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Biomedical subjects

M Ehrlich

Publications and source records attributed to M Ehrlich.

At least 19 recordsLinked to original sources
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↗
PubMed1992

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts.

The effects of methylation on plant viral DNA replication have been studied in Nicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m5C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m5C residues were substituted for cytosine residues in vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequences in vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.

Blotting, Southern↗
PubMed1992

Transfection of heteroduplexes containing uracil.guanine or thymine.guanine mispairs into plant cells.

We have compared the fate of U.G mispairs or analogous T.G mispairs in DNA heteroduplexes transfected into tobacco protoplasts. The heteroduplex DNA consisted of tomato golden mosaic virus DNA sequences in the Escherichia coli vectors pUC118 or pUC119. After transfection, the mismatched U residues were lost with an efficiency of greater than 95%, probably as a result of the uracil-DNA glycosylase pathway for excision of U residues in any sequence context. In contrast to the preferential removal of the mispaired U residues, biased removal of T residues from analogous heteroduplexes was not seen in the transfected plant cells. Also, we investigated the effect of extensively methylating one strand of the heteroduplex DNA used for transfection. Surprisingly, such methylation resulted in highly biased loss of the mismatched base from the 5-methylcytosine-rich strand of T.G-containing heteroduplexes.

Base Composition↗
PubMed1992

Increasing the activity of affinity-purified DNA-binding proteins by adding high concentrations of nonspecific proteins.

A large decrease in the activity of two sequence-specific DNA-binding proteins implicated in transcription control was seen when these were affinity purified and assayed under standard conditions in electrophoretic mobility shift assays. Increasing the concentration of bovine serum albumin in the reaction mixtures from 0.1 to 5 mg/ml stimulated the DNA-binding activity of these affinity-purified proteins, human CREB (cyclic AMP response element binding protein) and MDBP (methylated DNA-binding protein), approximately 5-to more than 20-fold. In the case of affinity-purified MDBP, adding back the affinity flow-through fraction to the assay mixture gave similar extents of stimulation at much lower final protein concentrations. The specific DNA-binding activity of the affinity-purified CREB, but not that of MDBP, was also increased by adding a nonionic detergent to the binding reaction buffer although not as much. The large increase in the amount of MDBP.DNA complex seen upon supplementation of the affinity-purified MDBP with the affinity flow-through fraction or 5 mg/ml of BSA was shown to be due to stimulation, by nonspecific proteins, of specific complex formation and not to prevention of activity losses by adsorption or denaturation during the assay.

Base Sequence↗
PubMed1991

End-filling of an oligonucleotide duplex containing an MDBP site in the human HSP70 promoter inhibits protein-DNA complex formation.

A site from the promoter region of the human hsp70 gene binds with a high affinity to the ubiquitous mammalian protein called methylated DNA-binding protein (MDBP) when it is present in a CpG-methylated oligonucleotide duplex with only 14 base-pairs. Binding to this site is dependent upon CpG methylation. Surprisingly, when the same methylated sequence is present in a duplex that has 22 or more base-pairs, binding to this protein is greatly inhibited. Such a requirement for a short duplex region is seen only in certain of the cytosine methylation-dependent binding sites for this protein and is proposed to reflect differences in the conformation of the duplex due to small differences in the nucleotide sequence.

Base 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

Comparison of gene expression of the dopamine D-2 receptor and DARPP-32 in rat brain, pituitary and adrenal gland.

In situ hybridization histochemistry was used to compare the distribution of dopamine D-2 receptor mRNA and DARPP-32 mRNA in rat brain, pituitary and adrenal gland. In many regions such as the caudate nucleus, nucleus accumbens and olfactory tubercle, there was an excellent correlation with dopamine receptor binding studies, whereas in some discrete brain regions mismatches were observed.

Adenylyl Cyclases↗
PubMed1990

Distribution and cellular localization of DARPP-32 mRNA in rat brain.

In situ hybridization histochemistry has been used to determine the regional distribution and cellular localization of DARPP-32 mRNA in the rat brain. Results support the concept that DARPP-32 is present primarily in cells expressing the dopamine D1 subtype receptor, and that DARPP-32 is not synthesized in dopamine-containing cells. Strongly labelled neuronal cell bodies were found in the caudate nucleus, nucleus accumbens, olfactory tubercle, parts of the bed nucleus of the stria terminalis, and the amygdaloid complex. In addition large amounts of DARPP-32 mRNA were visualized in the medial habenula and around the third ventricle, in ependymal cells and tanycytes, and in the cerebellar Purkinje cells. A less pronounced activity was seen in layers II-III and VI throughout the cerebral cortex. The present studies together with previous biochemical and immunocytochemical studies demonstrate that DARPP-32 gene expression is greatest primarily in D1 dopaminoceptive cells, although there are exceptions. In situ hybridization may thus be used to quantitate regulation of DARPP-32 mRNA in discrete brain regions.

Animals↗
PubMed1990

Chronic treatment of rats with SCH-23390 or raclopride does not affect the concentrations of DARPP-32 or its mRNA in dopamine-innervated brain regions.

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a neuronal phosphoprotein that is enriched in neurons which possess dopamine D1 receptors, particularly striatonigral neurons. In rat brain slices, the phosphorylation state of DARPP-32 is regulated by dopamine, acting through the dopamine D1 receptor and the adenylyl cyclase system. This study reports that chronic blockade (21 days) of either dopamine D1 receptors by SCH-23390 or dopamine D2 receptors by raclopride does not affect the concentrations of DARPP-32 in specific rat brain regions (striatum, thalamus, hippocampus, frontal cerebral cortical pole). Northern blot analysis indicates that the steady-state level of DARPP-32 mRNA in striatum is also unchanged by these treatments.

Animals↗
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

Dopamine- and cAMP-regulated phosphoprotein (DARPP-32) and dopamine DA1 agonist-sensitive Na+,K+-ATPase in renal tubule cells.

The cellular localization of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 that appears to mediate certain actions of dopamine in the mammalian brain by acting as an inhibitor of protein phosphatase 1, was studied in the kidney of several species. DARPP-32 mRNA and DARPP-32-like immunoreactivity were found in the cytoplasm of cells in the thick ascending limb of the loop of Henle. The specific dopamine DA1 agonist SKF 82526 caused a dose-dependent inhibition of Na+,K+-ATPase activity, which could be blocked by SCH 23390, a specific DA1 antagonist, and by PKI-(5-24) amide, a specific inhibitor of cAMP-dependent protein kinase. The results indicate that DA1 dopamine receptors and DARPP-32, an intracellular third messenger for dopamine, are part of the signal-transduction process for dopamine acting on renal tubule cells.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