Rapid isolation of specific DNA-binding proteins and their DNA-binding domains.
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Amino Acid Sequence↗
Biomedical subjects
C Brack
Publications and source records attributed to C Brack.
A survey of protein-DNA interaction sites within the murine immunoglobulin heavy chain locus reveals a particularly complex pattern around the DQ52 element.
The expression of immunoglobulin (Ig) genes is regulated at two levels: rearrangement of individual gene segments and transcription of continuous genes. To find transacting factors involved in mediating locus- and segment specific gene activation and expression, we surveyed a 3600-bp genomic region of the murine Ig heavy chain locus, spanning from the DQ52 element to the Ig heavy chain intron enhancer. We discovered nine, previously undescribed, protein-DNA complexes and estimated their individual binding-affinity preferences (Kr) by quantitative gel shift measurements. We observed one novel protein DNA interaction at the enhancer, two in the JH1 region and six within a 300-bp region immediately 5' to the DQ52 locus. The latter show a complex and specific binding pattern when comparing nuclear extracts derived from pre-B cells and fibroblasts. Further characterization of the interactions at the DQ52 locus by electron microscopy revealed the preferential formation of a protein complex binding to the DQ52 locus with pre B cell extracts. This behavior and the clustering of interaction sites 5' to the DQ52 element suggest that this region is involved in the regulation of heavy chain gene assembly.
Animals↗
[Acyl coenzyme A dehydrogenase deficiency of medium-chain fatty acids in a 9-year-old boy with adymia. A rare mitochondrial cytopathy which may be more common than previously assumed].
The medium-chain acyl-CoA dehydrogenase (MCAD) deficiency of mitochondrial beta oxidation has been identified in a nine-year old boy with a very bland course and easy fatigue as the main symptom. Repeated low frequency stimulation test and EMG for excluding a myasthenia gravis, and screening for urinary organic acid excretion were helpful for the diagnosis. The EMG test at the m. trapezius by stimulation of the n. accessorius showed an extreme decrease of muscle power down to 49%. After i.v. injection of Edrophonium the loss of power of 20% was still significant, so that we could exclude a myasthenia gravis, but we had found signs of a generalised defect in cell chemistry. The diagnosis could be confirmed by a positive 3-phenylpropionic acid-test and moleculargenetic proof of the Adenine to Guanine mutation at position 985 in the MCAD cDNA (G985) with the polymerase chain reaction. The incidence of this organic aciduria is probably 1:60,000 in Germany, but with more attention to this disease and diagnosis of cases with bland courses the incidence will be higher. The MCAD-defect should be considered in the differential diagnosis of patients with Reye syndrome-like encephalopathies, non-ketotic hypoglycaemia or sudden unexpected deaths in infancy.
Acyl-CoA Dehydrogenase↗
Gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata.
As a first step to investigate whether gonadotropin releasing hormone (GnRH) analogs might be able to modulate directly the proliferation of human epithelial ovarian carcinomata, we checked if binding sites for GnRH are present in these malignancies. Specific binding of [125I][D-Ala6-des Gly10]-GnRH-ethylamide (GnRH agonist = GnRH-A) could be demonstrated in plasma membranes from 32 out of 40 ovarian carcinomata tested. This binding was dependent on temperature, time and plasma membrane concentration. Mathematical analysis of the binding data showed that the interaction of GnRH-A with the binding sites was consistent with a single class of low affinity, high capacity binding sites (Ka = 1.42 +/- 0.14 X 10(5) M-1; range: 0.3-3.8 X 10(5) M-1; R = 209 +/- 69 X 10(-12) M/mg membrane protein; range 16-400 X 10(-12) M/mg MP; means +/- S.E., n = 32). Native GnRH and the GnRH antagonist [D-p-Glu1, D-Phe2, D-Trp3,6]-GnRH had Ka values comparable to those of the GnRH-A used. [125I]GnRH-A binding could not be displaced by oxytocin, thyrotropin releasing hormone and corticotropin releasing factor in concentrations up to 10(-4) M. Somatostatin cross-reacted with binding sites from some carcinomata, while it did not displace GnRH-A binding in membranes from others. Though the functional role of this specific binding site for GnRH in human epithelial ovarian carcinomata is still obscure, it might be part of an autocrine regulatory system and provide a possible point of attack for therapeutic approaches using GnRH analogs in this malignancy.
