bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of DNA: protein interactions that mediate HSV-1 immediate early gene activation by VP16.
Authors: Triezenberg S J , LaMarco K L , McKnight S L
Published: June 2007

The viral genes first expressed upon lytic infection by herpes simplex virus type 1 (HSV-1) encode the five immediate early (IE) proteins. IE gene expression is potently and specifically induced by a virion protein termed VP16. Previous studies have shown that the activating properties of VP16 are IE gene specific and mediated by upstream regulatory elements common to each IE gene. Paradoxically, however, VP16 does not appear to be a sequence-specific DNA-binding protein. To understand the specificity of VP16 activation, we identified the cis-regulatory sequences of an IE gene that mediate VP16 response. Two distinct DNA sequence motifs enable the ICP4 gene to respond to VP16. Biochemical fractionation of nuclear proteins from uninfected cells revealed the existence of cellular proteins that bind directly to each of these VP16 cis-response elements. These observations, in concert with the identification of functional domains of the VP16 protein, lead to the hypothesis that VP16 achieves activation specificity via protein: protein, rather than protein: DNA, interactions.
Splice site selection, rate of splicing, and alternative splicing on nascent transcripts.
Authors: Beyer A L , Osheim Y N
Published: June 2007

Based on ultrastructural analysis of actively transcribing genes seen in electron micrographs, we present evidence that pre-mRNA splicing occurs with a reasonable frequency on the nascent transcripts of early Drosophila embryo genes and that splice site selection may generally precede polyadenylation. The details of the process observed are in agreement with results from in vitro splicing systems but differ in the more rapid completion of in vivo splicing. For those introns that are removed cotranscriptionally, a series of events is initiated following 3' splice site synthesis, beginning with ribonucleoprotein (RNP) particle formation at the 3' splice site within 48 sec, intron loop formation within 2 min, and splicing within 3 min. The initiation of the process is correlated with 3' splice site synthesis but is independent of 5' splice site synthesis, the position of the intron within the transcript, and the age or length of the transcript. In some cases, introns are removed from the 5' end of a transcript before introns are synthesized at the 3' end, supporting a possible role for the order of transcription in splice site pairing. In general, our observations are consistent with the 'first-come-first-served' principle of splice site selection, although an observed example of exon skipping indicates that alternative splicing possibilities can be accommodated within this general framework.
Flexibility of the yeast alpha 2 repressor enables it to occupy the ends of its operator, leaving the center free.
Authors: Sauer R T , Smith D L , Johnson A D
Published: June 2007

The yeast alpha 2 protein, the product of the MAT alpha 2 gene, is a regulator of yeast cell type; it turns off transcription of the a-specific genes by binding to an operator located upstream of each gene. In this paper we describe the domain structure, subunit organization, and some unusual features of the way this protein contacts its operator. We show that the protein is folded into two domains. The carboxy-terminal domain binds specifically to the operator; the amino-terminal domain contains dimerization contacts. The alpha 2 dimer differs from those of the phage repressors in that it is flexible and therefore is able to bind tightly to differently spaced operator half-sites. In the natural operator, the centers of the operator half-sites are two and one-half turns of DNA apart, exposing them on opposite sides of the DNA helix. We show that the design of alpha 2 allows a dimer to reach across its operator such that it occupies the two half-sites but leaves the middle of the operator available to other proteins.
The scid gene encodes a trans-acting factor that mediates the rejoining event of Ig gene rearrangement.
Authors: Hendrickson E A , Schatz D G , Weaver D T
Published: June 2007

Homozygous mutation at the scid locus in the mouse impairs lymphoid development and results in animals deficient in B and T cells. We found that immunoglobulin heavy-chain gene rearrangement was blocked at the D-JH stage in Abelson-transformed scid pre-B cell lines. Examination of the recombinational junctions indicated that the correct gene elements (D and JH) were assembled, as shown by the presence of D region and JH-region DNA on the breakpoint restriction fragments cloned from the genome of the scid cell lines. All rearrangement events were accompanied by deletions of varying sizes such that none of the rearrangements resulted in the production of functional immunoglobulins. The breakpoints of the rearrangement events did not correspond to the utilization of a novel heptamer-nonamer recombination signal but probably arose by nonspecific deletion from distal JH and D heptamer-nonamer signals in the process of recombination. scid pre-B cell lines were infected with a recombinant retrovirus (DGR) containing Ig joining signals. Aberrant rearrangements were observed in DGR DNA that was integrated randomly throughout the mouse genome, which suggested that the mutation in scid mice encodes a trans-acting factor that is part of the lymphoid gene recombination machinery.
polyhomeotic: a gene required for the embryonic development of axon pathways in the central nervous system of Drosophila.
Authors: Smouse D , Goodman C , Mahowald A , Perrimon N
Published: June 2007

