least five distinct nuclear proteins, of which one is found only in cells that support proximal lck promoter activity and a second appears only in nonexpressing cells. Interestingly, the transcribed region of the lck gene contains positive control elements that can substantially boost expression from minimal (-130 bp) proximal promoter constructs. These results provide a basis for the biochemical dissection of transcriptional regulators that act at defined points during T-cell development.
Isolation of telomerelike sequences from Cryptococcus neoformans and their use in high-efficiency transformation.
Authors: Edman J C
Published: October 2015

Development of a transformation system for the fungal human pathogen Cryptococcus neoformans is an important prerequisite for the identification of genes involved in virulence. It has previously been reported that low-efficiency transformation can be achieved by using the cloned C. neoformans URA5 gene and ura5 mutants. The introduction of linearized URA5 vectors into C. neoformans resulted in unstable transformants which apparently harbored linear extrachromosomal DNA molecules. In this paper, the nature of these molecules is confirmed to be linear by exonuclease digestion. Recovery of the extrachromosomal DNA in Escherichia coli and sequence analysis demonstrates that repeats characteristic of telomeric DNA have been added to the ends of the introduced DNA. The recovered plasmids are capable of transforming at much higher efficiencies either in the supercoiled state (up to 200 transformants per microgram) or the linear state (up to 90,000 transformants per microgram).
Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation.
Authors: Thukral S K , Morrison M L , Young E T
Published: October 2015

ADR1 is a yeast transcription factor that contains two zinc fingers of the Cys-2-His-2 (C2H2) class. Mutations that change the specificity of DNA binding of ADR1 to its target site, upstream activation sequence 1 (UAS1), have been identified at three positions in the first zinc finger. Mutations Arg-115 to Gln, His-118 to Thr, and Arg-121 to Asn led to new specificities of DNA binding at adjacent positions 10, 9, and 8 (3'-GAG-5') in UAS1. Arg-115 is at the finger tip, and His-118 and Arg-121 are at positions 3 and 6, respectively, in the alpha helix of finger 1. One double mutant displayed the binding specificity expected from the properties of its constituent new-specificity mutations. Mutations in the second finger that allowed its binding site to be identified through loss-of-contact phenotypes were made. These mutations imply a tail-to-tail orientation of the two ADR1 monomers on their adjacent binding sites. Finger 1 is aligned on UAS1 in an amino-to-carboxyl-terminal orientation along the guanine-rich strand in a 3'-to-5' direction. One of the ADR1 mutants was functional in vivo with both its cognate binding site and wild-type UAS1, but the other two mutants were defective in transactivation despite their ability to bind with high affinity to their cognate binding sites.
Identification of a novel enhancer element mediating calcium-dependent induction of gene expression in response to either epidermal growth factor or activation of protein kinase C.
Authors: Lenormand P , Pribnow D , Rodland K D , Magun B E
Published: October 2015

The VL30 family of defective murine retroviruses consists of 100 to 200 members, of which fewer than 5% appear to be transcriptionally active. A genomic clone of the transcriptionally active VL30 element RVL-3 was identified and sequenced. Genetic analysis indicated that a triple-repeat sequence within the RVL-3 long terminal repeat is capable of functioning as an inducible enhancer element responding to a variety of agonists. In Rat-1 fibroblasts, the ability of the RVL-3 enhancer to mediate induction of gene expression from a heterologous promoter in response to either epidermal growth factor (EGF) or phorbol ester treatment required coelevation of intracellular calcium. Two CArG boxes present in the triple-repeat sequence appeared to exert a negative effect on gene expression, as mutation of these sequences elevated the basal level of expression observed without altering the fold induction in response to either EGF or protein kinase C activation. In the presence of these CArG elements, mutation of AP-1-like sites adjacent to the CArG elements significantly inhibited the ability of either EGF or phorbol esters to induce gene expression. The effect of mutating these AP-1-like sites was relieved by simultaneous mutation of the CArG sites, indicating that interactions among these sites modulate RVL-3 expression. Mutational analysis and gel mobility shift experiments have identified a third sequence within the VL30 triple-repeat element that is required for the induction of gene expression and serves as a binding site for nuclear proteins. Sequence comparisons indicate that this enhancer element has not been described previously.
High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors.
Authors: Thomas K R , Deng C , Capecchi M R
Published: October 2015

