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Biomedical subjects

E L Winnacker

Publications and source records attributed to E L Winnacker.

At least 55 records · Page 3Linked to original sources
PubMed1989

Clusters of nuclear factor I binding sites identify enhancers of several papillomaviruses but alone are not sufficient for enhancer function.

The long control region (LCR) of human papillomaviruses (HPV) encompasses 5-12% of the viral genome and contains an intricate network of cis responsive elements. In the LCR of seven unrelated HPV-types, namely HPV-1, 6, 8, 11, 16, 18 and 33, we have identified clusters of 4 to 7 5-TTGGC-3 motifs suggesting nuclear factor I (NFI) binding sites. We randomly selected 20 (out of a total of 38) of these motifs and showed that pure NFI from porcine liver protects virtually the same nucleotides as a factor present in crude HeLa nuclear extracts. The footprints obtained with HeLa extracts in the LCR of HPV-16 are eliminated in competition experiments by an oligonucleotide representing the palindromic adenovirus NFI binding site. Restriction fragments from the genome of HPV-11, 16 and 18, which contain this cluster of NFI binding sites associated with binding sites of unrelated transcription factors, function as transcriptional enhancers. In contrast, a fragment from HPV-8 exhibiting exclusively NFI binding sites, or polymerized NFI sites from HPV-16, are functionally inactive. NFI seems to be necessary but not sufficient for HPV enhancer activation.

Base Sequence↗
PubMed1989

Isolation and DNA-binding characteristics of a protein involved in transcription activation of two divergently transcribed, essential yeast genes.

We have identified a protein, BAF1, which has two oppositely oriented, partially overlapping binding sites within a symmetrical sequence located midway between and upstream of the divergently transcribed YPT1 and TUB2 genes of the yeast Saccharomyces cerevisiae. The 120 kd BAF1 protein was purified to near homogeneity and used to delineate the two binding sites and to identify apparent protein contact sites by the missing contact technique, methylation interference and by site-directed mutagenesis. The BAF1-recognition sequence contains a conserved TCN7ACG element recently identified at autonomously replicating sequences (ARS) and in the 5' and 3' flanking region of other yeast genes. The symmetrical sequence of the YPT1/TUB2 intergene region seems not to be involved in DNA replication but activates transcription in an orientation-independent fashion.

Base Sequence↗
PubMed1989

Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific gamma-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase).

An enzyme has been discovered and characterized from Silene cucubalus cell suspension cultures that catalyzes the transfer of the gamma-glutamylcysteine dipeptide moiety of glutathione to an acceptor glutathione molecule or a growing chain of [Glu(-Cys)](n)-Gly oligomers, thus synthesizing phytochelatins, the metal-binding peptides of higher plants and select fungi. The enzyme was named gamma-glutamylcysteine dipeptidyl transpeptidase and given the trivial name phytochelatin synthase. The primary reaction catalyzed is [Glu(-Cys)]-Gly + [Glu(-Cys)](n)-Gly --> [Glu(-Cys)](n+1)-Gly + Gly. The enzyme is isoelectric near pH 4.8 and has temperature and pH optima at 35 degrees C and 7.9, respectively. Phytochelatin synthase is constitutively present in cell cultures of various plant species and its formation is not noticeably induced by heavy metal ions in the growth medium. The enzyme (M(r)95,000) seems to be composed of four subunits, the dimer (M(r)50,000) being also catalytically active. Cd(2+) is by far the best metal activator of the enzyme followed by Ag(+), Bi(3+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), and Au(+). The K(m) for glutathione is 6.7 mM. The enzyme activity seems to be self-regulated in that the product of the reaction (the phytochelatins) chelates the enzyme-activating metal, thus terminating the enzyme reaction. The molar ratio of the gamma-glutamylcysteine dipeptide in phytochelatin to Cd(2+) in the newly formed complex was 2:1.

Journal Article↗
PubMed1989

Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter.