Hypomorphic alleles of the locus polyhomeotic (ph) produce multiple, homeotic-like transformations in adult flies that mimic dominant mutations in the Antennapedia and Bithorax complexes. Analysis of null alleles of ph has revealed a complex, embryonically lethal phenotype that includes cell death of the ventral epidermis and abnormalities in the patterns of expression of homeotic and segmentation genes. There is also a dramatic alteration in the pattern of axon pathways in the central nervous system, such that the wild-type array of segmentally repeated commissures and connectives is replaced by bundles of axons confined to the hemiganglia of origin. It is possible that this axonal phenotype is the result of loss of neuronal identity caused by abnormal homeotic and segmentation gene expression.
The maternal sex determination gene daughterless has zygotic activity necessary for the formation of peripheral neurons in Drosophila.
Authors: Caudy M , Grell E H , Dambly-Chaudière C , Ghysen A , Jan L Y , Jan Y N
Published: June 2007

The daughterless (da) gene is known to have separate maternal and zygotic functions: Maternally supplied daughterless activity is required for proper sex determination and dosage compensation in female embryos, whereas loss of zygotically supplied da+ activity causes embryonic lethality in both male and female embryos. We have found that the zygotic da+ activity is necessary for neural development: The use of neuron-specific antibodies and beta-galactosidase-marked X chromosomes has revealed that in both male and female embryos deletions or strong mutations of the da gene remove all peripheral neurons and associated sensory structures without disrupting the epithelium from which they derive. Partial da+ function causes partial removal of peripheral neurons. Our results indicate that da+ is required for the formation of peripheral neurons and their associated sensory structures.
An erythrocyte-specific protein that binds to the poly(dG) region of the chicken beta-globin gene promoter.
Authors: Lewis C D , Clark S P , Felsenfeld G , Gould H
Published: June 2007

The promoter region of the chicken adult beta-globin gene contains a sequence of 16 deoxyguanosine residues located at a nucleosome boundary in tissues where the gene is inactive. In definitive erythrocytes that express the beta-globin gene, the nucleosome is displaced, the G-string and adjacent sequences are occupied by sequence-specific DNA-binding proteins, and a nuclease hypersensitive domain is generated in this region. To gain insight into the role of the G-string in this series of events, we have examined the proteins that bind to it. Using the gel mobility shift assay and a monoclonal antibody that blocks specific binding to the G-string, we have identified a specific protein, BGP1, that is found only in chicken erythroid cells and appears at the same time, or shortly before, the changes in chromatin structure. The antibody interacts strongly with BGP1 and cross-reacts weakly with Sp1. Although both BGP1 and Sp1 require Zn2+ for their DNA-binding activity, these proteins differ in their binding-site specificities, chromatographic properties, and molecular weights. In contrast to Sp1, which is found in a wide variety of cell types, BGP1 is restricted to erythrocytes and is most abundant in definitive erythrocytes. Thus, its presence corresponds to the tissue- and stage-specific occupancy of the G-string in vivo.
An excised SV40 intron accumulates and is stable in Xenopus laevis oocytes.
Authors: Michaeli T , Pan Z Q , Prives C
Published: June 2007

Xenopus laevis oocytes injected with simian virus 40 (SV40) DNA synthesize abundant quantities of viral late region RNA. In a previous analysis of the 5' ends of oocyte SV40 late RNAs, it was observed that, in contrast to the majority of the late RNA species, an abundant class of viral late RNAs, whose 5' ends mapped at or near nucleotide 294, was not polyadenylated. The structure of this RNA class has now been characterized further. We have shown that this species consists of a class of small uncapped RNA molecules with heterogeneous 3' ends mapping between nucleotides 417 and 433. This corresponds well with the position of a 139-nucleotide intron within the leader region of late 16S RNA (nucleotides 294-433). The identification of this RNA class as an excised intron was strongly supported by the fact that it displayed anomalous mobilities on different percentage polyacrylamide gels, a property of lariat introns. Furthermore, incubation of oocyte RNA with a HeLa cell extract with lariat debranching activity converted the small RNA to a class that now migrated as less than or equal to 140 nucleotides in length in 8% gels, consistent with the size of the linear intraleader intron. Additional analysis of this RNA showed that it is primarily nuclear in localization and is probably the most stable viral RNA species in the oocyte. These data suggest that oocytes accumulate large quantities of the 16S intraleader intron because of their failure to debranch this RNA efficiently.
Paternal DNA strands segregate to both trophectoderm and inner cell mass of the developing mouse embryo.
Authors: Ito K , McGhee J D , Schultz G A
Published: June 2007