Adenocarcinoma↗
A homologous in vitro system to analyze transcription of a mouse immunoglobulin mu heavy-chain gene.
In order to investigate the molecular mechanisms of the regulation of immunoglobulin (Ig) gene transcription, a cell-free system was developed in which a cloned mouse Ig mu heavy-chain gene was transcribed using nuclear extracts prepared from a mouse B cell hybridoma line. To monitor transcription, an RNA.RNA hybridization assay was developed in which a 32P-labeled, SP6-synthesized RNA probe complementary to Ig mu RNA was hybridized to unlabeled RNA transcribed in the nuclear extract. Accurate initiation of transcription, which resulted in the protection of the RNA probe from digestion with nuclease S1, was detected by the separation of the products on denaturing polyacrylamide gels, followed by autoradiography. Using this assay, an in-vitro-synthesized RNA was detected. The 5' end of the in-vitro-transcribed Ig mu RNA maps exactly to the same position as the 5' end of the corresponding in vivo mRNA and its formation was sensitive to the addition of low levels of alpha-amanitin (1 microgram/ml), indicating transcription by RNA polymerase II. It was shown by competition experiments with oligonucleotides containing the 'decamer recognition site' that this sequence interacts with (a) decamer-binding factor(s) and plays a positive role in transcription. The competition effects of the decamer-containing oligonucleotide appeared to be restricted to the decamer motif present in the promoter region. No effects of the enhancer region were detectable in vitro. Little or no transcriptional activity was found in transcription experiments using the Ig mu promoter and nuclear extracts prepared from HeLa cells. This suggests that tissue-specific factors involved in Ig mu heavy-chain gene transcription are present in the mouse B cell extracts.
Amanitins↗
Modulation of gonadotropin-releasing hormone receptor concentration in cultured female rat pituitary cells by estradiol treatment.
Short-term (0.5-4 h) treatment of rat pituitary cells in culture with estradiol (E2) results in a significant decrease of Gonadotropin-Releasing Hormone (GnRH) induced LH-release. We studied whether changes in the concentrations of GnRH-receptors (GnRH-R) might account for this phenomenon: pituitary cells from adult female rats were incubated for 4 or 24 h in the presence or absence of 10(-9) M E2. Then saturation curves of D-Ala6-des-Gly10-GnRH ethylamide binding were obtained. In addition, binding studies were carried out in cultures incubated for 0.5, 1, 2 or 4 h with or without 10(-9) M E2 using a near saturating concentration of GnRH-analog. No changes of GnRH-R affinity occurred (4 h experiments: Ka in vehicle treated cells: 0.94 +/- 0.2 x 10(9) M-1, Ka in E2 treated cells: 1.06 +/- 0.3 x 10(9) M-1; 24 h experiments: Ka vehicle: 0.95 +/- 0.2 x 10(9) M-1, Ka E2: 0.82 +/- 0.3 x 10(9) M-1). The GnRH-R concentrations, however, were significantly reduced (44 +/- 3%; P less than 0.001) by 4 h E2 treatment and increased (by 68 +/- 8%; P less than 0.01) by 24 h of E2 treatment. The GnRH induced LH-release in aliquots of the same cell preparations was significantly reduced after 4 h and markedly increased after 24 h of E2 treatment. The experiments on the time-course of the reduction of D-Ala6-GnRH-binding by E2 treatment showed that the number of GnRH-R was significantly decreased (24 +/- 1%; P less than 0.05) already after 0.5 h of exposure to the estrogen. This is also the time period after which the negative E2-effect on GnRH-induced LH-release becomes significant. These data provide first evidence that the short-term negative E2-effect on GnRH induced LH-release by rat pituitary cells in culture could be mediated via a reduction of available GnRH-R.
Animals↗
Modulation of LH-secretion in ovariectomized ewes by constant infusions of oestradiol and 4-hydroxyoestradiol: effects of varying infusion times and of high oestrogen doses.
