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At least 523 records · Page 29Linked to original sources
PubMed2001

Probing DNA single strands for single-base bulges with neocarzinostatin chromophore.

Neocarzinostatin chromophore (NCS-Chrom) induces strong cleavage at a single site (C3) in the single-stranded and 5' (32)P-end-labeled 13-mer GCCAGATTTGAGC in a reaction dependent on a thiol. By contrast, in the duplex form of the same 13-mer, strand cleavage occurs only at the T and A residues, and C3 is not cleaved. To determine the minimal structural requirement(s) for C3 cleavage in the single-stranded oligomer, several deletions and mutations were made in the 13-mer. A 10-mer (GCCAGAGAGC) derived from the 13-mer by deletion of the three T residues was also cleaved exclusively at C3 by NCS-Chrom, generating fragments having 5' phosphate ends. That the cleavage at C3 is initiated by abstraction of its 5' hydrogen is confirmed in experiments using 3' (32)P-end-labeled 10-mer. The competent 13-mer and 10-mer were assigned hairpin structures with a stem loop and a single bulged out A base, placing C3 across from and 3' to the bulge. Removal of the bulged A base from the 13-mer and the 10-mer resulted in complete loss of cutting activity, proving that it is the essential determinant in competent substrates. Studies of thiol post-activated NCS-Chrom binding to the DNA oligomers show that the drug binds to the bulge-containing 13-mer (K(d) = 0.78 microM) and the 10-mer (K(d) = 1.11 microM), much more strongly than to the 12-mer (K(d) = 20 microM) and the 9-mer (K(d) = 41 microM), lacking the single-base bulge. A mutually induced-fit between NCS-Chrom and the oligomer resulting in optimal stabilization of the drug-DNA complex is proposed to account for the site-specific cleavage at C3. These studies establish the usefulness of NCS-Chrom as a probe for single-base bulges in DNA.

Antibiotics, Antineoplastic↗
PubMed2005

The adenomatous polyposis coli protein (APC) exists in two distinct soluble complexes with different functions.

Mutations resulting in the truncation of the adenomatous polyposis coli (APC) protein are common to most colonic tumours. The APC protein has emerged as a multifunctional protein that contributes to cytoskeletal organisation and is involved in the regulation of beta-catenin. Both, changes in transcription due to increases in beta-catenin, as well as defects in directed cell migration and cell division contribute to cancer when APC is mutated. Little is known about how separate functions of APC are coordinated. In this study, we identified two distinct soluble protein pools containing APC. We found that one of these pools represents the fully assembled beta-catenin-targeting complex. The second pool contained at least two different forms of APC: APC that was bound to partially assembled beta-catenin-targeting complexes and APC that could bind microtubules. Consistent with the previously proposed role for glycogen synthase kinase 3beta (GSK3beta) in modulating the assembly and activity of the beta-catenin-targeting complex, formation of the fully assembled complex was reduced by inhibitors of GSK3beta. Similarly, tumour cells with truncated APC only contained the partially assembly beta-catenin-targeting complex. We also found that highly elevated levels of beta-catenin in tumour cells containing wild-type APC correlated with a decrease in the ability of the endogenous APC protein to bind microtubules. Additionally, APC lacking the direct microtubule binding site was more effective at downregulating beta-catenin. Together, our data suggest that the interaction of APC with microtubules and the beta-catenin-targeting complex are mutually exclusive, and indicate that the distribution of endogenous APC between different pools is dynamic, which allows cells to distribute it as required.

Adenomatous Polyposis Coli Protein↗
PubMedDate not supplied

p53 overexpression and human papillomavirus (HPV) infection in oesophageal squamous cell carcinomas derived from a high-incidence area in China.