The localization of sperm DNA strands was examined in preimplantation mouse embryos. Male mice were treated with bromodeoxyuridine (BrdU) to label germ-cell-line DNA and were then mated with unlabeled females. Sperm DNA strands in early embryos derived from these matings could be detected by means of a fluorescent antibody specific to BrdU. The position and number of fluorescent spots detected in the developing embryos are consistent with a model in which paternal DNA strands segregate at random into both the trophectoderm and the inner cell mass. Although we could not follow the segregation of individual paternal chromosomes, we could detect no overall segregation pattern of the sperm DNA strands that could be obviously related to chromosome imprinting.
Identification and purification of EBP1: a HeLa cell protein that binds to a region overlapping the 'core' of the SV40 enhancer.
Authors: Clark L , Pollock R M , Hay R T
Published: June 2007

The SV40 enhancer consists of multiple DNA sequence motifs that are recognized by a variety of trans-acting factors. Using DNase I protection and a gel electrophoresis DNA-binding assay, we identified a HeLa cell protein (EBP1) that binds to the 'core' region of the SV40 enhancer. A short double-stranded synthetic oligonucleotide containing the binding site for EBP1 was used to assay for EBP1 activity and to purify a 57,000-m.w. polypeptide by recognition site affinity chromatography. Bromodeoxyuracil cross-linking identified a 60,000-m.w. species as the polypeptide responsible for the DNA-binding activity. Analysis of the DNA sequences required for EBP1 binding indicated that EBP1 could be distinguished from a number of recently characterized proteins (EBP20, AP-2, and AP-3) by its binding to a variety of mutant templates. Correlation of the in vivo transcriptional activity of wild-type and mutated enhancers with EBP1 binding indicates that this protein may be important for SV40 enhancer activity because mutations that abolish EBP1 binding also have a severe deleterious effect on transcription.
Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo.
Authors: Calzone F J , Thézé N , Thiebaud P , Hill R L , Britten R J , Davidson E H
Published: June 2007

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (Kr) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of Kr range from approximately 2 x 10(4) to approximately 2 x 10(6) for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10(5) to 2 x 10(6) molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., less than or equal to 10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage.
The human beta-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein.
Authors: Wall L , deBoer E , Grosveld F
Published: June 2007

We have shown that the minimal enhancer fragment present in the 3'-flanking region of the human beta-globin gene contains four regions that bind nuclear proteins in vitro. By using gel mobility shift and DNase I footprinting assays, we were able to show that each of these regions binds an erythroid-cell-specific nuclear factor which we name NF-E1. This factor is present in erythroid cells at different developmental stages of globin gene expression. The recognition sequence of this protein (A/C Py T/A ATC A/T Py) is also present in the intragenic enhancer and the promoter of the beta-globin gene as well as in the promoter of other erythroid-cell specific genes. In addition to NF-E1, each of the four binding regions interacts with at least one other protein factor.
Structural arrangements of transcription control domains within the 5'-untranslated leader regions of the HIV-1 and HIV-2 promoters.
Authors: Jones K A , Luciw P A , Duchange N
Published: June 2007

Promoter-proximal downstream regions of the human immunodeficiency viruses (HIV-1 and HIV-2) mediate the action of the viral transcription activator protein, Tat. We demonstrate here that the downstream domain of each virus interacts with two RNA polymerase II transcription factors. One of these, CTF/NF I, is a multifunctional protein associated previously with activation of transcription and DNA replication. The other cellular protein, designated LBP-1 (leader-binding protein-1), recognizes repeated elements within an extended region of DNA corresponding to part of the 5'-untranslated leader. Analysis of clustered point mutants in the HIV-1 leader for DNA-binding and transcription activity in vitro and in vivo suggests a role for LBP-1 as part of the basal promoter. A complex overlapping arrangement is observed between sequences required for the interaction of LBP-1 and CTF/NF I proteins and those defined previously for regulation by the HIV-1 Tat protein.
Hormone-mediated repression: a negative glucocorticoid response element from the bovine prolactin gene.
Authors: Sakai D D , Helms S , Carlstedt-Duke J , Gustafsson J A , Rottman F M , Yamamoto K R
Published: June 2007

We have defined and characterized a region upstream of the bovine prolactin gene that confers repression by glucocorticoids. This 'negative glucocorticoid response element' (nGRE) contains multiple footprinting sites for purified glucocorticoid receptor protein between -51 and -562 bp. A strong consensus sequence for receptor binding within the nGRE has not yet been defined, but it is apparent that nGRE sequences differ from the GRE consensus elements that confer positive glucocorticoid regulation. Unlike 'positive' GREs, the nGRE enhances promoter activity in the absence of glucocorticoids or receptor, presumably through the action of a protein that binds in the same region and activates transcription. The hormone-receptor complex appears to negate this enhancement by competing or inactivating the second factor. As with positive GREs, nGRE sequences confer hormonal regulation upon linked heterologous promoters within various cell types; a 34-bp subfragment containing a single receptor binding site is sufficient for nGRE activity. We speculate that nGRE sequences might alter the structure of bound receptor, thereby preventing it from functioning as a positive regulator when bound at those sites.
Presplicing complex formation requires two proteins and U2 snRNP.
Authors: Krämer A
Published: June 2007

