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Biomedical subjects

W E Wright

Publications and source records attributed to W E Wright.

At least 19 recordsLinked to original sources
PubMed1992

Cyclic amplification and selection of targets for multicomponent complexes: myogenin interacts with factors recognizing binding sites for basic helix-loop-helix, nuclear factor 1, myocyte-specific enhancer-binding factor 2, and COMP1 factor.

Myogenin is one of four muscle-specific basic helix-loop-helix regulatory factors involved in controlling myogenesis. We here describe various protein complexes that increase the affinity of myogenin for DNA. We mixed an oligonucleotide containing a degenerate center large enough to accommodate multiple binding sites with crude myotube nuclear extracts and used cyclic amplification and selection of targets with an antimyogenin monoclonal antibody to isolate protein-DNA complexes. Since each cycle of selection results in the enrichment for the sequences with the highest affinity, we isolated multicomponent sites in which myogenin binding was increased by its interaction with other DNA binding proteins. Myogenin interacts with members of the nuclear factor 1 family, the muscle-specific factor myocyte-specific enhancer-binding factor 2, and another factor, COMP1 (cooperates with myogenic proteins 1), that binds to the sequence TGATTGAC. Myogenin also exhibits cooperative binding with other proteins that recognize CANNTG motifs, and various constraints on spacing and orientation were observed. The application of this approach to other transcription factors should not only help identify the different functions of myogenin versus other members of the muscle basic helix-loop-helix regulatory family but also help define the general combinatorial mechanisms involved in eukaryotic gene regulation.

Base Sequence↗
PubMed1992

Alternative multimeric structures affect myogenin DNA binding activity.

The native molecular weight of the basic helix-loop-helix (bHLH) proteins myogenin, MyoD, and E12 was calculated from their mobilities on sucrose gradients and molecular sieve chromatography. The muscle bHLH proteins associate to form a variety of higher order complexes, most of which are larger than dimers. Homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer whereas homotetramers and larger forms do not recognize this DNA sequence. The ubiquitous bHLH protein E12 forms monomers or homodimers with little evidence for higher order complexes. Mixtures of myogenin and E12 show some heterodimeric structures, but most of the myogenin remains in large complexes. This result using purified proteins is also obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin is present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein containing only the myogenin HLH region forms large homomeric complexes. A model is presented in which each helix associates with a different subunit to form chains or ring structures to explain these observations. The partition of myogenin in nuclear extracts into dimers that recognize known DNA sequences and higher order complexes that do not raises important new issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.

Animals↗
PubMed1992

Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes.

The activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoD1 and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoD1 and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precursor cells in muscle regenerating in vivo.

Animals↗
PubMed1992

Cellular and molecular advances in elucidating p53 function.

The finding that in many human tumors there is allelic loss and/or mutations in p53, in combination with recognition that these events may play a role in multi-stage carcinogenesis, has focused considerable interest on this gene. To help keep abreast of this rapidly expanding field, recent experiments on the role and potential regulation of p53 are described: these include discussions of p53 as an anti-proliferative agent, the p53 mutations found in human tumors and tumor cell lines, the conformational states of p53, phosphorylation of p53 by p34cdc2, and signals for the nuclear localization of p53. p53 may act as a transcriptional activator and the specific DNA sequences to which p53 protein binds are also discussed as is the importance of abrogation of p53 function in overcoming cellular senescence.

Base Sequence↗
PubMed1992

Telomere positional effects and the regulation of cellular senescence.

Normal cells have a limited capacity to proliferate but the molecular clock that regulates the onset of cellular senescence remains unidentified. The ends of chromosomes--telomeres--have been shown to shorten progressively with age in normal cells. Here, we present a working model of how telomeric shortening may induce programmed changes in the regulation of cellular proliferation.

Cell Death↗
PubMed1992

Muscle basic helix-loop-helix proteins and the regulation of myogenesis.

Significant progress has been made in defining the structural motifs that distinguish the muscle-specific from other basic helix-loop-helix proteins. Evidence is accumulating for multiple levels of regulation of the expression and action of the muscle basic helix-loop-helix factors.

Animals↗
PubMed1992

MyoD, myogenin independent differentiation of primordial myoblasts in mouse somites.