Mutations were targeted to the Hprt locus in murine embryonic stem cells by using sequence replacement vectors. When the vector was designed such that the mutated sequences were flanked on both sides by several kilobases of DNA homologous to the target locus, replacement of chromosomal sequences with the exogenous DNA occurred with precision. If, on the other hand, the target-homologous DNA on one arm of the vector was reduced to below 1 kb in length, the fidelity of recombination was diminished.
U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation.
Authors: You Y , Chen C Y , Shyu A B
Published: October 2015

Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.
Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.
Authors: van Ooij C , Snyder R C , Paeper B W , Duester G
Published: October 2015

The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver.
Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro.
Authors: Bardwell L , Cooper A J , Friedberg E C
Published: October 2015

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are two of at least seven genes which are known to be required for damage-specific recognition and/or damage-specific incision of DNA during nucleotide excision repair. RAD1 and RAD10 are also involved in a specialized mitotic recombination pathway. We have previously reported the purification of the RAD10 protein to homogeneity (L. Bardwell, H. Burtscher, W. A. Weiss, C. M. Nicolet, and E. C. Friedberg, Biochemistry 29:3119-3126, 1990). In the present studies we show that the RAD1 protein, produced by in vitro transcription and translation of the cloned gene, specifically coimmunoprecipitates with the RAD10 protein translated in vitro or purified from yeast. Conversely, in vitro-translated RAD10 protein specifically coimmunoprecipitates with the RAD1 protein. The sites of this stable and specific interaction have been mapped to the C-terminal regions of both polypeptides. This portion of RAD10 protein is evolutionarily conserved. These results are the first biochemical evidence of a specific association between any eukaryotic proteins genetically identified as belonging to a recombination or DNA repair pathway and suggest that the RAD1 and RAD10 proteins act at the same or consecutive biochemical steps in both nucleotide excision repair and mitotic recombination.
Binding properties of replication protein A from human and yeast cells.
Authors: Kim C , Snyder R O , Wold M S
Published: October 2015

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.
Induction of the POU domain transcription factor Oct-2 during T-cell activation by cognate antigen.
Authors: Kang S M , Tsang W , Doll S , Scherle P , Ko H S , Tran A C , Lenardo M J , Staudt L M
Published: October 2015

Oct-2 is a transcription factor that binds specifically to octamer DNA motifs in the promoters of immunoglobulin and interleukin-2 genes. All tumor cell lines from the B-cell lineage and a few from the T-cell lineage express Oct-2. To address the role of Oct-2 in the T-cell lineage, we studied the expression of Oct-2 mRNA and protein in nontransformed human and mouse T cells. Oct-2 was found in CD4+ and CD8+ T cells prepared from human peripheral blood and in mouse lymph node T cells. In a T-cell clone specific for pigeon cytochrome c in the context of I-Ek, Oct-2 was induced by antigen stimulation, with the increase in Oct-2 protein seen first at 3 h after activation and continuing for at least 24 h. Oct-2 mRNA induction during antigen-driven T-cell activation was blocked by cyclosporin A, as well as by protein synthesis inhibitors. These results suggest that Oct-2 participates in transcriptional regulation during T-cell activation. The relatively delayed kinetics of Oct-2 induction suggests that Oct-2 mediates the changes in gene expression which occur many hours or days following antigen stimulation of T lymphocytes.
Differential binding of zinc fingers from Xenopus TFIIIA and p43 to 5S RNA and the 5S RNA gene.
Authors: Darby M K , Joho K E
Published: October 2015

Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.
Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro.
Authors: Bennett M , Piñol-Roma S , Staknis D , Dreyfuss G , Reed R
Published: October 2015

We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.
Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells.
Authors: Li W , Deanin G G , Margolis B , Schlessinger J , Oliver J M
Published: October 2015

In basophils, mast cells, and the RBL-2H3 tumor mast cell line, cross-linking the high-affinity immunoglobulin E receptor (Fc epsilon R1) stimulates a series of responses, particularly the activation of phospholipase C (PLC), that lead to allergic and other immediate hypersensitivity reactions. The mechanism of activation of PLC, however, is not clear. Here, we show that cross-linking Fc epsilon R1 on RBL-2H3 cells causes the tyrosine phosphorylation of at least 12 cellular proteins, including PLC gamma 1 (PLC gamma 1) and the receptor beta and gamma subunits. 32P-labeled PLC gamma 1 can be detected by anti-phosphotyrosine antibody as early as 10 s after the addition of antigen. The tyrosine-phosphorylated 33-kDa beta subunit and 9- to 11-kDa gamma subunit of the Fc epsilon R1 are additionally phosphorylated on serine and theonine residues, respectively, and are found as complexes with other phosphotyrosine-containing proteins in antigen-stimulated cells. Our results indicate a means by which the Fc epsilon R1 may control PLC activity in RBL-2H3 cells and raise the possibility that other receptor-mediated signalling events in mast cells may also be controlled through protein tyrosine phosphorylation.
Transcriptional regulation of the apolipoprotein B100 gene: purification and characterization of trans-acting factor BRF-2.
Authors: Zhuang H , Chuang S S , Das H K
Published: October 2015