Six fractions derived from a HeLa cell nuclear extract are necessary for the generation of spliced mRNA in vitro. To establish a function for the protein factors present in these fractions, their role in the formation of splicing complexes was analyzed by electrophoresis in native polyacrylamide gels. Two of the fractions are sufficient to assemble the adenovirus major late mRNA precursor into a presplicing complex with characteristics similar to the presplicing complex assembled in nuclear extract. One fraction supplies splicing factor (SF) 1 and at least one small nuclear ribonucleoprotein particle, U2 snRNP. The other fraction contains SF3. Extensive fractionation of this protein has revealed that it is essential for presplicing complex assembly and the splicing reaction.
Regulatory genes in the thermoregulation of Escherichia coli pili gene transcription.
Authors: Göransson M , Forsman K , Uhlin B E
Published: June 2007

Expression of several different pilus adhesins by Escherichia coli is subject to thermoregulation. The surface-located fimbrial structures are present during growth at 37 degrees C but are not produced by cells grown at lower temperatures, such as 25 degrees C. As a step toward understanding the molecular mechanism, we have studied the role of different cistrons of a cloned pilus adhesin gene cluster (pap) from a uropathogenic E. coli isolate. By promoter cloning, mRNA analysis, and expression of subcloned genes in trans, we have identified the papI gene as the mediator of thermoregulation at the level of pilus adhesin gene transcription. Expression of the major pilus subunit gene (papA) and several other pilus protein cistrons appeared to be dependent on stimulation by the papB and papI gene products. Constructs carrying different pap DNA regions indicated that none of the known Pap proteins acts directly as thermosensor. The chromosomal rpoH gene and RpoH sigma factor did not appear to be required for pap transcription, and the thermoregulation of pilus gene transcription must be different from that of the heat shock regulon. By overexpressing the papI gene product from an expression plasmid in trans, we could circumvent the temperature regulation and turn on production of pilus adhesin at low temperature. Our results suggest that the level of mRNA encoding the PapI activator is limiting at low growth temperatures and that thermoregulation is due to a determinant in the papI-papB intercistronic region.
The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.
Authors: Martínez-Salas E , Linney E , Hassell J , DePamphilis M L
Published: June 2007

Microinjection of the firefly luciferase gene coupled to a thymidine kinase (tk) promoter provided a quantitative assay to evaluate the requirements for gene expression in individual mouse oocytes and embryos. Polyoma virus (PyV) enhancers had no effect on the level of gene expression or competition for transcription factors as long as the DNA remained either in the oocyte germinal vesicle or the pronuclei of one-cell embryos. Expression of injected genes could be observed in pronuclei because the signal that normally triggers zygotic gene expression in two-cell embryos still occurred in one-cell embryos arrested in S phase. However, when the tk promoter was injected into zygotic nuclei of two-cell embryos, enhancers increased the number of embryos that expressed luciferase as well as the level of luciferase activity per embryo. PyV enhancer mutation F101, selected for growth in mouse embryonal carcinoma F9 cells, stimulated expression in developing two-cell embryos about seven times better than the wild-type PyV enhancer and competed effectively for factors required for transcription. These results were consistent with the fact that enhancers are required to activate the PyV origin of DNA replication in developing two-cell embryos but not in one-cell embryos. The maximum levels of gene expression in oocytes, one-cell embryos, and developing two-cell embryos (1:67:21) were inversely related to the extent of chromatin assembly, but the need for enhancers was independent of chromatin assembly. Therefore, it appears that the need for enhancers to activate promoters or origins of replication results from some negative regulatory factor that first appears as a component of zygotic nuclear structure.
Spatial regulation of zerknüllt: a dorsal-ventral patterning gene in Drosophila.
Authors: Doyle H J , Kraut R , Levine M
Published: June 2007

zerknüllt (zen) is one of approximately 10 zygotically active genes that control the differentiation of the dorsal-ventral (D/V) pattern during early embryogenesis in Drosophila. Past genetic analyses suggest that maternal factors repress the expression of zen in ventral regions, thereby restricting zen products to dorsal and dorsal-lateral regions of precellular embryos. Subsequent interactions with other zygotic D/V regulatory genes refine the zen pattern, restricting expression to the dorsal-most ectoderm. Here we describe the use of zen promoter fusions and P-element transformation to identify cis elements that are responsible for the complex spatial pattern of zen expression. The zen promoter shows a two-tier organization: Distal sequences mediate its initial response to maternal factors, whereas proximal sequences are responsible for the refinement of the pattern in older embryos. The distal regulatory element has the property of a silencer (or anti-enhancer) element and can act over a distance to repress ventral expression of a heterologous promoter. Also, we discuss evidence that proximal promoter sequences interact with factors that may be modulated by a cell-cell communication pathway.
High-level, erythroid-specific expression of the human alpha-globin gene in transgenic mice and the production of human hemoglobin in murine erythrocytes.
Authors: Hanscombe O , Vidal M , Kaeda J , Luzzatto L , Greaves D R , Grosveld F
Published: June 2007