Transcription from one of the two initiation sites, P1 and P2, of the dual human MYC promoter seems to be essential in all proliferating cells. To identify proteins and target structures for MYC regulation, a DNA region was analyzed that is critical for P2 promoter activity. Here, we show that a nuclear factor binds to a DNA element within P2, which is conserved perfectly between mouse and man and displays a striking homology to the E1a-inducible E2 promoter of adenovirus type 5 (Ad5). We demonstrate that the same transcription factor, defined recently as E2F, which plays an essential role in the activation of adenovirus early promoters and enhancers, also interacts as a dominant nuclear factor with the MYC promoter. The presence of an intact E2F binding site is required for basic expression and for trans-activation of the P2 promoter by E1a proteins. The human MYC promoter is the first cellular target described for E2F. The results suggest that expression of MYC might be regulated via modulation of E2F by cellular 'E1a-like' factors.

Base Sequence↗
PubMed1988

Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI)--generation of protein sequence information.

The paper describes a potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins. This procedure exploits the sequence-specific DNA-binding affinity of such proteins for their enrichment, comparable to recognition site DNA affinity chromatography. The method was employed to obtain a pure preparation of nuclear factor I (NFI) from porcine liver from which sequences of partial peptides could be obtained. Oligonucleotide probes derived from these amino-acid sequences were used to identify genomic and cDNA clones of NFI.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of the porcine nuclear factor I (NFI) gene.

This study describes the isolation of a major portion of the gene for nuclear factor I (NFI) including its 5'-flanking region with transcriptional start sites. We screened a porcine liver, genomic DNA library in phage EMBL3A with synthetic oligonucleotides derived from tryptic and cyanogen-bromide peptide sequences obtained from purified NFI protein. The NFI gene is present as a single copy in porcine DNA.

Amino Acid Sequence↗
PubMed1988

A quantitative analysis of nuclear factor I/DNA interactions.

Nuclear factor I (NFI) was purified to homogeneity from porcine liver by DNA-affinity chromatography and displays a single band with a molecular weight of 36 kDa in SDS-polyacrylamide gels. The purified protein was used to determine absolute equilibrium binding constants by gel retardation techniques for a variety of DNA fragments with genuine or mutated NFI binding sites and a number of DNA fragments derived from various eukaryotic promoters carrying the CCAAT-box as a half-site for NFI binding. We present a model which allows prediction of the functional significance of mutated NFI binding-sites from sequence data. The data suggest that the single molecular species of NFI from porcine liver may not be able to recognize and activate the -CCAAT- promoter element in vivo without additional interactions, e.g. with other proteins.

Adenoviridae↗
PubMed1987

Phytochelatins, a class of heavy-metal-binding peptides from plants, are functionally analogous to metallothioneins.

Phytochelatins are a class of heavy-metal-binding peptides previously isolated from cell suspension cultures of several dicotyledonous and monocotyledonous plants. These peptides consist of repetitive gamma-glutamylcysteine units with a carboxyl-terminal glycine and range from 5 to 17 amino acids in length. In the present paper we show that all plants tested synthesized phytochelatins upon exposure to heavy metal ions. No evidence for the occurrence of metallothionein-like proteins was found. All data so far obtained indicate that phytochelatins are involved in detoxification and homeostasis of heavy metals in plants and thus serve functions analogous to those of metallothioneins in animals and some fungi. Phytochelatins are induced by a wide range of metal anions and cations. Phytochelatin synthesis in suspension cultures was inhibited by buthionine sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2). This finding and kinetic studies of phytochelatin induction point to a synthesis from glutathione or its precursor, gamma-glutamylcysteine, in a sequential manner, thereby generating the set of homologous peptides.

Journal Article↗
PubMed1987

Expression of hepatitis A virus cDNA in Escherichia coli: antigenic VP1 recombinant protein.

The genome of hepatitis A virus (HAV) was reverse transcribed into cDNA and molecularly cloned. cDNA clones coding for the capsid protein VP1 that carries the major HAV antigen were cloned into the expression vector pUR290 and expressed in Escherichia coli. The recombinant fusion protein reacted in an immunoblot with rabbit anti-HAV serum, suggesting that it possesses HAV antigenicity.