The accumulation of two myogenic regulatory proteins, MyoD and myogenin, was investigated by double-immunocytochemistry and correlated with myosin heavy chain expression in different classes of myoblasts in culture and during early myogenesis in vivo. During in vitro differentiation of fetal myoblasts, MyoD-positive cells were detected first, followed by the appearance of cells positive for both MyoD and myogenin and finally by the appearance of differentiated myocytes and myotubes expressing myosin heavy chain (MHC). A similar pattern of expression was observed in cultures of embryonic and satellite cells. In contrast, most myogenic cells isolated from newly formed somites, expressed MHC in the absence of detectable levels of myogenin or MyoD. In vivo, the appearance of both myogenin and MyoD proteins was only detected at 10.5 d postcoitum (d.p.c.), when terminally differentiated muscle cells could already be identified in the myotome. Parasagittal sections of the caudal myotomes of 10.5-d-old embryos showed that expression of contractile proteins preceded the expression of myogenin or MyoD and, when coexpressed, MHC and myogenin did not co-localize within all the cells of the myotome. In the limb bud, however, many myogenin (or MyoD) positive/MHC negative cells could be observed in the proximal region at day 11. During further embryonic development the expression of these proteins remained constant in all the muscle anlagens examined, decreasing to a low level during the late fetal period. Western and Northern analysis confirmed that the myogenin protein could only be detected after 10.5 d.p.c. while the corresponding message was clearly present at 9.5 d.p.c., strongly suggesting a posttranscriptional regulation of myogenin during this stage of embryonic development. These data show that the first myogenic cells which appear in the mouse myotome, and can be cultured from it, accumulate muscle structural proteins in their cytoplasm without expressing detectable levels of myogenin protein (although the message is clearly accumulated). Neither MyoD message or protein are detectable in these cells, which may represent a distinct myogenic population whose role in development remains to be established.

Animals↗
PubMed1992

A transcriptionally active DNA-binding site for human p53 protein complexes.

Recent studies have demonstrated transcriptional activation domains within the tumor suppressor protein p53, while others have described specific DNA-binding sites for p53, implying that the protein may act as a transcriptional regulatory factor. We have used a reiterative selection procedure (CASTing: cyclic amplification and selection of targets) to identify new specific binding sites for p53, using nuclear extracts from normal human fibroblasts as the source of p53 protein. The preferred consensus is the palindrome GGACATGCCCGGGCATGTCC. In vitro-translated p53 binds to this sequence only when mixed with nuclear extracts, suggesting that p53 may bind DNA after posttranslational modification or as a complex with other protein partners. When placed upstream of a reporter construct, this sequence promotes p53-dependent transcription in transient transfection assays.

Base Sequence↗
PubMed1992

Partial-digest DNA sequencing.

A technique called partial-digest sequencing that permits DNA of 4-6 kb in length to be sequenced without subcloning is described. The method exploits the specific cuts introduced by partial digestion with restriction endonucleases that have 4-base recognition sites to produce ordered ladders of PCR-amplified fragments. The staggered ends contain PCR primers and can thus be individually sequenced using conventional methods to yield overlapping sequences covering the entire region. This method should have significant impact on both large and small DNA sequencing projects and find many applications in general manipulations in which ordered sets of deletions need to be produced.

Animals↗
PubMed1992

Multimeric structures influence the binding activity of bHLH muscle regulatory factors.

Sucrose gradients and molecular sieve chromatography were used to determine the native molecular weight of the basic HLH proteins myogenin, MyoD and E12. The muscle bHLH proteins not only formed dimers but also associated in a variety of higher order complexes. Although homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer, homotetramers and larger forms do not recognize this DNA sequence. Little evidence for complexes larger than dimers was found for the ubiquitous bHLH protein E12. Most of the myogenin remains in large complexes when myogenin and E12 are mixed. The same result was obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin was found to be present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein that contains only the myogenin HLH region fused to glutathione-S-transferase also forms large homomeric complexes. A model to explain these results is that each helix of the HLH motif can associate with a different subunit to form chains or ring structures. The presence of myogenin in nuclear extracts as both dimers that recognize known DNA sequences as well as higher order complexes that do not raises significant issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.

Animals↗
PubMed1992

Complexes containing the retinoblastoma gene product recognize different DNA motifs related to the E2F binding site.

The retinoblastoma tumor-suppressor gene encodes a 105-kDa nuclear phosphoprotein (RB) that can associate with DRTF-1 and E2F. These two transcription factors can recognize the same DNA motif in the adenovirus E2A promoter and can bind to it by themselves or in association with RB. In the present report, we describe the use of CASTing (cyclic amplification and selection of targets) to determine the consensus binding site of RB-containing complexes. An anti-human RB antibody was used to isolate RB-containing complexes formed after mixing nuclear extracts obtained from human diploid fibroblasts with a pool of random oligonucleotides flanked with polymerase chain reaction (PCR) primers. After the immunoselection, the DNA was isolated, amplified, mixed with fresh nuclear extract and reselected. After six CASTing cycles, the DNA was cloned and sequenced. We found that the highest affinity motifs recognized by RB-containing complexes are related to the E2F/DRTF-1 binding site and fall into three classes: TTTTCCCGCCAAAA, TTTTCCCGCCTTTTTT or TTTTCCCGCGCTTTTTT. Competition experiments revealed that these three classes are functionally equivalent to each other and to the E2F/DRTF-1 binding site in the adenovirus E2A promoter. Screening these sequences against a DNA database identified their presence in non-coding regions of many oncogenes, growth factor genes and in the RB gene itself.