Using the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of the endogenous mouse alpha-globin genes. Transgenic fetuses with high-copy numbers of the transgene suffer severe anemia and die before birth. Using a construct with both the human alpha- and beta-globin genes and the beta-globin DCR, live mice with low-copy numbers were obtained. Both human globin genes are expressed at high levels in adult red cells to give human hemoglobin HbA in amounts equal to or greater than endogenous mouse hemoglobin. Expression of HbA in murine red cells is not accompanied by any increase in mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). However, these transgenic mice tend to have an increased number of reticulocytes in peripheral blood; consistent with some degree of hemolysis. Metabolic labeling experiments showed balanced mouse globin synthesis, but imbalanced human globin synthesis, with an alpha/beta biosynthetic ratio of approximately 0.6. Thus, these mice have mild anemia. These results are discussed with relation to the coordinate regulation of alpha- and beta-globin synthesis in erythroid tissues.
The cell-specific transcription factor PTF1 contains two different subunits that interact with the DNA.
Authors: Roux E , Strubin M , Hagenbüchle O , Wellauer P K
Published: June 2007

The cognate sequence of transcription factor PTF1, which plays a key role in pancreas-specific gene expression, has a bipartite organization. Two separate DNA domains, the A and the B boxes, are required for efficient binding of the factor. The structure of PTF1 was elucidated by cross-linking purified PTF1 to DNA templates that had been differentially substituted with azido-deoxyuridine (N3.dU). This site-directed UV cross-linking shows that PTF1 contains two DNA-binding proteins, distinct in size and sensitivity to Staphylococcus aureus V8 protease. A 64-kD protein is cross-linked with DNA containing N3.dU substitutions in the A box, and a 48-kD protein is cross-linked with DNA containing N3.dU substitutions in the B box. Both proteins bind simultaneously to the same DNA molecule. The data indicate that PTF1 is a heteromeric oligomer and that its cell-specific DNA-binding potential is the result of a concerted activity of two DNA-binding subunits.
Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.
Authors: Lyons K M , Pelton R W , Hogan B L
Published: June 2007

The murine Vgr-1 (Vg-related) and BMP-2a (bone morphogenetic protein 2a) genes are members of the decapentaplegic subgroup of the transforming growth factor-beta (TGF beta) superfamily. Although genetic and biochemical studies suggest that the members of this subgroup play important roles in development, little is known about their function in mammals. Therefore, we investigated the expression of Vgr-1 and BMP-2a RNAs in embryonic, newborn, and adult tissues by in situ hybridization. Vgr-1 RNA is maternally encoded in ovarian oocytes but declines in fertilized eggs and is undectable by the two- to four-cell stage. Only low levels of transcripts are seen in blastocysts and early postimplantation stages. From mid-gestation on, Vgr-1 RNA is expressed at high levels in developing skin, especially in the suprabasal cells of the proliferating epidermis but not in the dermis or hair follicles, both of which contain TGF beta 1 and/or TGF beta 2 RNAs. In contrast, BMP-2a transcripts are seen only in the hair follicles in the cells of the hair bulb cortex. Temporally and spatially distinct patterns of BMP-2a, Vgr-1, TGF beta 1, and TGF beta 2 expression are also seen in different populations of mesenchymal cells in the developing skeletal system (cartilage and bone). Our results suggest that the coordinated expression of several members of the TGF beta superfamily is required to control the progression of specific cell types through their differentiation pathways.
A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB.
Authors: Baeuerle P A , Baltimore D
Published: June 2007

The NF-kappaB transcription factor was affinity-purified from deoxycholate (DOC)-treated cytosol of HeLa cells and shown to contain both a 50-kappaD polypeptide (p50) with a DNA-binding specificity identical to that of nuclear NF-kappaB and a 65-kappaD protein (p65) lacking DNA binding activity. Electrophoretically purified p50, after renaturation, gave rise to a protein-DNA complex that migrated faster than that made by native NF-kappaB. Reconstitution of p50 and p65 together produced a protein that combined with DNA to form a complex with electrophoretic mobility indistinguishable from that of the complex formed by nuclear extracts and DOC-treated cytosolic fractions. Sedimentation and gel filtration analyses indicate that alone, the p50 protein exists as a dimer; two molecules of p65 bind to it to form a heterotetramer. Unlike I kappaB, the specific inhibitor of NF-kappaB, p65 displayed no inhibitor activity and was not released from NF-kappaB by DOC. p65 did not change the DNA binding specificity or the stimulatory effect of GTP on the p50 homodimer. Surprisingly, NF-kappaB could only be inactivated by I kappaB when p65 was bound. It would appear that one function of p65 is to make NF-kappaB susceptible to inhibition by I kappaB.
The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex.
Authors: Huang M M , Hearing P
Published: June 2007

