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D Levens

Publications and source records attributed to D Levens.

At least 19 recordsLinked to original sources
PubMed1995

Cellular nucleic acid binding protein regulates the CT element of the human c-myc protooncogene.

The CT element of the c-myc gene is required for promoter P1 usage and can drive expression of a heterologous promoter. Both double strand (Sp1) and single strand (hnRNP K) CT-binding proteins have been implicated as mediators of CT action. Although significant levels of CT activity persisted following Sp1 immunodepletion, EGTA totally abolished transactivation, thus implicating another metal requiring factor in CT element activity. As hnRNP K binds to one strand of the CT element, but has no metal requirement, the opposite (purine-rich strand) was examined as a target for a metal-dependent protein. A zinc-requiring purine strand binding activity was identified as cellular nucleic acid binding protein (CNBP), a protein previously implicated in the regulation of sterol responsive genes. Two forms of CNBP differed in their relative binding to the CT- or sterol-response elements. CNBP was shown to be a bona fide regulator of the CT element by cotransfection of a CNBP expression vector that stimulated expression of a CT-driven but not an AP1-dependent reporter. These data suggest that hnRNP K and CNBP bind to opposite strands and co-regulate the CT element.

Amino Acid Sequence↗
PubMed1995

Targeted melting and binding of a DNA regulatory element by a transactivator of c-myc.

A far upstream element (FUSE) of c-myc stimulates promoter activity when bound by a newly identified trans-acting protein, which is expressed in cycling cells. Since FUSE binding protein (FBP) binds only the noncoding strand (NCS) of its regulatory element in a sequence-specific manner, and not double-stranded (ds) DNA, formation of the protein DNA complex in vivo first requires unwinding of the DNA helix. In this report, we show evidence that FBP forces strand separation of short stretches of linear dsDNA. Because FUSE is contained within a region of helical instability that is partially unwound in negatively supercoiled DNA, it is a target for more extensive duplex strand separation by FBP, which first exposes and then selectively binds its NCS cognate sequence. In contrast, other single-stranded DNA binding proteins (SSBs) do not demonstrate this FUSE targeting activity. The novel linkage of regional dsDNA melting with cis-element binding by a transcriptional activator has broad implications in the regulation of eukaryotic gene expression.

Base Sequence↗
PubMed1990

A far upstream element stimulates c-myc expression in undifferentiated leukemia cells.

A sensitive exonuclease assay revealed multiple sites for interaction, in vitro, of sequence specific factors with c-myc upstream elements. At one site, more than 1500 base pairs upstream of the c-myc promoter P1, binding activity was lost as dimethyl sulfoxide (Me2SO) induced shut-off of c-myc expression in HL-60 and U-937 leukemia cells. The disappearance of other specific binding activities was not noted. In addition, the binding activity was noted to be cell-line specific. The sequence binding the Me2SO-regulated factor was precisely located allowing confirmation of the temporal pattern of regulation by electrophoretic mobility shift analysis. Because the binding activity was most abundant before the decrease of c-myc expression during differentiation, it was inferred that the far upstream element (FUSE) served a positive role, potentiating c-myc expression. A 4-base pair deletion which eliminated binding to FUSE also reduced expression of a transfected, chimeric c-myc-CAT gene in untreated, but not in Me2SO-treated U-937 cells. FUSE and its binding protein may contribute to cell line- and differentiation-specific modes of c-myc regulation.

Base Sequence↗
PubMed1990

Phosphorylation-dependent binding of a 138-kDa myc intron factor to a regulatory element in the first intron of the c-myc gene.

A 138-kDa nuclear protein was identified from HeLa cell extracts as a factor which binds to a previously described 20-base pair cis element located in the intron I of the c-myc gene. This myc intron factor (MIF) binds to the wild type c-myc sequence but does not bind under similar conditions to c-myc from Burkitt's lymphoma which contain point mutations in this binding region. We have demonstrated that the 138-kDa MIF is a phosphoprotein and that treatment of the purified MIF with potato acid phosphatase abolished binding to its 20-base pair c-myc recognition sequence; binding activity was protected by inclusion of phosphatase inhibitors. These results suggest that phosphorylation is required for the specific DNA-MIF interaction in vitro and that the phosphorylation state of MIF may be an important factor in controlling c-myc expression in vivo.

Acid Phosphatase↗
PubMed1989

fos/jun and octamer-binding protein interact with a common site in a negative element of the human c-myc gene.