Apolipoprotein B100 (apoB), the only protein of low-density lipoprotein, is produced primarily in the liver and serves as a ligand for the low-density lipoprotein receptor. Hepatic cell-specific expression of the human apoB gene is controlled by at least two cis-acting positive elements located between positions-128 and -70 (H. K. Das, T. Leff, and J.L. Breslow, J. Biol. Chem. 263:11452-11458, 1988). The distal element (-128 to -85) appears to be liver specific since it shows positive activity in HepG2 cells and negative activity in HeLa cells. The proximal element (-84 to -70) acts as a positive element in both these cell lines, and two rat liver nuclear proteins, BRF-1 and C/EBP, bind to two overlapping sites (-84 to -60 and -70 to -50, respectively). By gel mobility shift assay, we have identified a rat liver nuclear protein (BRF-2) which binds to the distal element (-128 to -85) of the apoB gene. This putative trans-acting factor has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. The purified BRF-2 has an apparent molecular mass of 120 kDa and was found to specifically recognize sequence -128 to -85; BRF-2 also produced a strong hypersensitive site at nucleotide position -95 with copper-orthophenanthroline reagent. A double-stranded oligonucleotide (-128 to -85) containing a 3-nucleotide (TTC) insertion between position -95 and -94 was found to abolish DNA binding by BRF-2. This result suggests that the region surrounding the hypersensitive site -95 is important for protein-DNA interaction. By using apoB promoter fragments containing various internal deletions as templates for gel mobility shift assay, the region between -104 and -85 was identified to be crucial for binding by BRF-2. We propose that BRF-2 may play an important role in the tissue-specific regulation of apoB gene transcription.
In vivo splicing of the beta tropomyosin pre-mRNA: a role for branch point and donor site competition.
Authors: Libri D , Balvay L , Fiszman M Y
Published: October 2015

The chicken beta tropomyosin gene contains two sets of alternatively spliced, mutually exclusive exons whose utilization is developmentally regulated. Exons 6A and 6B are used in nonmuscle cells (or undifferentiated muscle cells) and skeletal muscle cells, respectively. A complex arrangement of cis-acting sequence elements is involved in alternative splicing regulation. We have performed an extensive mutational analysis on the sequence spanning the region from exon 6A to the constitutive exon 7. A large number of mutant minigenes have been tested in transfection assays of cultured myogenic cells, and the splicing products have been analyzed by cDNA polymerase chain reaction. We demonstrate that in undifferentiated myoblasts, exon 6B is skipped as a result of a negative control on its selection, while exon 6A is spliced as a default choice. We provide evidence that the focal point of such a regulation is localized in the intron upstream of exon 6B and probably involves the blockage of its associated branch point. In differentiated myotubes, in contrast, both exons are accessible to the splicing machinery. We show that the preferential choice of exon 6B in this splicing environment depends on the existence of a competition between the two exons for the flanking constitutive splice sites. We demonstrate that both the donors and the branch points of the two exons are involved in this competition.
Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal sequence element of small nuclear RNA genes.
Authors: Murphy S , Yoon J B , Gerster T , Roeder R G
Published: October 2015

The promoters of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA (snRNA) genes contain an essential and highly conserved proximal sequence element (PSE) approximately 55 bp upstream from the transcription start site. In addition, the upstream enhancers of all snRNA genes contain binding sites for octamer-binding transcription factors (Octs), and functional studies have indicated that the PSE and octamer elements work cooperatively. The present study has identified and characterized a novel transcription factor (designated PTF) which specifically binds to the PSE sequence of both RNA polymerase II- and RNA polymerase III-transcribed snRNA genes. PTF binding is markedly potentiated by Oct binding to an adjacent octamer site. This potentiation is effected by Oct-1, Oct-2, or the conserved POU domain of these factors. In agreement with these results and despite the independent binding of Octs to the promoter, PTF and Oct-1 enhance transcription from the 7SK promoter in an interdependent manner. Moreover, the POU domain of Oct-1 is sufficient for significant in vitro activity in the presence of PTF. These results suggest that essential activation domains reside in PTF and that the potentiation of PTF binding by Octs plays a key role in the function of octamer-containing snRNA gene enhancers.
Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters.
Authors: van Wijnen A J , van den Ent F M , Lian J B , Stein J L , Stein G S
Published: October 2015