Nuclear factor E2F is a cellular protein that binds to the adenovirus E2 promoter and E1A enhancer regions and to the cellular c-myc P2 promoter region. The DNA binding activity of E2F, detected in vitro using nuclear extracts prepared from HeLa cells, is increased by adenovirus infection (termed E2F induction). We demonstrate here that a 19.5-kD protein, encoded by adenovirus early region 4 (E4) open reading frame (ORF) 6/7, is primarily responsible for the induction of E2F DNA binding activity to the E2 promoter region. Viral mutants that contain frame-shift mutations in E4 ORF 6/7 failed to induce E2F binding activity; a virus that carries an E4 ORF 6/7 cDNA in place of the E4-coding sequences induced E2F efficiently. Using gel mobility shift assays, we demonstrate that the E4 ORF 6/7 product induces the binding of E2F to the E2 promoter via a direct complex. The addition of a peptide-specific antiserum, directed against the E4 ORF 6/7 protein, to an in vitro E2F-binding reaction resulted in the formation of a DNA-protein complex with reduced gel mobility compared to the normal, adenovirus-induced E2F-E2 promoter complex. The formation of the E2F-E2 promoter-antibody complex was blocked by the addition of the cognate peptide used to generate the antiserum but not by a nonspecific peptide. Nuclear extracts prepared from adenovirus-infected HeLa cells were cleared of E2F binding activity using the ORF 6/7 peptide-specific serum, but not the preimmune serum, suggesting that E2F and the E4 ORF 6/7 product form a protein-protein complex in solution. The adenovirus E1A proteins are not absolutely required for the induction of E2F binding activity because the infection of HeLa cells with an E1A mutant, dl312, at high multiplicity resulted in E2F induction. Under these conditions of infection, the E4 ORF 6/7 product was synthesized. E2F binding activity was induced, but inefficiently, in cells infected with E4 ORF 6/7 mutants, indicating that an additional pathway may lead to E2F induction.
Four factors are required for 3'-end cleavage of pre-mRNAs.
Authors: Takagaki Y , Ryner L C , Manley J L
Published: June 2007

We reported previously that authentic polyadenylation of pre-mRNAs in vitro requires at least two factors: a cleavage/specificity factor (CSF) and a fraction containing nonspecific poly(A) polymerase activity. To study the molecular mechanisms underlying 3' cleavage of pre-mRNAs, we fractionated CSF further and show that it consists of four separable subunits. One of these, called specificity factor (SF; Mr, approximately 290,000), is required for both specific cleavage and for specific polyadenylation and thus appears responsible for the specificity of the reaction. Although SF has not been purified to homogeneity, several lines of evidence suggest that it may not contain an essential RNA component. Two other factors, designated cleavage factors I (CFI; Mr, approximately 130,000) and II (CFII; Mr, approximately 110,000), are sufficient to reconstitute accurate cleavage when mixed with SF. A fourth factor, termed cleavage stimulation factor (CstF; Mr, approximately 200,000), enhances cleavage efficiency significantly when added to a mixture of the three other factors. CFI, CFII, and CstF do not contain RNA components, nor do they affect specific polyadenylation in the absence of cleavage. Although these four factors are necessary and sufficient to reconstitute efficient cleavage of one pre-RNA tested, poly(A) polymerase is also required to cleave several others. A model suggesting how these factors interact with the pre-mRNA and with each other is discussed.
In situ nucleoprotein structure at the SV40 major late promoter: melted and wrapped DNA flank the start site.
Authors: Zhang L , Gralla J D
Published: June 2007

New in situ probing methods have been developed and used to probe the nucleoprotein structures at the SV40 major late promoter in infected monkey cells. The region that contains the three proximal transcription elements was probed with DNase I and micrococcal nuclease in transcriptionally active, permeabilized cells, and with the single-strand selective reagent KMnO4 in intact cells. The downstream element is included in a region of enhanced DNase I reactivity at 10- to 11-bp intervals for approximately 140 bp, presumably because of DNA wrapping around a specifically positioned nucleosome particle. The two other proximal DNA elements appear to be mostly melted, with a protecting factor bound primarily to the template DNA strand. The protecting factor directly borders the wrapped particle. These observations provide an initial description of parts of the biological transcription machinery and suggest that the SV40 major late promoter elements are part of a higher order nucleoprotein complex that involves wrapped and melted DNA.
Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis.
Authors: Sun D X , Stragier P , Setlow P
Published: June 2007