Oesophageal epithelium is frequently exposed to various carcinogens and mutagens, many of which may cause p53 gene mutations. The epithelium can also be infected with human papillomavirus (HPV), the E6 protein of which may complex with p53 protein and facilitate its degradation. To identify HPV infection and p53 overexpression in oesophageal cancer, we performed immunohistochemical analysis using CM-1 anti-p53 antibody and DNA in situ hybridization with biotinylated HPV DNA probes on paraffin-embedded sections in 36 patients with oesophageal squamous cell carcinomas derived from a high-incidence area in northern China. Samples from cancer tissue, adjacent epithelia, regional lymph nodes as well as resection margins were examined. p53 protein accumulation was detected in 55.6% (20/36) of cancer samples, in 20% (1/5) of hyperplastic epithelium, in 20% (2/10) of dysplastic lesions as well as in 67% (2/3) of carcinoma in situ lesions adjacent to invasive carcinomas. HPV DNA sequences were demonstrated in 3 patients (8.3% of the total). Two of these HPV-positive carcinomas were immunohistochemically negative for p53 and one was weakly positive. Our results suggest that p53 overexpression is frequently found in oesophageal carcinomas and that p53 alteration may be an early event in esophageal carcinogenesis. HPV and elevated p53 are not mutually exclusive events, instead they can coexist in some oesophageal squamous cell carcinomas.

Adult↗
PubMed2000

A new look at the microtubule binding patterns of dimeric kinesins.

The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.

Adenosine Triphosphate↗
PubMed1990

A family of constitutive C/EBP-like DNA binding proteins attenuate the IL-1 alpha induced, NF kappa B mediated trans-activation of the angiotensinogen gene acute-phase response element.

The gene encoding angiotensinogen, the glycoprotein precursor of the potent vasopressor peptide angiotensin II, is transcriptionally activated in hepatocytes during the acute-phase response through interactions of mutually cooperative glucocorticoid receptors and proteins that bind to an acute-phase response element (APRE) 5'-AGTTGGGATTTCCCAACC-3'. The APRE binds a family of constitutive proteins (BPcs) and a cytokine inducible protein (BPi) that is indistinguishable from nuclear factor kappa B (NF kappa B). The interactions of purified proteins with the APRE were studied by in vitro binding and in vivo transcriptional trans-activation assays. BPc is a family of heat-stable DNA binding proteins, the different sized members of which are capable of forming heterodimers. BPcs are recognized by anti-C/EBP antiserum and produce a footprint similar to bacterially expressed C/EBP on the APRE. BPi has a 4- to 5-fold greater affinity for the APRE than the BPcs, and contacts guanosine residues distinct from those contacted by the BPcs, demonstrating that these two classes of proteins contain functionally distinct DNA binding domains. Assays of APRE-luciferase reporter plasmids transfected into HepG2 cells show that a mutated APRE that binds only BPi functions as an IL-1 alpha inducible enhancer, whereas a mutated APRE that binds only BPc does not. The APRE mutant that binds the C/EBP-like BPcs to the exclusion of BPi functions as an uninducible basal enhancer both in the native context of the angiotensinogen gene and when multimerized and placed upstream of a minimal angiotensinogen promoter. The wild-type APRE that binds both BPi and BPc is less inducible by IL-1 alpha than the mutated APRE that binds only BPi. Gel shift competition assays demonstrate in vitro that the mechanism of transcriptional regulation by the APRE involves a competition between BPc and the inducible BPi for binding to the APRE. IL-1 alpha stimulation of hepatocytes leads to nuclear translocation of the NF kappa B-like BPi which competes with the constitutive C/EBP-like BPcs for overlapping binding sites on the APRE and thereby replaces weak transcriptional activators with a stronger one.

Angiotensinogen↗
PubMed1995

pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line.

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.

Animals↗
PubMed1995

Binding sites for transcription factor NTF-1/Elf-1 contribute to the ventral repression of decapentaplegic.

The Dorsal morphogen is a transcription factor that activates some genes and represses others to establish multiple domains of gene expression along the dorsal/ventral axis of the early Drosophila embryo. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). We have identified a corepression element in decapentaplegic (dpp), a zygotically active gene that is repressed by the Dorsal morphogen. This dpp repression element (DRE) is located within a previously identified VRR and close to essential Dorsal-binding sites. We have purified a factor from Drosophila embryo extracts that binds to the DRE but not to mutant forms of the DRE that fail to support efficient repression. This protein also binds to an apparently essential region in a VRR associated with the zerknüllt (zen) gene. One of the DREs in the dpp VRR overlaps the binding site for a potential activator protein suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. We have found the DRE-binding protein to be identical to NTF-1 (equivalent to Elf-1, the product of the grainyhead gene), a factor originally identified as an activator of the Ultrabithorax and Dopa decarboxylase promoters. NTF-1 mRNA is synthesized during oogenesis and deposited in the developing oocyte where it is available to contribute to ventral repression during early embryogenesis. Previous studies have shown that overexpression of NTF-1 in the postblastoderm embryo results in a phenotype that is consistent with a role for this factor in the repression of dpp later in embryogenesis.