Antigens, Viral↗
PubMed1986

A sensitive and rapid gel retention assay for nuclear factor I and other DNA-binding proteins in crude nuclear extracts.

The paper describes a rapid and sensitive assay for DNA binding proteins which interact with specific and defined binding sites. It exploits the observation that complexes of proteins and small synthetic DNA fragments (40 bp) containing the protein/DNA binding site can enter native polyacrylamide gels and remain stably associated during electrophoresis under non-denaturing conditions. The assay was applied to nuclear factor I, to its identification and purification from porcine liver, to an analysis of its binding site on adenovirus type 5 DNA and to an exploration of other potential binding sites for DNA binding proteins within the inverted terminal repetition of adenovirus DNA. The extreme sensitivity of the assay which surpasses that of conventional footprint assays by at least two orders of magnitude permitted the identification of nuclear factor I-like activities in Saccharomyces cerevisiae.

Animals↗
PubMed1984

A size analysis of the adenovirus replicon.

The linear double-stranded genome of adenovirus DNA replicates semiconservatively from two origins of replication at either of the two molecular ends. Using an in vitro replication system which is able to initiate de novo DNA synthesis we have mapped the origin of DNA replication within the terminal 19 bp of the viral genome. Our conclusions are based on the use of different natural DNA templates, i.e., adenovirus type 2 and mouse adenovirus Fl DNA. In addition, we have employed linearized plasmid DNA templates which contain cloned terminal restriction enzyme fragments as well as chemically synthesized adenovirus termini of different length.

Adenoviridae↗
PubMed1983

Improvements in the phosphoramidite procedure for the synthesis of oligodeoxyribonucleotides.

The paper describes an improved method for the synthesis of oligodeoxyribonucleotides using phosphoramidite chemistry. Our procedure relies on novel phosphoramidite intermediates, the deoxyribonucleoside-3'-morpholine-methoxyphosphins. These compounds are extremely stable and can be purified readily. Condensation reactions during solid-phase synthesis can thus be performed with high efficiency and result in a high yield synthesis of long chain oligodeoxyribonucleotides.

Chemical Phenomena↗
PubMed1982

Defective expression of mouse adenovirus Fl in human cells.

HeLa cells infected with mouse adenovirus strain Fl ( AdFl ) produce at least 2000 times less virus than permissive mouse 3T3 cells. Viral DNA synthesis, however, proceeds unimpaired. The defect in virion production was linked to a dramatic reduction in the synthesis of AdFl structural proteins, in particular the hexon. The identity of the AdFl hexon gene product was recognized through its immunogenic reactivity towards an antiserum raised against the human adenovirus type 2 (Ad2) hexon gene product. This cross-reactivity is reflected in an extensive DNA sequence homology between AdFl and Ad2 DNA at position 53-60, the locus of the hexon gene, on the Ad2 physical map. Through hybridization at different formamide concentrations, the present study identifies one additional, highly conserved region at map positions 14-15 on the Ad2 genome.

Adenoviridae↗
PubMed1982

The nucleotide sequence of cDNA coding for preproinsulin from the primate Macaca fascicularis.

DNA complementary to preproinsulin messenger RNA from the primate Macaca fascicularis has been cloned into the PstI endonuclease site of the plasmid pBR322. One clone contains the entire preproinsulin coding region as well as 59 nucleotides of the 5'-untranslated region. The results predict an amino acid sequence for the Macaca fascicularis preproinsulin and establish for the first time that the primary structures of human and primate insulins are identical. The two amino acid exchanges between human and primate preproinsulins are restricted to the pre- and the C-peptide, respectively.

Amino Acid Sequence↗
PubMed1981

Cloning of hepatitis A virus genome.

Hepatitis A virus (HAV) was highly purified from faeces. The genomic RNA was transcribed to cDNA and this DNA was then cloned into plasmid pBR 322 at the Pst I site, and clones were selected in presence of tetracycline. Most clones contained inserts which hybridized to HAV-specific RNA isolated from HAV-infected cell cultures derived from a human hepatocellular carcinoma. Two clones expressed low amounts of viral antigens.

Base Sequence↗