Characterization of BCE-1, a transcriptional enhancer regulated by prolactin and extracellular matrix and modulated by the state of histone acetylation.

We have previously described a 160-bp enhancer (BCE-1) in the bovine beta-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-beta and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin- and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-beta but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.

A peptide isolated by phage display binds to ICAM-1 and inhibits binding to LFA-1.

A mixed phage library containing random peptides from four to eight residues in length flanked by cysteine residues was screened using a recombinant soluble, form of human ICAM-1, which included residues 1-453, (ICAM-1(1-453)). Phage bound to immobilized ICAM-1(1-453) were eluted by three methods: (1) soluble ICAM-1(1-453), (2) neutralizing murine monoclonal antibody, (anti-ICAM-1, M174F5B7), (3) acidic conditions. After three rounds of binding and elution, a single, unique ICAM-1 binding phage bearing the peptide EWCEYLGGYLRCYA was isolated; the identical phage was selected with each method of elution. Attempts to isolate phage from non-constrained (i.e., not containing cysteines) libraries did not yield a phage that bound to ICAM-1. Phage displaying EWCEYLGGYLRCYA bound to immobilized ICAM-1(1-453) and to ICAM-1(1-185), a recombinant ICAM-1, which contains only the two amino-terminal immunoglobulin domains residing within residues 1-185. This is the region of the ICAM-1 that is bound by LFA-1. The phage did not bind to proteins other than ICAM-1. The phage bound to two ICAM-1 mutants, which contained amino acid substitutions that dramatically decreased or eliminated the binding to LFA-1. Studies were also performed with the corresponding synthetic peptide. The linear form of the synthetic EWCEYLGGYLRCYA peptide was found to inhibit LFA-1 binding to immobilized ICAM-1(1-453) in a protein-protein binding assay. By contrast, the disulfide, cyclized, form of the peptide was inactive. The EWCEYL portion of the sequence is homologous to the EWPEYL sequence found within rhinovirus coat protein 14, a nonintegrin protein that binds to ICAM-1. Taken together, the results suggests that the EWCEYLGGYLRCYA sequence is capable to binding to immobilized ICAM-1. Phage display appears to represent a new approach for the identification of peptides that interfere with ICAM-1 binding to beta 2 integrins.

Transcriptional activation by viral enhancers: critical dependence on extracellular matrix-cell interactions in mammary epithelial cells.

Extracellular matrix (ECM)-cell interactions are essential for the regulation of many genes in differentiated cell types. A number of expression vectors that work well in cells cultured on tissue-culture plastic appear to be inactive or sporadically active in vivo. We reasoned that these responses also may be influenced by the ECM. We therefore examined three commonly used viral enhancers and found that they all responded either positively or negatively to the presence of exogenous ECM. Using mouse mammary epithelial cells, we found that a mouse mammary tumor virus enhancer linked to its own promoter or to a truncated (and by itself inactive) beta-casein promoter drove transcription efficiently only when the cells were in contact with an ECM (more than a 100-fold induction over tissue-culture plastic). Similarly, the cytomegalovirus enhancer was more active in cells in contact with ECM. In contrast, the simian virus 40 enhancer, linked to the beta-casein promoter, was 12-fold more active in cells on tissue-culture plastic. This activity was strongly reduced when the cells interacted with ECM. Thus, we conclude that different enhancers can respond to ECM by either activating or suppressing transcription. This observation has important implications for understanding the mechanisms of promoter action and for designing expression systems for use in gene therapy.

A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression.

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate beta-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of beta-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes beta-casein unidirectionally into a lumen. We have further shown that ECM- and prolactin-dependent regulations of beta-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine beta-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the beta-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin- and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive beta-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin- and ECM-mediated regulation.

Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells.

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

Adenylyl cyclase in yeast: antibodies and mutations identify a regulatory domain.