Animals↗
PubMed2004

Novel isoforms of the sodium channels Nav1.8 and Nav1.5 are produced by a conserved mechanism in mouse and rat.

The voltage-gated sodium channel Na(v)1.8 is only expressed in subsets of neurons in dorsal root ganglia (DRG) and trigeminal and nodose ganglia. We have isolated mouse partial length Na(v)1.8 cDNA clones spanning the exon 17 sequence, which have 17 nucleotide substitutions and 12 predicted amino acid differences from the published sequence. The absence of a mutually exclusive alternative exon 17 was confirmed by sequencing 4.1 kilobases of genomic DNA spanning exons 16-18 of Scn10a. A novel cDNA isoform was identified, designated Na(v)1.8c, which results from alternative 3'-splice site selection at a CAG/CAG motif to exclude the codon for glutamine 1031 within the interdomain cytoplasmic loop IDII/III. The ratio of Na(v)1.8c (CAG-skipped) to Na(v)1.8 (CAG-inclusive) mRNA in mouse is approximately 2:1 in adult DRG, trigeminal ganglion, and neonatal DRG. A Na(v)1.8c isoform also occurs in rat DRG, but is less common. Of the two other tetrodotoxin-resistant channels, no analogous alternative splicing of mouse Na(v)1.9 was detected, whereas rare alternative splicing of Na(v)1.5 at a CAG/CAG motif resulted in the introduction of a CAG trinucleotide. This isoform, designated Na(v)1.5c, is conserved in rat and encodes an additional glutamine residue that disrupts a putative CK2 phosphorylation site. In summary, novel isoforms of Na(v)1.8 and Na(v)1.5 are each generated by alternative splicing at CAG/CAG motifs, which result in the absence or presence of predicted glutamine residues within the interdomain cytoplasmic loop IDII/III. Mutations of sodium channels within this cytoplasmic loop have previously been demonstrated to alter electrophysiological properties and cause cardiac arrhythmias and epilepsy.

Alternative Splicing↗
PubMed1993

Asbestos-induced lung disease.

This review attempts to deal with two major questions concerning asbestos-induced lung disease: How does inhaled asbestos cause cell proliferation and fibrosis? and Will there continue to be risk from exposure to asbestos in schools and public buildings? The first is a scientific question that has spawned many interesting new experiments over the past 10 years, and there appear to be two hypothetical schemes which could explain, at least in part, the fibroproliferative effects of asbestos fibers. One supports the view that toxic oxygen radicals generated on fiber surfaces and/or intracellularly are the central mediators of disease. The second hypothesis is not mutually exclusive of the first, but, in my opinion, may be integral to it, i.e., the cellular injury induced by oxygen radicals stimulates the elaboration of multiple varieties of growth factors and cytokines that mediate the pathogenesis of asbestosis. There is increasing evidence that molecules such as platelet-derived growth factor and transforming growth factor beta, both synthesized and secreted by activated lung macrophages, are responsible, respectively, for the increased interstitial cell populations and extracellular matrix proteins that are the hallmarks of asbestos-induced fibrosis. The challenge today is to establish which combinations of the many factors released actually are playing a role in disease pathogenesis. The issue of continued risk currently is more a question of policy and perception than science because a sufficient database has not yet been established to allow full knowledge of the circumstances under which asbestos in buildings constitutes an ongoing health hazard. The litigious nature of this question does not help its resolution. In as much as public policy statements and risk assessment are not within my purview, I have focused on the state-of-the-art of asbestos as a complete carcinogen. It appears to be generally nongenotoxic, but all asbestos fiber types can induce chromosomal mutations and aneuploidy, perhaps through their ability to disrupt normal chromosome segregation.

Asbestos↗
PubMed1992

Interference of pathway specific transcription factors.