During sporulation of Bacillus subtilis, two identical genomes segregate in two compartments, the forespore and mother cell. These genomes are expressed differentially, with some genes such as sspE turned on only in the forespore. In vitro transcription of sspE was obtained only with RNA polymerase extracted from sporulating cells. Fractionation of factors associated with this enzyme and reconstitution with core RNA polymerase from vegetative cells generated an enzyme accurately transcribing sspE in vitro and led to purification of a polypeptide with the amino-terminal sequence of the spoIIIG product. Inactivation of spoIIIG abolished expression of sspE and five other forespore-specific genes, whereas synthesis of the spoIIIG product in vegetative cells rapidly turned these genes on. Therefore, spoIIIG encodes a sigma-factor, sigma G, which controls the expression of multiple genes in the forespore compartment.
The product of a fos-related gene, fra-1, binds cooperatively to the AP-1 site with Jun: transcription factor AP-1 is comprised of multiple protein complexes.
Authors: Cohen D R , Ferreira P C , Gentz R , Franza B R , Curran T
Published: June 2007

fra-1 encodes a serum-inducible protein (Fra-1) that is antigenically related to Fos. We have characterized Fra-1 expression in serum-stimulated cells using antibodies raised against several regions of this protein. Fra-1, expressed transiently in COS cells or in serum-stimulated rat fibroblasts, undergoes extensive post-translational modification, primarily by phosphorylation of serine residues. It is present in both the nucleus and the cytoplasm and participates in a protein complex with Jun. Using proteins synthesized in reticulocyte lysates, we have shown that Fra-1, like Fos, binds to the AP-1 recognition element cooperatively with Jun. A truncated Fra-1 protein that contains the leucine zipper region but not an adjacent basic amino acid domain, complexes with Jun in vitro but fails to bind AP-1 oligonucleotides. These results demonstrate that Fra-1 contributes to the DNA-binding activity ascribed to transcription factor AP-1.
Position and density effects on repression by stationary and mobile DNA-binding proteins.
Authors: Elledge S J , Davis R W
Published: June 2007

We have investigated the effects of two types of DNA-binding proteins on bacterial repression. First, the effects of operator positioning on repression by stationary DNA-binding proteins, the Lac repressor and the Trp repressor, were examined in vivo. Both operator number and positioning play a role in determining in vivo levels of repression. Operators located within a promoter are more efficient regulators than those positioned at the start of transcription. Second, we investigated the effects of DNA-binding protein density on repression using a mobile DNA-binding protein, Escherichia coli RNA polymerase. We employed a transcriptional interference assay using convergent transcriptional units. The strong synthetic promoter conI and its derivatives were observed to interfere with expression of the aadA gene, which confers spectinomycin resistance upon its host. Transcriptional interference by RNA polymerase occurred only in cis and had a strong dependence on polymerase density that was modulated by varying the promoter strengths. A change in the density of approximately fourfold completely abolished the observed transcriptional interference. Several models are discussed to explain the repression patterns observed for stationary and mobile DNA-binding proteins.
Multiple sequence elements in the c-fos promoter mediate induction by cAMP.
Authors: Fisch T M , Prywes R , Simon M C , Roeder R G
Published: June 2007

Induction of c-fos by cyclic AMP in NIH-3T3 cells is distinct from induction by serum. Whereas induction by serum is mediated by the serum response element (SRE), induction by cAMP does not require this element. In fact, no single sequence element in the c-fos promoter/enhancer is stringently required for the cAMP response. Rather, multiple sequence elements in the c-fos promoter/enhancer can mediate induction by cAMP independently. These elements are: (1) the region from -72 to -54, which contains a binding site for a cellular activating transcription factor (ATF); (2) the region from -225 to -99; (3) the region from -303 to -281, which is homologous to the consensus binding site for the transcription factor AP1; and (4) the region from -317 to -298, which contains the SRE. These sequence elements convey cAMP inducibility when fused to the cAMP-unresponsive 'minimal' fos promoter (-53 to +42). In addition, the c-fos regions from -700 to -63 and from -71 to -48 can confer cAMP inducibility to a heterologous promoter.
3'-end formation of mouse pre-rRNA involves both transcription termination and a specific processing reaction.
Authors: Kuhn A , Grummt I
Published: June 2007

We have studied the sequence requirements for 3'-end formation of rDNA transcripts in a cell-free system and show that the generation of correct ends of mouse pre-rRNA is brought about by a two-step process that involves a bona fide termination reaction, followed by a specific trimming of the primary transcript by 10 nucleotides. We show that termination of mouse ribosomal gene transcription by RNA polymerase I (pol I) takes place in front of an 18-bp DNA sequence element (the 'Sal box'), which was previously shown to function as termination signal. Termination of pol I transcription occurs at a fixed distance (11 bp) upstream of the Sal box, independent of the sequence of adjacent gene regions. The processing reaction, however, is strongly influenced by sequences flanking the termination signal at the 5' site. Substitution of a cluster of T residues by guanines within the region of 3'-end formation abolishes the 3'-terminal trimming of the primary transcript. Interestingly, this 3'-terminal processing event, which can be uncoupled from the termination reaction, requires both a correct 3' end and specific sequences in the 3'-terminal region of the primary transcript. Read-through transcripts generated in the extract system or by SP6 RNA polymerase are no substrate for the processing nuclease(s). Because the termination and processing activity can be separated chromatographically, the nucleolytic activity does not reside in TTF-I, the factor that binds to the Sal box and directs transcription termination.
Overlapping genes of Drosophila melanogaster: organization of the z600-gonadal-Eip28/29 gene cluster.
Authors: Schulz R A , Butler B A
Published: June 2007