The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have obtained rabbit antibodies that recognize the CYR1 protein. Antibodies were raised against synthetic oligopeptides and against a recombinant beta-galactosidase/CYR1 fusion protein. These antibodies have allowed the identification of the CYR1 gene product as a 205 kDa protein. Treatment with trypsin (2 micrograms/ml) reduced the size of the CYR1 protein from 205 to 155 kDa and produced an activated enzyme which no longer responded to guanine nucleotides. This result is consistent with a model in which adenylyl cyclase activity is regulated by an inhibitory domain near the amino-terminus of the CYR1 protein. This model is further supported by the finding that adenylyl cyclase activity is also markedly elevated and unresponsive to guanine nucleotides in mutant yeast strains that express only the carboxy-terminal half of the CYR1 protein. Treatment with high trypsin concentrations (greater than 10 micrograms/ml) caused release of adenylyl cyclase activity from the membrane. Comparison of immunoreactive CYR1 fragments released by trypsin and membrane bound genetically altered proteins suggests that the CYR1 protein is attached to the membrane via a separate trypsin sensitive anchoring protein rather than via a membrane anchoring domain.

Adenylyl cyclase in yeast. Hydrodynamic properties and activation by trypsin.

The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have determined hydrodynamic properties of yeast adenylyl cyclase in taurocholate extracts of wild type and RAS-deficient membranes. In taurocholate extracts of both kinds of membranes, the enzyme is insensitive to guanine nucleotide stimulation; in the presence of 0.5 M NaCl, the taurocholate-solubilized enzyme has a sedimentation coefficient of 12.5 S and a Stokes radius of 11 nm, consistent with a molecular weight of 594,000 for the protein-detergent complex. Treatment of particulate fractions with trypsin (less than 10 micrograms/ml) markedly activates membrane-bound adenylyl cyclase activity, abolishes stimulation by guanine nucleotides, and reduces the sedimentation coefficient of the detergent-solubilized enzyme; higher concentrations of trypsin release a still smaller water-soluble enzyme complex (7.5 S, 6.1 nm Stokes radius, calculated Mr = 190,000) from the membrane. In combination with genetic evidence (Kataoka, T., Broek, D., and Wigler M., (1985) Cell 43, 493-505), our data are consistent with a structural and functional model of yeast adenylyl cyclase in which GTP-activated RAS proteins stimulate cAMP synthesis by relieving an inhibitory constraint on the activity of the CYR1 gene product. This constraint may be mediated by the amino-terminal portion of the CYR1 polypeptide.

[Effect of 2-ethyl-2-ene-1-al and analogous compounds on the growth and ultrastructure of fungi]. / Wirkung von 2-Ethyl-2-en-1-al und verwandter Verbindungen auf Wachstum und Ultrastruktur von Pilzen.

Analogs of the natural substance (E)-hex-2-en-1-al which occurs naturally in green plants and is known to have microbiocidal properties were synthesized and studied with respect to their antifungal properties. Saturated and unsaturated aldehydes, alcohols, carbon acids and enolacetate were tested against various fungi by applying these compounds either over the gaseous phase or in suspension. The following ranking was observed on the basis of fungicidal activity: enolacetate of 2-ethylhex-2-enoic acid greater than 2-ethylhex-2-en-1-al greater than 2-ethylhex-2-en-1-al greater than 2-ethylhex-2-en-1-ol. Saturated analogs showed the same order of activity as the unsaturated ones, however, they were less efficient. The effects on the ultrastructural changes were analyzed in Mucor mucedo exposed to concentrations of the analogs that inhibited growth. Main effects were observed on mitochondrial and nuclear membranes. The kind of destruction caused by enolacetate of 2-ethylhex-2-en-1-al differed from the other substances tested. Aspects of the mode of action are discussed.

Isolation of the gene encoding adenylate cyclase in Saccharomyces cerevisiae.