A negative element has previously been localized to a 57-base pair segment approximately 300 base pairs upstream of the human c-myc promoter P1. Within this element, a 26-base pair region was protected in vitro from DNase I digestion with a HeLa cell nuclear factor(s). Two specific DNA-protein complexes were identified in gel retardation assays using HeLa cell nuclear extracts and an oligonucleotide probe spanning the footprinted region. Exonuclease and chemical footprint analyses suggested that the binding sites for both complexes are almost entirely overlapping. One of the complexes was eliminated by oligonucleotide competitors possessing known AP-1 binding sites. This same complex reacted strongly with anti-fos immunoglobulin suggesting a role for c-fos in governing c-myc expression. Precipitation of fos protein bound to c-myc DNA that was immobilized on beads confirmed the involvement of c-fos in a specific complex with the c-myc upstream sequence. In contrast, the other complex seen by the c-myc probe could not be competitively inhibited by AP-1 binding sites and was not affected by anti-fos antibody. Instead, this complex was efficiently eliminated by unlabeled oligonucleotides containing the octamer DNA motif found in immunoglobulin gene promoters. Purified octamer-binding proteins formed stable complexes with the 26-base pair c-myc sequences. These results demonstrate that degeneracy in the consensus recognition sequences of these distinct factors allows each of them to bind the c-myc negative element. The interaction of known transcriptional activators with a negative element suggests that the same factors can mediate both transcriptional activation and repression.

Base Sequence↗
PubMed1989

Distinct factors bind the AP-1 consensus sites in gibbon ape leukemia virus and simian virus 40 enhancers.

We have demonstrated that the gibbon ape leukemia virus (GALV) enhancer AP-1 element and the simian virus 40 AP-1 enhancer element bind different factors in HeLa nuclear extracts. A 39-kilodalton HeLa nuclear protein and the c-fos protein bind to the GALV element. Antibodies to c-fos abolish binding to the GALV AP-1 site. In contrast, anti-c-fos immunoglobulin fails to inhibit formation of the simian virus 40-specific complex from extracts of HeLa cells. Thus, AP-1-binding complexes are subject to compositional variation at different binding sites.

Animals↗
PubMed1989

Multiple components are required for sequence recognition of the AP1 site in the gibbon ape leukemia virus enhancer.

At least two subunits contributed to the formation in vitro of a specific complex binding to the AP1 consensus sequence (TGAGTCA) in the gibbon ape leukemia virus (GALV) enhancer in MLA144 cells. This complex can be dissociated on a monomeric GALV oligonucleotide affinity column. One protein, termed the core protein, was retained on the oligonucleotide affinity column. The second protein flowed through the oligonucleotide affinity column and, when alone, did not bind to DNA; however, when present with the core protein, it bound strongly and very specifically to the GALV sequence. MLA144 cells contained only trace amounts of c-fos and c-jun by immunoblot analysis, suggesting that the proteins specifically binding to the GALV AP1 site were distinct from c-fos and c-jun. In addition to the major complex that recognized the GALV element, MLA144 cells contained a minor complex that is chromatographically different from and antigenically related to c-fos. The factor in the flowthrough complemented a human T-cell nuclear extract (Jurkat cell line), which, when alone, had no assayable complex that specifically bound to the GALV enhancer; this complementation gave rise to a specific complex similar to that seen in MLA144 cells. Together, these results suggest that the GALV enhancer can interact with multicomponent protein complexes in a cell-line-specific manner.

Animals↗
PubMed1988

A point mutation in the c-myc locus of a Burkitt lymphoma abolishes binding of a nuclear protein.

A 20-base pair region in the first intron of the human c-myc gene was identified as the binding site of a nuclear protein. This binding site is mutated in five out of seven Burkitt lymphomas sequenced to date. To investigate the protein-recognition region in greater detail, the abnormal c-myc allele from a Burkitt lymphoma line (PA682) that carries a t(8;22) chromosomal translocation was used. A point mutation in the binding region of the PA682 c-myc DNA abolished binding of this nuclear protein. This protein may be an important factor for control of c-myc expression, and mutations in its recognition sequence may be associated with c-myc activation in many cases of Burkitt lymphoma.

Base Sequence↗
PubMed1987

Regulatory elements that modulate expression of human c-myc.

Regulation of transcription from the proto-oncogene c-myc apparently plays an important part in cellular proliferation and the genesis of diverse tumors. Here, we report that the abundance of transcripts from the two principal promoters for human c-myc (P1 and P2) is governed by a composite of positive and negative regulators, located within a 2.3-kb domain upstream of the gene. In actively proliferating cells, the action of the positive elements is apparently dominant over that of the single negative regulator that we have identified. Nuclear proteins bind specifically to nucleotide sequences within the negative regulator and at least one of the positive regulators. The cooperative and counteracting actions of the regulatory elements described here presumably contribute to the plasticity of transcription from c-myc and may be affected by the tumorigenic damage that sometimes afflicts c-myc.

Animals↗
PubMed1987

B-cell nuclear proteins binding in vitro to the human immunoglobulin kappa enhancer: localization by exonuclease protection.

