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K C Ehrlich

Publications and source records attributed to K C Ehrlich.

At least 37 records · Page 2Linked to original sources
PubMed1994

An acid phosphatase from Aspergillus ficuum has homology to Penicillium chrysogenum PhoA.

Three secreted acid phosphatases had previously been characterized from Aspergillus ficuum grown under conditions of limited phosphate. One of these could not be readily separated from AFPhyB, a pH 2.5 optimum acid phosphatase with phytase activity. From extensive protein sequence analysis and subsequent cloning of the gene, we have shown that the AFPhyB protein fraction contains a fourth secreted acid phosphatase (AFPhoA) that has 64% homology to a phosphate-repressible acid phosphatase from Penicillium chrysogenum. Garnier plot analysis revealed that the putative phosphate catalytic domain of AFPhoA at His215Asp216 is similar to those of other acid phosphatases, but that AFPhoA lacks the phosphate-binding motif RHGXRXP of known histidine phosphatases.

Acid Phosphatase↗
PubMed1993

Identification and cloning of a second phytase gene (phyB) from Aspergillus niger (ficuum).

An Aspergillus niger (ficuum) genomic DNA lambda EMBL3 library was probed with a 354-bp DNA fragment obtained by polymerase chain reaction of A. niger DNA with oligonucleotides based on partial amino acid sequence of a pH 2.5 optimum acid phosphatase. A clone containing a 1605 bp segment (phyB) encoding the 479 amino acid enzyme was isolated and found to contain four exons. Global alignment revealed 23.5% homology to Aspergillus niger phytase (PhyA); four regions of extensive homology were identified. Some of these regions may contain catalytic sites for phosphatase function.

6-Phytase↗
PubMed1993

Characterization of DBPm, a plant protein that binds to DNA containing 5-methylcytosine.

A protein (DBPm) has been isolated from nuclear extracts of soybean seeds, cauliflower florets, corn seed, wheat germ, and pea hypocotyl, seeds, apices, roots, and leaves that specifically binds to double-strand DNA containing 5-methylcytosine residues. In electrophoretic mobility shift assays, non-methylated duplex DNAs competed only slightly, while methylated DNAs were strong competitors. Specific binding still occurred after partial proteolysis of DBPm, but not after heating at 45 degrees C. By ultraviolet light-crosslinking and sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration, the size of pea seed DBPm was estimated to be in the range 70-90 kDa. From equilibrium binding studies the equilibrium constant for binding of pea seed DBPm to a 34 bp duplex deoxyoligonucleotide containing 12 5-methylcytosine residues was 1.2 x 10(9) M-1. The binding properties of DBPm make it a good candidate for a plant protein capable of mediating the effects of DNA methylation on the activity of some plant genes.

5-Methylcytosine↗
PubMed1993

Partial purification of a pea seed DNA-binding protein that specifically recognizes 5-methylcytosine.

Previously, a DNA-binding protein (DBPm) was identified in plant nuclei that may mediate the effects of DNA methylation on chromatin structure and transcription. In the present report, DBPm was partially purified from germinated pea (Pisum sativum) seed nuclear extracts by DEAE-cellulose, phenylsepharose, heparin-sepharose chromatography, and preparative mobility shift on polyacrylamide gels. The purified activity showed a band at approximately 50 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as by Sephadex G100 chromatography, suggesting that DBPm is present as a monomer.

5-Methylcytosine↗
PubMed1992

A broad bean cDNA clone encoding a DNA-binding protein resembling mammalian CREB in its sequence specificity and DNA methylation sensitivity.

A plant cDNA has been cloned that encodes a DNA-binding protein displaying a nucleotide (nt) sequence specificity similar to that of the mammalian cyclic AMP response element-binding protein/activating transcription factor (CREB/ATF) family of mammalian proteins. This cDNA was cloned in Escherichia coli from a broad bean (Vicia faba) cDNA expression library using a recognition site probe. The deduced amino acid (aa) sequence of the recombinant cDNA-encoded protein, called VBP1, has a basic region adjacent to a leucine zipper motif, of the type seen in the DNA-binding domains of many eukaryotic DNA-binding proteins, including mammalian CREB/ATF. Although this aa sequence has homology to regions of deduced aa sequences of other cloned plant cDNAs, it is distinct in both the derived primary structure and in its nt sequence specificity. VBP1, as well as proteins in nuclear extracts of V. faba with similar nt sequence specificity, have their binding to DNA suppressed more than tenfold by cytosine methylation at the CREB/ATF consensus sequence.

Amino Acid Sequence↗
PubMed1991

CpG methylation inhibits binding of several sequence-specific DNA-binding proteins from pea, wheat, soybean and cauliflower.