Adenosine Triphosphate↗
PubMed1991

Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo.

In this report we provide four lines of evidence indicating that E12/E47-like proteins interact in vivo with the myogenic HLH proteins MyoD and myogenin. First, cotransfection of MyoD and E47 in COS cells indicates that these factors synergistically enhance transcription of a reporter gene containing an oligomerized MyoD-binding site. Second, mobility-shift assays of muscle cell nuclear extracts, "double shifted" with specific antisera, have identified complexes binding to the MEF1 site that contain either MyoD or myogenin in association with E12/E47-like proteins. Third, association with E47 alters the phosphorylation state of MyoD. Fourth, C3H10T1/2 cells expressing antisense E2A transcripts contain low levels of E2A gene products and display less terminal muscle differentiation when infected with retroviral MyoD or when challenged to differentiate with 5-azacytidine treatment. In addition we demonstrate that MyoD, in conjunction with E12/E47-like proteins, is functioning as a regulatory nodal point for activation of several other downstream muscle regulators.

Adenovirus Early Proteins↗
PubMed1991

Baculovirus-expressed myogenic determination factors require E12 complex formation for binding to the myosin-light-chain enhancer.

Two recombinant baculoviruses BcV-myf4 and BcV-myf5 have been constructed to synthesize the human myogenic determination factors myogenin (myf4) and myf5 in eucaryotic cells. Both recombinant proteins are localized to the nucleus of virus-infected Spodoroptera frugiperda (sf) insect cells and can be recovered as soluble factors. The virus-produced proteins exhibit high-affinity binding to a muscle-specific DNA sequence in the presence of the ubiquitous helix-loop-helix (HLH) protein E12, but only marginal binding in unsupplemented sf nuclear extracts. Both baculovirus-encoded myogenic factors are able to heterooligomerize with E12 in the absence of DNA-binding sites. We conclude from our results that these muscle-specific HLH proteins produced in eucaryotic cells largely depend on dimerization with E12 or similar HLH proteins to recognize the myosin-light-chain-enhancer-MEF-1-binding site. We have no evidence for intracellular protein modifications exerting major effects on the interaction between these factors and DNA.

Baculoviridae↗
PubMed1991

Defining the molecular mechanisms of human cell immortalization.

Although the immortalization of human cells is a key step in oncogenic progression, the molecular mechanisms underlying this event are poorly understood. After reviewing the use of chemicals, physical agents, oncogenes and DNA tumor viruses as immortalizing agents, we consider the importance of negative regulators of cell growth (RB and p53), their inactivation, as well as genomic instability in the pathogenesis of cancer. Finally, a molecular model for human cell immortalization that integrates many of the above observations is presented along with supporting evidence.

Cell Transformation, Neoplastic↗
PubMed1991

A role for both RB and p53 in the regulation of human cellular senescence.

We present evidence for the possible involvement of both the RB and p53 proteins in the regulation of cellular senescence. Human fibroblasts immortalized with an inducible SV40 T-antigen become senescent following the de-induction of T-antigen. Plasmids expressing an alternative source of intact T-antigen restore proliferation but T-antigen deletion mutants lacking either the RB or p53 binding domains are unable to do so. Similarly, combinations of adenovirus E1A + E1B or human papillomavirus E6 + E7 genes are able to replace T-antigen functions and permit cell proliferation, whereas the individual genes do not. These results are discussed in terms of a two-stage model for the escape from in vitro cellular senescence.

Adenoviruses, Human↗
PubMed1991

Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site.

The consensus binding site for the muscle regulatory factor myogenin was determined from an unbiased set of degenerate oligonucleotides using CASTing (cyclic amplification and selection of targets). Stretches of totally random sequence flanked by polymerase chain reaction priming sequences were mixed with purified myogenin or myotube nuclear extracts, DNA-protein complexes were immunoprecipitated with an antimyogenin antibody, and the DNA was amplified by polymerase chain reaction. Specific binding was obtained after four to six cycles of CASTing. The population of selected binding sites was then cloned, and a consensus was determined from sequencing individual isolates. Starting from a pool with 14 random bases, purified myogenin yielded a consensus binding site of AACAG[T/C]TGTT, while nuclear extracts retrieved the sequence TTGCACCTGTTNNTT from a pool containing 35 random bases. The latter sequence is consistent with that predicted from combining an E12/E47 half-site (N[not T]CAC) with the purified myogenin half-site ([T/C] TGTT). The presence of paired E boxes in many of the sequences isolated following CASTing with nuclear extracts proves that myogenin can bind cooperatively with other E-box-binding factors.

Animals↗