The Drosophila gonadal (gdl) gene is a member of a gene cluster that maps cytogenetically to the 71CD interval of chromosome 3. The gene is bordered distally by z600, a gene expressed predominantly during early embryogenesis, and proximally by Eip28/29, a gene regulated by ecdysone in Drosophila cell lines. gdl can be expressed in either of two modes in adults: gdlF expression leads to the transcripts 1300 and 1000, which are found in the ovaries, whereas gdlM expression leads to the transcripts 1500 and 1200, which are found in the testes. In situ hybridization analysis reveals that this expression occurs in the germ line during oogenesis and spermatogenesis. Structural studies identify an unusual gdl sequence organization. The ovarian and testes transcripts differ at their 5' ends because of the utilization of different transcription initiation sites. This result indicates that alternative promoter usage is responsible for sex-specific gdl expression. Within an expression mode, the two transcripts differ at their 3' ends as a result of multiple polyadenylation site usage; one of these sites resides within the 5' exon of the Eip28/29 gene. gdl is overlapped by z600 as well because the z600 transcript is polyadenylated at position +91 of the gdlM transcripts. An analysis of germ-line transformants reveals that gdl can be expressed properly outside the overlapping gene environment because a 1.8-kb DNA region contains all the sequences necessary for gdl sex-specific expression.
Embryonic expression of a Drosophila src gene: alternate forms of the protein are expressed in segmental stripes and in the nervous system.
Authors: Vincent W S , Gregory R J , Wadsworth S C
Published: June 2007

Expression of the Drosophila src-related gene, Dsrc28C has been investigated at the protein level using monoclonal antibodies. This analysis has revealed that the Dsrc28C gene encodes two protein forms: a 66-kD doublet predicted from the sequence of a cDNA clone and an additional 55-kD form. The 66-kD protein doublet is observed first at the cellular blastoderm stage and is not detectable in embryos after 12 hr of development. Expression of the 55-kD protein lags behind that of the 66-kD doublet and then persists throughout embryogenesis. Indirect immunofluorescence microscopy reveals that Dsrc28C protein is localized to the cell periphery during cellular blastoderm and gastrulation. The cell periphery-associated staining is then resolved into ectodermal stripes along the fully extended germ band. After the stripes fade, cytoplasmic staining of the majority of cells within the central and peripheral nervous system is observed. The 66-kD protein was shown to represent the cell periphery-associated form of the protein through antibody staining of larval salivary glands expressing a heat shock promoter-driven, full-length Dsrc28C cDNA. Staining of embryos with a monoclonal antibody specific for the 66-kD protein indicates that the 55-kD protein is the nervous system form. Thus, the 66- and 55-kD proteins are products of the Dsrc28C gene, which exhibit different temporal and spatial patterns of expression in the embryo.
Alternative splicing of the neural cell adhesion molecule gene generates variant extracellular domain structure in skeletal muscle and brain.
Authors: Thompson J , Dickson G , Moore S E , Gower H J , Putt W , Kenimer J G , Barton C H , Walsh F S
Published: June 2007

Myotube mRNA isoforms of the neural cell adhesion molecule (N-CAM) contain a novel sequence block termed muscle-specific domain 1 (MSD1), which is inserted within the extracellular coding region. Here, we report a characterization of the genomic organization of MSD1 and its pattern of expression within cellular N-CAM RNA and polypeptide species. S1 nuclease protection analyses and sequence analysis of an N-CAM human genomic clone containing MSD1 sequences indicated that MSD1 is comprised of three discrete exons of 15, 48, and 42 bp, designated MSD1a, MSD1b, and MSD1c, respectively. Although the MSD1a exon was present in a small proportion of mRNAs from both brain and muscle cells, the entire MSD1 sequence occurred predominantly in mRNAs from differentiated myotube cells. In addition, antiserum raised to a synthetic, MSD1b-encoded peptide sequence was found to stain the cell surface of human skeletal myotubes in culture, whereas myoblasts, fibroblasts, and neural cells were negative. MSD1a, MSD1b, and MSD1c sequences thus arise collectively in N-CAM mRNA and polypeptide isoforms as a result of muscle tissue-specific and developmentally regulated alternative mRNA splicing events. In addition, the occurrence of brain and muscle mRNAs containing only MSD1a indicate that alternative splicing may occur within the MSD region itself to generate further diversity.