A mechanism for reversibility of cellular decisions, e.g., proliferation or differentiation, is mediated by direct interference of pathway-specific transcription factors. Due to the relative activity of the transcription factors, genetic programs are reversibly switched on and off. In the case of the transcription factors AP-1 and the family of steroid hormone receptors (including retinoic acid receptor, thyroid hormone and vitamin D receptors) two different mechanisms account for negative interference. In one case overlapping DNA binding sites are suggestive of mutually exclusive binding of transcription factors to account for repression. In the other case, a DNA bound transcription factor is attacked by another one via protein-protein interaction, without the need of the latter factor binding to DNA. Although it is not excluded, that also in the case of overlapping DNA binding elements protein-protein interaction might in fact be responsible for repression instead of substitution, the two situations can be clearly distinguished by specific mutants in the glucocorticoid receptor and in Jun and Fos. For the interaction mechanism, we favor the interpretation that the binding of one transcription factor to another one interferes with transactivation directly without influence on DNA binding. The fact that at least two different mechanisms are established to guarantee effective interference, demonstrates how important this type of transcription factor crosstalk is.

Amino Acid Sequence↗
PubMed2002

Lack of synergy for inhibitors targeting a multi-drug-resistant HIV-1 protease.

The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to approximately 2.2 A resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.

Anti-HIV Agents↗
PubMed2025

Molecular analysis of lung adenocarcinomas from the SAFIR02-Lung cohort reveals new metastasis-associated copy-number alterations including frequent mutant-specific KRAS-allelic imbalance and identifies CDKN2A homozygous deletions as an independent biomarker of poor prognosis.

BACKGROUND: Identifying molecular alterations specific to advanced lung adenocarcinomas could provide insights into tumour progression and dissemination mechanisms. METHOD: We analysed tumour samples, either from locoregional lesions or distant metastases, from patients with advanced lung adenocarcinoma from the SAFIR02-Lung trial by targeted sequencing of 45 cancer genes and comparative genomic hybridisation array and compared them to early tumours samples from The Cancer Genome Atlas. RESULTS: Differences in copy-number alterations frequencies suggest the involvement in tumour progression of LAMB3, TNN/KIAA0040/TNR, KRAS, DAB2, MYC, EPHA3 and VIPR2, and in metastatic dissemination of AREG, ZNF503, PAX8, MMP13, JAM3, and MTURN. Conversely, no meaningful difference was found in pathogenic single-nucleotide variant frequencies, reinforcing the notion that they are early events in tumorigenesis. CDKN2A homozygous deletion was linked to poor clinical outcome in patients with early tumours (overall survival hazard ratio 2.17, 95% CI: 1.43-3.28, corrected p-value = 0.01). Furthermore, we found that KRAS mutant allele specific imbalance, i.e. focal amplification of the mutant allele, is more prevalent in locoregional or distant samples of metastatic patients than in early lesions (8.4%, 13% and 2.8% respectively). This observation was replicated in three public cohorts. Tumours with KRAS mutant allele specific imbalance show specific patterns of co-occurrence and mutual exclusion with alterations in key cancer genes like CDKN2A, TP53, STK11 and NKX2-1, often in a tumour type dependent manner. CONCLUSION: Advanced LUAD tumours exhibit higher copy-number alteration burden, with distinct alterations associated with tumour progression and metastasis. CDKN2A homozygous deletions predict poor prognosis in early disease, while KRAS mutant allele-specific imbalance is enriched in advanced tumours.

Humans↗
PubMed1992

Intronic sequence with both negative and positive effects on the regulation of alternative transcripts of the chicken beta tropomyosin transcripts.

The chicken beta tropomyosin gene generates three major transcripts by alternative splicing. A pair of internal exons are spliced in a mutually exclusive manner and their utilisation is developmentally regulated. Exon 6A and exon 6B are used respectively in myoblasts and myotubes during the process of differentiation of muscle cells. We have previously reported that, in myoblasts, exon 6B is skipped because of a negative regulation which involves intron as well as exon sequences. In this report, we describe a previously uncharacterized intronic element which is involved in the regulation of the splicing of both exons 6A and 6B. This cis-element is localized 37nt downstream of exon 6A and is approximately 30nt long. Its deletion, as well as modification of its sequence, results in the activation of the use of exon 6B and, at the same time, in the inhibition of the use of exon 6A. The mechanisms by which this region could act are further discussed.