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.
Characterization of cis-acting elements regulating transcription from the promoter of a constitutively active rice actin gene.
Authors: Wang Y , Zhang W , Cao J , McElroy D , Wu R
Published: October 2015

The promoter of the constitutively expressed rice (Oryza sativa) actin 1 gene (Act1) is highly active in transformed rice plants (W. Zhang, D. McElroy, and R. Wu, Plant Cell 3:1150-1160, 1991). A region 834 bp upstream of the Act1 transcription initiation site contains all the regulatory elements necessary for maximal gene expression in transformed rice protoplasts (D. McElroy, W. Zhang, J. Cao, and R. Wu, Plant Cell 2:163-171, 1990). We have constructed a series of Act1 promoter deletions fused to a bacterial beta-glucuronidase reporter sequence (Gus). Transient expression assays in transformed rice protoplasts, as well as transformed maize cells and tissues, identified two distinct cis-acting regulatory elements in the Act1 promoter. A 38-bp poly(dA-dT) region was found to be a positive regulator of Act1 promoter activity. Deletion of the poly(dA-dT) element lowered Gus expression by at least threefold compared with expression produced by the full-length Act1 promoter. By gel retardation and footprinting, we identified a ubiquitous rice protein which specifically recognizes this poly(dA-dT) element in the constitutively active Act1 promoter. A CCCAA pentamer repeat-containing region was found to be a negative regulator of the Act1 promoter in transformed rice protoplasts. Transient expression assays in different maize cells and tissues with use of the Act1 deletion constructs suggested that the CCCAA pentamer repeat region functions in a complex tissue-specific manner. A CCCAA-binding protein was detected only in root extracts.
Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes.
Authors: Yoshida Y , Kawata M , Miura Y , Musha T , Sasaki T , Kikuchi A , Takai Y
Published: October 2015

Microinjection of either Ki-rasVal-12 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rap1/Krev-1 p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA synthesis but only in the presence of insulin. Either the GDP-bound form of Ki-ras p21 or the same form of smg p21B alone was inactive, but smg GDS alone was slightly active only in the presence of insulin. The morphology of the cells was analyzed by scanning electron, phase-contrast, and confocal laser scanning microscopies. Ki-rasVal-12 p21 induced membrane ruffling irrespective of the presence or absence of insulin. The GTP gamma S-bound form of smg p21B showed the same effect only in the presence of insulin. Either the GDP-bound form of Ki-ras p21, the same form of smg p21B, or smg GDS alone was inactive. Upon microinjection of Ki-rasVal-12 p21, stress fibers markedly decreased and the cells became round and piled up. In contrast, upon microinjection of the GTP gamma S-bound form of smg p21B, stress fibers did not markedly decrease and the cells neither became round nor piled up. These results indicate that both ras p21 and smg p21 are mitogenic in Swiss 3T3 cells but that their actions are slightly different.
Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells.
Authors: Kavanaugh W M , Klippel A , Escobedo J A , Williams L T
Published: October 2015

The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.
The functional importance of a cap site-proximal region of the human prointerleukin 1 beta gene is defined by viral protein trans-activation.
Authors: Hunninghake G W , Monks B G , Geist L J , Monick M M , Monroy M A , Stinski M F , Webb A C , Dayer J M , Auron P E , Fenton M J
Published: October 2015

Prointerleukin 1 beta (IL-1 beta) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1 beta transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1 beta gene. We identified a protein, termed NFIL-1 beta A (NF beta A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1 beta genes. The IL-1 alpha gene, which lacks a TATA motif, does not possess an NF beta A-binding sequence within the promoter region, suggesting that NF beta A may selectively regulate IL-1 beta expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varies rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF beta A was assessed in transient transfection assays. These data indicate that NF beta A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF beta A in order to trans-activate the proximal IL-1 beta promoter in a monocytic cell line. We propose that NF beta A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation.
Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA.
Authors: Chen J , Moore C
Published: October 2015

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.