Ovariectomized ewes were infused for different times (2-24 h) during the breeding season (September to February) with oestradiol (E2, 2 micrograms/h) or the catecholoestrogen 4-hydroxyoestradiol (4-OHE2, 10 micrograms/h). At these infusion rates comparable plasma levels of E2 and 4-OHE2 were obtained when steady state was reached after 3 h (E2: 20 +/- 4 pg/ml; 4-OHE2: 22 +/- 3 pg/ml). When E2 was infused for at least 6 h, all animals had significant LH-surges, starting 14-16 h after the beginning of oestrogen treatment, even when E2 was infused for up to 24 h. 4-OHE2, however, had only to be infused for 4 h to induce significant LH-surges in all animals tested. When E2 was infused for 12 h at a rate of 100 micrograms/h, the LH-surges in these ewes were significantly lower than the LH-surges in the same animals treated for 12 h with E2 at a rate of 2 micrograms/h. These data indicate: Once E2 has been administered at a specific infusion rate for a critical time period of 6 h, LH-surges occur, no matter whether the E2-infusion is continued or stopped. For the catecholoestrogen 4-OHE2 this critical time period amounts only to 4 h, if comparable plasma levels of either oestrogen are achieved. This might hint at a prolonged intracellular action of 4-OHE2 as compared to E2. At extremely high infusion rates of E2 (100 micrograms/h for 12 h) the positive oestrogen effect is significantly impaired, a finding supporting the concept of a bell-shaped dose-response relationship between oestrogens and their positive effect on LH-secretion.
Animals↗
Cyclosporin A is a differential inhibitor of eukaryotic RNA polymerases.
In order to investigate the molecular mechanism of biological action of the drug cyclosporin A (CsA), we have studied its effect on eukaryotic RNA polymerase activities in two systems: (a) in vitro transcription; (b) injection into frog oocytes. The electron microscopic transcription R-loop method [1] has been used to assay for specific transcription of an immunoglobulin gene clone in HeLa cell lysates. In this system, CsA inhibits efficiently the transcription of immunoglobulin genes. Dilution experiments show that RNA polymerase II activity is inhibited by nanomolar concentrations (i.e. amounts comparable to the ones needed for the inhibition of lymphocyte stimulation), whereas polymerase III is inhibited at micromolar concentrations. In vitro transcription with E. coli RNA polymerase is not inhibited. Parallel experiments with the mushroom toxin alpha-amanitin have shown that the eukaryotic RNA polymerase II displays similar sensitivity towards the two cyclic peptides CsA and alpha-amanitin in vitro. When CsA is injected into frog oocytes, together with a cloned Xenopus laevis U1 small nuclear RNA gene, transcription of this gene by endogenous RNA polymerase II is inhibited by 60%. RNA polymerase III and RNA polymerase I transcription is not inhibited in the oocyte. Various possibilities for the selective action of CsA on stimulated lymphocytes are discussed.
Amanitins↗
Effects of continuous infusions of 4-hydroxyoestradiol and oestradiol on plasma LH-levels in the ovariectomized ewe.
Ovariectomized ewes were infused over a period of 12 h at constant rates with different doses of the catecholoestrogen 4-hydroxyoestradiol (4-OHE2) or the primary oestrogen oestradiol (E2) via a catheter placed in the right atrium. Blood samples were drawn every hour for a total period of 48 h starting 1 h before the beginning of the steroid infusion. Luteinizing hormone (LH), 4-OHE2 and E2 concentrations were measured in these samples by specific radioimmunoassays. Infusions of low doses E2 (0.5 microgram/h) or 4-OHE2 (2 micrograms/h) caused only a suppression of LH-secretion. At doses of 1 microgram E2/h or 5 micrograms 4-OHE2/h this negative effect was followed by inconsistent elevations of plasma LH. Beyond this threshold dose, E2 at infusion rates of 2, 5 and 10 micrograms/h and 4-OHE2 at infusion rates of 10, 25 and 50 micrograms/h produced the negative effect and massive LH-surges. At still higher infusion rates (E2: 20 micrograms/h, 4-OHE2: 100 micrograms/h) lower elevations of plasma LH levels were observed. 4-OHE2 had to be infused at 4-5 times higher rates than E2 to obtain comparable plasma concentrations of either oestrogen. Under this condition the effects of 4-OHE2 and E2 were similar indicating that 4-OHE2 has the same potency as E2 at central target sites.
Animals↗
Protein component from Drosophila larval nuclei showing sequence specificity for a short region near a major heat-shock protein gene.
Using a nitrocellulose filter binding assay we have detected a protein component in extracts prepared from the nuclei of D. melanogaster third instar larvae which shows sequence-specific binding to a short region on a cloned D. melanogaster DNA segment containing a copy of the major heat-shock protein gene. The specificity of binding was confirmed by direct visualization of the protein DNA complexes in the electron microscope. Protein blotting experiments demonstrated that three polypeptides in the extract were capable of such sequence-specific binding. The protein binding site detected in this cloned sequence lies 800-1000 bp upstream from the presumptive transcription start.