By complementation of the cyr1-1 mutation in Saccharomyces cerevisiae, we have isolated yeast genomic DNA containing the structural gene that encodes the catalytic unit of adenylate cyclase (EC 4.6.1.1). The isolated DNA restored adenylate cyclase activity to cyr1-1 mutants and directed integration at the CYR1 locus. Wild-type strains transformed with CYR1 DNA on the high copy number vector YEp24 contained 4- to 6-fold more adenylate cyclase activity than strains carrying the plasmid with no insert. This result suggests that expression of the CYR1 gene product, rather than that of other polypeptide components of the adenylate cyclase system, limits total adenylate cyclase activity in S. cerevisiae. CYR1-containing plasmids also complemented the temperature-sensitive growth defect of the cell division cycle mutation cdc35-1, which confers a phenotype under restrictive conditions similar to that of cyr1-1 and maps to the same locus. Further, cdc35-1 cam mutants, which contain mutations that enable them to take up cAMP from the medium, grew at the restrictive temperature in the presence of exogenous cAMP. These observations support the view that CDC35 and CYR1 are allelic and confirm the hypothesis that cAMP synthesis is required for cells to pass through the "start" position of the cell division cycle.

[Use of cytochemical methods for diagnosis in fungicide research]. / Einsatz zytochemischer Methoden zur Diagnostik in der Fungizidforschung.

Cytochemical localization of acid and alkaline phosphatases, cytochrom c-oxidase, peroxidases and catalase was carried out on Phytophthora infestans (Mont.) de By. and Mucor mucedo (L.) Fres. First results are obtained about the influence of sublethal dosages of fungicidal compounds on the demonstration of these enzymes in the electron-microscope.

A guanine nucleotide-sensitive adenylate cyclase in the yeast Saccharomyces cerevisiae.

Adenylate cyclase in particulate extracts of Saccharomyces cerevisiae utilized either MnATP or MgATP as substrate. A mutation in the CYR1 gene, which codes for the catalytic unit of yeast adenylate cyclase (Matsumoto, K., Uno, I., and Ishikawa, T. (1983) Cell 32, 417-423), eliminated utilization of both MgATP and MnATP, indicating that a single enzyme was responsible for both activities. GTP and guanylyl-5'-imidodiphosphate stimulated yeast adenylate cyclase, while a GDP analog, guanosine-5'-O-(2-thiodiphosphate), competitively inhibited this stimulation. Thermal inactivation studies distinguished putative guanine-nucleotide regulatory protein (N) from the catalytic unit (C) of yeast adenylate cyclase. Yeast N, which conferred guanine nucleotide regulation and the ability to utilize MgATP on yeast C, was quickly inactivated by incubation of particulate extracts at 30 degrees C. In contrast, yeast C, which apparently utilized MnATP as substrate in the absence of a functional N protein, resisted inactivation at 30 degrees C. These observations suggested that physically distinct protein components mediated the catalytic activity of yeast adenylate cyclase and its regulation by guanine nucleotides. These findings indicate a striking homology between the adenylate cyclase systems of S. cerevisiae and those of vertebrate cells.

Specificity of anti-DNA antibodies in SLE-I. Definition and gross specificity of antibody populations in human SLE plasma.

Populations of anti-DNA antibodies in two SLE plasma were defined based on their patterns of reactivity in inhibition assays with single and double-stranded DNA as well as mono- and oligonucleotides. Two populations of anti-DNA antibodies were seen in both plasma tested. The first population reacted specifically with ssDNA and was inhibited by relatively low concentrations of free nucleotides indicating that it recognized the nucleotide bases in ssDNA. The second population bound both ss and ds calf thymus DNA with apparent equal affinity. The cross-reactive anti-DNA antibodies were inhibited by mononucleotides (at high concentrations) and by single-stranded oligonucleotides (average length tetranucleotides). For one of the plasma tested (PS), pBR322 plasmid DNA (54% G + C) was a significantly more effective inhibitor than calf thymus DNA (39% G + C). These results suggested that nucleotide bases contributed to dsDNA binding by cross-reactive anti-DNA antibodies.