To elucidate how methylation of specific sites in plant DNA might control transcription, we examined the effect of DNA methylation at CpG sequences on the binding of plant nuclear factors to an oligonucleotide duplex containing the consensus sequence for mammalian CREB (cAMP response element binding protein). CREB is part of the ATF (activating transcription factor) family of mammalian proteins specifically binding to 5'-TGACGTCA-3' and related sequences. Proteins recognizing the CREB-specific ligand were identified in nuclear extracts of pea seeds, wheat germ, cauliflower, and soybean leaves using electrophoretic mobility shift assays. Cytosine methylation inhibited binding of this protein in all these extracts, and so this sequence-specific DNA-binding activity is referred to as methylation-inhibited binding protein 1 (MIB-1). Sites somewhat similar to that of the CREB ligand are found in the upstream regions of a wheat histone H3 gene and tomato and pea ribulose 1,5-bisphosphate carboxylase genes. These sites were bound preferentially by distinct proteins that may be related to the previously described plant proteins HBP-1, HSBF, ASF-1, or GBF. Methylation of cytosine residues at these sites and at a site for MIB-1 located upstream of a soybean proline-rich protein gene also reduced specific binding with all the nuclear extracts tested. Similarly, substitution of the central CpG dinucleotide with TpG decreased binding.

Base Sequence↗
PubMed1991

Three MDBP sites in the immediate-early enhancer-promoter region of human cytomegalovirus.

MDBP, a mammalian sequence-specific DNA-binding protein, was found to recognize two sites in the major immediate-early (IE) enhancer of human cytomegalovirus. The recognition sequence for MDBP at each of these sites was localized to 14 bp by studying the effects of limited G methylation, depurination, depyrimidination, or deoxyribose modification on the ability of these sites to bind to MDBP. In addition to the two high-affinity MDBP sites in the enhancer, one low-affinity MDBP site was detected 5 bp after the transcription initiating residue of this IE transcription unit. The possible biological significance of the two enhancer MDBP sites and the downstream MDBP site is discussed.

Base Sequence↗
PubMed1990

Binding sites in mammalian genes and viral gene regulatory regions recognized by methylated DNA-binding protein.

Methylated DNA-binding protein (MDBP), a ubiquitous mammalian protein, recognizes a variety of related DNA sequences. Some of these sequences require methylation of their CpG dinucleotides for binding and others do not. We report that MDBP binds, in a DNA methylation-independent fashion, to two sites in the mouse polyomavirus enhancer, one in the enhancer of the human hepatitis B virus, and to one in the long terminal repeat of equine infectious anemia proviral DNA. We have also found a number of MDBP sites in human and rodent DNAs which bind much better to MDBP when they are methylated at CpG dinucleotides within the recognition site. These include sites at the beginning of the human genes for hypoxanthine phosphoribosyl transferase, HLA-A2, -A3, and -A25 antigens, and alpha-galactosidase A. In the case of methylation-responsive MDBP sites, changes in their methylation status during differentiation or DNA replication could help drive development by modulating transcription.

Animals↗
PubMed1990

An MDBP site in the first intron of the human c-myc gene.

The expression of the human c-myc protooncogene is subject to many levels and types of control. Evidence suggests that regulation of the expression of this gene involves elements within the gene as well as those upstream from the gene. We show that a ubiquitous mammalian sequence-specific DNA-binding protein, MDBP, binds specifically to a site in the beginning of the first intron of this gene. This protein, which binds to certain viral enhancers, may be helping to control expression of the c-myc gene. In some Burkitt lymphomas, which contain activated c-myc genes, the MDBP site is lost by chromosome rearrangement or by multiple spontaneous mutations. This might contribute to cancer-related activation of this protooncogene.

Base Sequence↗
PubMed1990

Highly repeated sites in the apolipoprotein(a) gene recognized by methylated DNA-binding protein, a sequence-specific DNA-binding protein.

Methylated DNA-binding protein (MDBP), a sequence-specific DNA-binding protein, was found to recognize more than 30 sites within an allele of the human apolipoprotein(a) gene. High plasma levels of apolipoprotein(a), a risk factor for atherosclerosis, have been correlated with genetically inherited lower-molecular-mass isoforms of this protein. MDBP might help down modulate the expression of the apolipoprotein(a) gene in a manner dependent on the length of a given allele of the gene and the number of MDBP sites in it.

Alleles↗
PubMed1989

How different DNA sequences are recognized by a DNA-binding protein: effects of partial proteolysis.