Animals↗
Molecular analysis of the human interferon-alpha gene family.
Fifteen DNA clones containing sequences related to human interferon-alpha cDNA were isolated from a human chromosomal gene bank (Nagata et al., Nature 287 (1980) 401-408) and characterized by restriction mapping, R-loop and heteroduplex analysis. Nine distinct DNA segments hybridized strongly with interferon-alpha 1 cDNA and formed R-loops with poly(A) RNA from interferon-producing human leukocytes; most if not all of these segments represent functional interferon genes. Five segments hybridized weakly with the probe and did not form R-loops with the poly(A) RNA; one of these was characterized as an interferon-alpha pseudogene. Several DNA segments overlap and define a region of 36 kilobase pairs (kb) that contains three strongly and three weakly hybridizing sequences. From our data and those of Goeddel et al. (Nature 290 (1981) 20-25) we conclude that there exist at least 11 distinct genes of gene-like sequences of the interferon-alpha type in the human genome, of which most likely represents an allelic variant, and at least five pseudogenes distantly related to the interferon-alpha genes.
Chromosome Mapping↗
Partial mapping of ten genes of the human interferon- alpha family.
HulFN-alpha genes form a family of not less than 9-10 distinct members, at least three of which are arranged in tandem. In one case, two variants, believed to be alleles of the same gene, could be identified by sequence analysis. The three gene sequenced so far are devoid in introns.
Alleles↗
DNA electron microscopy.
In recent years DNA electron microscopy has become a tool of increasing interest in the fields of molecular genetics and molecular and cell biology. Together with the development of in vitro recombination and DNA cloning, new electron microscope techniques have been developed with the aim of studying the structural and functional organization of genetic material. The most important methods are based on nucleic acid hybridizations: DNA-DNA hybridization (heteroduplex, D-loop), RNA-DNA hybridization (R-loop), or combinations of both (R-hybrid). They allow both qualitative and quantitative analysis of gene organization, position and extension of homology regions, and characterization of transcription. The reproducibility and resolution of these methods make it possible to map a specific DNA region within 50 to 100 nucleotides. Therefore they have become a prerequisite for determining regions of interest for subsequent nucleotide sequencing. Special methods have been developed also for the analysis of protein-DNA interaction: e.g., direct visualization of specific protein-DNA complexes (enzymes, regulatory proteins), and analysis of structures with higher complexity (chromatin, transcription complexes).
Adsorption↗
Characterization of Drosophila DNA-binding protein DB-2: demonstration of its sequence-specific interaction with DNA.
A DNA-binding protein (DB-2) was isolated from unfertilized Drosophila eggs by DNA-cellulose chromatogrphy. In competition assays with DNA from other species, DB-2 preferentially binds to Drosophila DNA. This binding protein can also be isolated from pupal nuclei and comprises only a small fraction ( < 0.01%) of the total nonhistone chromosomal proteins. In order to investigate the specificity of the interaction between DB-2 and the DNA, we attempted to isolate the DNA sequences to which DB-2 binds. DB-2 was used as a probe to screen our gene bank established by inserting randomly sheared fragments of Drosophila DNA into bacterial plasmids. Groups of plasmids were tested for binding to DB-2 by a filter binding assay. The plasmids bound to the nitrocellulose filter were eluted and used for bacterial transformation. After several cycles of transformation and cloning, two plasmids, A17 and B10, were isolated that bind DB-2 specifically, as measured by filter binding and competition assays. In plasmid A17, binding of DB-2 protects two short DNA segments of approximately 13 and 30 base pairs from digestion by DNase I. By filter hybridization according to Southern, these sequences were mapped to a defined restriction fragment. Further evidence for the binding specificity was obtained by visualizing the protein-DNA complex in the electron microscope. In salivary gland giant chromosomes, A17 DNA hybridizes to a single site (95A/B) on chromosome 3.
Animals↗
Domains and the hinge region of an immunoglobulin heavy chain are encoded in separate DNA segments.
A 6.8-kilobase DNA fragment containing the sequence coding for the constant region of the mouse immunoglobulin gamma1 heavy chain was cloned from total cellular DNA. Electron microscopic and nucleotide sequencing studies showed that the three protein domains and the hinge region are encoded in separate DNA segments.