Proteins capable of interacting with the enhancer of the immunoglobulin kappa gene in vitro have been detected in extracts of nuclei from human B cells and from human, mouse, and rabbit spleens. The experiments, based on an exonuclease protection technique, demonstrate nuclear protein factors binding to a 30- to 35-base-pair domain containing both the simian virus 40 enhancer core element (TTTCCA) and the octamer CAGGTGGC that was previously identified as the consensus sequence for protein-binding sites in the murine immunoglobulin heavy-chain enhancer. This 30- to 35-base-pair domain in the human kappa enhancer is homologous to a site of protein binding detected in the murine kappa enhancer by other investigators using a gel retardation assay. Our results complement in vivo dimethyl sulfate footprinting studies of the human immunoglobulin kappa enhancer which demonstrated B cell-specific changes in guanine reactivity immediately 5' to the consensus octamer. Together, these findings suggest that DNA-binding proteins in B-cell nuclei interact with the 5' portion of the human kappa-gene enhancer. Such proteins could play a role in the B cell-specific transcription of the human immunoglobulin kappa gene.

B-Lymphocytes↗
PubMed1987

Binding of a cellular protein to the gibbon ape leukemia virus enhancer.

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.

Animals↗
PubMed1986

Improved survival of island flaps after prolonged ischemia by perfusion with superoxide dismutase.

Perfusion of rat groin flaps after 10 and 11 hours of complete ischemia with superoxide dismutase, an oxygen free-radical scavenger, significantly improved the survival of these flaps. This finding provides further evidence for the important role that oxygen-derived free radicals play in ischemic injury. The study also demonstrates that while restoration of blood supply alone is not enough to ensure tissue survival after prolonged ischemia, chemical agents can be utilized to achieve viable flaps beyond what was believed to be "a point of no return".

Animals↗
PubMed1985

Myelopathy following intrathecal chemotherapy in a patient with extensive Burkitt's lymphoma and altered immune status.

A 30-year-old homosexual man presented with widespread Burkitt's lymphoma. On the basis of immunologic and viral studies, he was suspected of having the acquired immune deficiency syndrome. Following chemotherapy that included intrathecal cytosine arabinoside and methotrexate, brain stem edema, paraplegia, and an elevated cerebrospinal fluid level of myelin basic protein developed. Autopsy revealed vacuolar demyelination of spinal cord, brain stem, and cerebellum. The pathologic findings were similar to those reported to occur in myelopathy associated with intrathecal chemotherapy, but far more extensive. The contribution of the suspected acquired immune deficiency syndrome is unknown.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

Base Sequence↗
PubMed1982

Transcriptional initiation and processing of the small ribosomal RNA of yeast mitochondria.

We have identified the nucleotide at which transcription initiates on the yeast mitochondrial small (14 S) rRNA gene by sequencing of RNA labeled at the 5' initiating triphosphate with vaccinia virus guanylyltransferase [alpha-32P]GTP (in vitro capping reaction). Initiation occurs within the stem of a 12-base palindromic repeat. The initiation sequence has homology with the large (21 S) ribosomal RNA initiation sequence that has been previously determined. We have also sequenced the 5' and 3' ends of the mature 14 S rRNA after labeling with T4 polynucleotide kinase and RNA ligase, respectively. These sequences demonstrate that about 80 nucleotides are cleaved from the 5' end of a precursor to produce the mature 14 S rRNA. This cleavage is imprecise in that the processing occurs at one of five adjacent nucleotides 77 to 81 nucleotides downstream from the 5' initiation site. The 3' ends of this precursor and the mature 14 S rRNA are unique and identical.

Base Sequence↗
PubMed1982

The biogenesis and regulation of yeast mitochondria RNA polymerase.

Yeast mitochondrial RNA polymerase is a nuclear-coded protein of approximately 90,000 daltons comprised of two 45,000-dalton subunits of pI 6.9 to 7.0. To investigate the nature of the initial translation product of the RNA polymerase, we have analyzed those products of a cell-free translation system directed by yeast RNA that are immunoreactive with antibodies to the 45,000-dalton peptide of polymerase. A precursor of one or more of the subunits of the polymerase, 2,000 daltons later than the mature product, has been characterized using immunoreaction, immunocompetition, and peptide digestion. The role of transcription of the polymerase gene in catabolite repression of mitochondrial development has been investigated by analyzing the changes in cell-free synthesis of the RNA polymerase precursor during glucose and raffinose growth. The results indicate an increase in precursor synthesis and probably in the corresponding transcript abundance during glucose derepression. In contrast, the precursor is present at high levels until stationary phase during raffinose growth. These data indicate the involvement of increased transcription of the polymerase gene in the process of derepression.

Cell Nucleus↗