MDBP is a sequence-specific DNA-binding protein from mammals that recognizes a variety of DNA sequences, all of which show much homology to a partially palindromic 14 base-pair consensus sequence. MDBP subjected to limited proteolysis and then incubated with various specific oligonucleotide duplexes yielded two types of complexes. The relative concentrations of these complexes varied greatly depending on how closely the MDBP site matched the consensus sequence. No such DNA sequence-specific differences in the types of complexes formed were seen with intact MDBP. Partial proteolysis also changed the relative affinity of MDBP for several of its binding sites. The nature of the two types of complexes formed from fragmented MDBP and DNA was studied by DNA competition assays, protein titration, site-directed mutagenesis, and dimethyl sulfate and missing base interference assays. The results suggest that, for some specific DNA sequences, half-site interactions with one MDBP subunit predominate and for others, strong interaction of two subunits with both half-sites readily occur.

Animals↗
PubMed1989

Related sites in human and herpesvirus DNA recognized by methylated DNA-binding protein from human placenta.

Methylated DNA-binding protein (MDBP) from mammalian cells binds specifically to six pBR322 and M13mp8 DNA sequences but only when they are methylated at their CpG dinucleotide pairs. We cloned three high-affinity MDBP recognition sites from the human genome on the basis of their binding to MDBP. These showed much homology to the previously characterized prokaryotic sites. However, the human sites exhibited methylation-independent binding apparently because of the replacement of m5C residues with T residues. We also identified three other MDBP sites in the herpes simplex virus type 1 genome, two of which require in vitro CpG methylation for binding and are in the upstream regions of viral genes. A comparison of MDBP sites leads to the following partially symmetrical consensus sequence for MDBP recognition sites: 5'-R T m5Y R Y Y A m5Y R G m5Y R A Y-3'; m5Y (m5C or T), R (A or G), Y (C or T). This consensus sequence displays an unusually high degree of degeneracy. Also, interesting deviations from this consensus sequence, including a one base-pair deletion in the middle, are sometimes observed in high-affinity MDBP sites.

Base Sequence↗
PubMed1989

Preparation of the Fusarium toxin, nivalenol, by oxidation of the putative biosynthetic precursor, 7-deoxynivalenol.

Nivalenol is a toxic trichothecene metabolite which is produced by a number of different Fusarium species. However, the nature of its immediate biosynthetic precursor is not known. Oxidation of 7-deoxynivalenol(3 alpha,4 beta,15-trihydroxy-12,13- epoxytrichothec-9-ene-8-one) to nivalenol occurred with reagents known to react by a free radical pathway, such as hydrogen peroxide-ferrous ion-ascorbic acid or lead tetracetate, but not with electrophilic reagents requiring prior formation of the enol. These results suggest that 7-deoxynivalenol or an acetylated derivative could be the biosynthetic precursor of nivalenol.

Fusarium↗
PubMed1989

A plant DNA-binding protein that recognizes 5-methylcytosine residues.

A novel, 5-methylcytosine-specific, DNA-binding protein, DBP-m, has been identified in nuclear extracts of peas. DBP-m specifically recognizes 5-methylcytosine residues in DNA without appreciable DNA sequence specificity, unlike a mammalian DNA-binding protein (MDBP), which recognizes 5-methylcytosine residues but only in a related family of 14-base-pair sequences.

5-Methylcytosine↗
PubMed1988

Human methylated DNA-binding protein. Determinants of a pBR322 recognition site.

Methylated DNA-binding protein (MDBP) from human placenta has a high affinity for a site in pBR322 (pB site 1) when that site is methylated at its CpG dinucleotides. Dimethyl sulfate interference analysis and experiments with ligands prepared by oligonucleotide-directed mutagenesis indicate that 15 contiguous base pairs, 14 of which exhibit hyphenated dyad symmetry, influence MDBP binding to pB site 1. These 14 base pairs, 5'-RTMGYCAMGG(M/T)GAY-3' (M, 5-methylcytosine), suffice for recognition by MDBP as demonstrated with a double-stranded, MpG-containing oligonucleotide used as a free ligand or cloned into M13mp19 and subsequently methylated. Seven single-site mutations at different positions of this 14-base pair region largely eliminated binding, and several others increased binding up to 2-fold when compared to the nonmutant, triply methylated sequence. However, MDBP recognizes a site in hemimethylated M13mp19 replicative form DNA, which was homology to pB site 1 at only 10 of 14 base pairs, and all four of these different base pairs are equivalent to transversions. Based upon the above data, a mixed oligonucleotide probe was constructed that contains variants of pB site 1 which should be recognized by MDBP. This 14-base probe hybridizes under stringent conditions to a number of discrete fragments in restriction digests of human DNA. this suggests that there are multiple pB site 1-related sequences in human DNA that might, when methylated, bind MDBP in vivo.

Amino Acid Sequence↗
PubMed1988

Methylated DNA-binding protein is present in various mammalian cell types.

A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m5C) residues at specific positions. We found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. From the extracts of rat and calf tissues, oligonucleotide protein complexes formed that also had the same specificity as human placental MDBP although they had a higher electrophoretic mobility probably due to digestion by proteases in the nuclear extracts. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.

5-Methylcytosine↗