Animals↗
Electron microscopic analysis of transcription: mapping of initiation sites and direction of transcription.
An electron microscope technique is described that allows rapid characterization of transcription in vitro. DNA is transcribed with Escherichia coli RNA polymerase in vitro, and the RNA is hybridized to its template. Measurement of the resulting transcription R-loop molecules allows accurate mapping of transcription initiation sites (promoter sites) and analysis of the direction and rate of transcription and the level of transcription from each initiation site. The two major early promoters pR and pL of bacteriophage lambda have been mapped within 0.1-0.3 map units of the known positions and three additional sites have been confirmed. Six transcription initiation sites have been preliminarily mapped on plasmid pSF2124 DNA.
Bacteriophage lambda↗
A complete immunoglobulin gene is created by somatic recombination.
Using a pCRI plasmid containing an enzymatically synthesized, full-length DNA transcript of immunoglobulin lambda chain mRNA as the hybridization probe in the Southern gel blotting experiments (Southern, 1975), we identified three DNA fragments of 8.6, 4.8 and 3.5 kb in Eco RI-digested total DNA from BALB/c mouse embryos. A fourth fragment of 7.4 kb was found in addition to these three fragments in similarly digested total DNA from a lambda chain-secreting myeloma (HOPC 2020). We have cloned the four DNA fragments in an EK-2 phage vector, lambdaWES, and characterized them with respect to size, type of lambda gene sequences contained and position of these sequences in the fragments, using agarose gel electrophoresis, the gel blotting technique and electron microscopic R loop mapping. The embryonic DNA clones Ig 99 lambda, Ig 25lambda and Ig 13lambda contain one copy each of V lambdaI, C lambdaI and V lambdaII sequences, respectively, while the myeloma DNA clone Ig 303lambda contains one copy each of V lambdaI and C lambdaI sequences that are separated by a 1.2 kb nontranslated DNA segment. Ig 25lambda was also shown to contain a DNA segment of approximately 40 base pairs (bp) (J sequence) that lies 1.2 kb away from the C lambdaI sequence and is homologous to the V-C junction region of a lambdaI mRNA. Heteroduplex analysis of the three lambdaI DNA clones revealed that Ig 303lambda DNA is composed of two parts, one of which is entirely homologous to one end of Ig 99lambda, and the other to one end of Ig 25lambda DNA. The sequence arrangement observed in the cloned DNA is the same as that in the corresponding cellular DNA. This was shown by identifying certain restriction enzyme cleavage sites on the cloned DNAs and demonstrating the presence of these sites in the total cellular DNA by the gel blotting technique. The site of the homology switch is at the boundary of the V sequence and the 1.2 kb nontranslated DNA segment, and corresponds to the position of the J sequence on the Ig 25lambda DNA. We consider the above experimental results the most direct evidence for somatic rearrangement in immunoglobulin genes. We discuss the significance of these findings for the origin of genes in the evolution of higher organisms and in cell differentiation.
Animals↗
DNA clones containing mouse immunoglobulin kappa chain genes isolated by in vitro packaging into phage lambda coats.
Endonuclease EcoRI-digested DNAs from BALB/c mouse embryos and MOPC 321 (a kappa chain secretor) myeloma were fractionated by agarose gel electrophoresis, and the DNA fragments containing part or all of the MOPC 321 kappa chain structural gene sequences were visualized by the Southern gel blotting technique using as the hybridization probes pCRI plasmids containing all or part of the enzymatically synthesized cDNA transcripts of the MOPC 321 kappa chain mRNA. The clear differences observed in the hybridization patterns of the two DNAs are in agreement with our previously reported results obtained with endonuclease BamHI and confirms that the sequence arrangement of kappa chain genes is different in the embryo and myeloma cells. We have cloned most of the kappa-sequence-positive EcoRI DNA fragments in Charon 4A phage by using the highly efficient in vitro phage lambda DNA packaging method, and we have characterized the cloned mouse DNA sequences by agarose gel blotting and R-loop mapping in electron microscopy. These studies identified, among others, one EcoRI DNA fragment which contains both variable and constant immunoglobulin kappa-gene sequences and is present only in the myeloma DNA. The two sequences are separated by a 2.8-kbase intron. We tentatively conclude that the kappa gene sequences on this DNA fragment underwent somatic rearrangement.
Animals↗