Animals↗
PubMed2000

A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent Escherichia coli 113/3 cells.

The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343-418. The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12-dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive.

Amino Acid Sequence↗
PubMed1996

Mitogenic signaling from the egf receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback mechanism.

We recently demonstrated that epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level. How these two mutually exclusive cell responses cross-communicate is not known. We investigated a possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis. Inhibition of PLC gamma activation by U73122 (1 microM) augmented the EGF-induced [3H]thymidine incorporation by 23-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis was observed in parallel. The time dependence of this increase revealed that it was due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrated augmented mitogenic responses by 25-68% when compared with control cells. PLCz- or U73122-augmented mitogenesis was not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 microM) resulted in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine654 was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrated EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells were treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response.

Animals↗
PubMed1996

oriP is essential for EBNA gene promoter activity in Epstein-Barr virus-immortalized lymphoblastoid cell lines.

During Epstein-Barr virus latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities are mutually exclusive. Upon infection, Wp is initially active, followed by a switch to Cp for the duration of latency. In this study, the region upstream of Cp was analyzed for the presence of cis elements involved in regulating the activities of the EBNA gene promoters in established in vitro immortalized lymphoblastoid cell lines (LCLs). It was determined that oriP, the origin for episomal maintenance during latency, is essential for efficient transcription initiation from either Cp or Wp in LCLs, as well as in some Burkitt's lymphoma cell lines. Deletion of the EBNA2-dependent enhancer located upstream of Cp resulted in a ca. two- to fivefold reduction in Cp activity in the LCLs assayed. More extensive deletion of sequences upstream of Cp, including the EBNA2-dependent enhancer, resulted in nearly complete loss of Cp activity. This loss of activity was shown to correlate with deletion of two CCAAT boxes, a proximal CCAAT box located at bp -61 to -65 and a distal CCAAT box located at bp -253 to -257, upstream of Cp. Site-directed mutagenesis of these cis elements demonstrated that Cp activity is highly dependent on the presence of a properly positioned CCAAT box, with the dependence on the distal CCAAT box apparent only when the proximal CCAAT box was deleted or mutated. Deletion of the glucocorticoid response elements located at ca. bp -850 upstream of Cp did not result in a significant loss in activity. In general, deletions which diminished Cp activity resulted in induction of Wp activity, consistent with suppression of Wp activity by transcriptional interference from Cp. The identification of oriP and the EBNA2-dependent enhancer as the major positive cis elements involved in regulating Cp activity in LCL suggests that EBNA gene transcription is largely autoregulated by EBNA 1 and EBNA 2.

Animals↗
PubMed1994

Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6.

We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro. We found that mutations in two regions of the pre-mRNA led to activation of the 3'-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3'-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5'-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3'-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5'-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3'-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5'-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro, such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5'-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection.

Alternative Splicing↗
PubMed1994

Characterization of epitopes defining two major subclasses of polytropic murine leukemia viruses (MuLVs) which are differentially expressed in mice infected with different ecotropic MuLVs.

Polytropic murine leukemia viruses (MuLVs) arise in mice by recombination of ecotropic MuLVs with endogenous retroviral envelope genes and have been implicated in the induction of hematopoietic proliferative diseases. Inbred mouse strains contain many endogenous sequences which are homologous to the polytropic env genes; however, the extent to which particular sequences participate in the generation of the recombinants is unknown. Previous studies have established antigenic heterogeneity among the env genes of polytropic MuLVs, which may reflect recombination with distinct endogenous genes. In the present study, we have examined many polytropic MuLVs and found that nearly all isolates fall into two mutually exclusive antigenic subclasses on the basis of the ability of their SU proteins to react with one of two monoclonal antibodies, termed Hy 7 and MAb 516. Epitope-mapping studies revealed that reactivity to the two antibodies is dependent on the identity of a single amino acid residue encoded in a variable region of the receptor-binding domain of the env gene. This indicated that the two antigenic subclasses of MuLVs arose by recombination with distinct sets of endogenous genes. Evaluation of polytropic MuLVs in mice revealed distinctly different ratios of the two subclasses after inoculation of different ecotropic MuLVs, suggesting that individual ecotropic MuLVs preferentially recombine with distinct sets of endogenous polytropic env genes.

3T3 Cells↗