Specificity of anti-DNA antibodies in SLE--II. Relative contribution of backbone, secondary structure and nucleotide sequence to DNA binding.

Fine specificity of a population of anti-DNA antibodies which bound both ssDNA and dsDNA with apparently equal affinity was studied in two SLE plasma. Sensitivity of DNA binding to increasing sodium chloride concentration indicated that electrostatic interactions occurred between antibody and phosphate moieties of DNA. Secondary nucleic acid structure was important to DNA binding as double-stranded synthetic deoxynucleotide polymers were more effective inhibitors than their substituent single-stranded polymers. Nucleotide bases were also found to play a role in recognition of DNA by these cross-reactive antibodies, as ssDNA binding was sensitive to increasing temperature which caused unstacking of the nucleotide bases. Differing patterns of reactivity with synthetic deoxynucleotide polymers with similar secondary structures but different nucleotide compositions further indicated the importance of nucleotide bases to dsDNA binding by cross-reactive anti-DNA antibodies in SLE plasma.

[Effect of pentachloronitrobenzene (PCNB) on the ultrastructure of Mucor mucedo and Phytophthora cactorum]. / Wirkung von Pentachlornitrobenzol (PCNB) auf die Ultra-struktur von Mucor mucedo und Phytophthora cactorum.

The effect of PCNB in various concentrations on the ultrastructure of Mucor mucedo and phytophthora cactorum was analyzed after an incubation period of 2 hours. The most striking effect in both fungi was a diffuse lysis of the internal structure of the mitochondria which differs markedly from the lysis induced by etridiazol (terrazol). Moreover an enlargement of the perinuclear space and an increased formation of vacuoles was observed. In Mucor mucedo, but not in Phytophthora cactorum a pathological thickening of the cell wall was observed. Although after 2 hours incubation with PCNB Phytophthora gave similar ultrastructural reactions in the mitochondria as Mucor, in growth experiments on agar dishes this species was 5-10 times less sensitive to PCNB compared to Mucor.

Anomalous cell wall synthesis in Mucor mucedo (L.) Fres. induced by some fungicides and other compounds related to the problem of dimorphism.

An anomalous cell wall thickening in Mucor mucedo is induced already within 60-120 min by some fungicides (etridiazol, chloroneb, pentachloronitrobenzene, dicloran, drazoxolon, biphenyl) as well as with a N2-atmosphere or high concentrations of glucose, but not with 2,4-dinitrophenol, chlorinated phenols, dichlofluanid and antimycin A. This effect seems to be identical to the change from the mycelial (M-) to the yeast (Y-) form in dimorphic fungi, which can be achieved by culture conditions as well as by addition of chemicals. The cause seems to be a specific, complex change in the metabolic state. A scheme of regulation is presented which explains most of the experimental results described till now.

Colicin E1 plasmid as a probe for detection and study of anti-dna activity in SLE sera.

Tritiated colicin E1 plasmid DNA ([3H]DNA) was purified, characterized and developed as a test antigen for study of anti-DNA antibody activity in a modified Farr assay. The homogeneous molecular weight (4.2 X 10(6) daltons), physical state (intact covalently closed circles), and capacity for intrinsic radioactive labeling to a suitable specific activity were all important properties of the plasmid probe. In addition, the ability of the Farr assay to measure primary antigen/antibody interactions made quantitative determinations of anti-double-stranded DNA (dsDNA) antibody activity possible. Sonication of the plasmid resulted in definable fragments which were thermally denatured and used in the measurement of anti-single-stranded DNA (ssDNA) antibody activity. Results from studies in which unlabeled dsDNA, ssDNA, amd free 2'-deoxyribonucleotides were employed to inhibit binding of [3H]DNA by anti-DNA antibodies indicated the presence of 3 distinct anti-DNA specificities in SLE sera.