Purification and cDNA cloning of maize Poly(ADP)-ribose polymerase.

Poly(ADP)-ribose polymerase (PADPRP) has been purified to apparent homogeneity from suspension cultures of the maize (Zea mays) callus line. The purified enzyme is a single polypeptide of approximately 115 kD, which appears to dimerize through an S-S linkage. The catalytic properties of the maize enzyme are very similar to those of its animal counterpart. The amino acid sequences of three tryptic peptides were obtained by microsequencing. Antibodies raised against peptides from maize PADPRP cross-reacted specifically with the maize enzyme but not with the enzyme from human cells, and vice versa. We have also characterized a 3.45-kb expressed-sequence-tag clone that contains a full-length cDNA for maize PADPRP. An open reading frame of 2943 bp within this clone encodes a protein of 980 amino acids. The deduced amino acid sequence of the maize PADPRP shows 40% to 42% identity and about 50% similarity to the known vertebrate PADPRP sequences. All important features of the modular structure of the PADPRP molecule, such as two zinc fingers, a putative nuclear localization signal, the automodification domain, and the NAD+-binding domain, are conserved in the maize enzyme. Northern-blot analysis indicated that the cDNA probe hybridizes to a message of about 4 kb.

Modulation of transcription of rRNA genes by rapamycin.

Lymphosarcoma P1798 cells undergo growth arrest when exponentially growing cultures are exposed to 1 micrograms/ml of Rapamycin (Rapa). This growth arrest is accompanied by inhibition of RNA biosynthesis as measured by incorporation of 3H-uridine into the newly synthesized RNA. Approximately 50% inhibition of 3H-uridine incorporation was observed, upon exposure of P1798 cells to 1 microgram/ml Rapa for 24 h. Run-on transcription experiments using nuclei from Rapa-treated cells indicated a dose-dependent inhibition of transcription or rRNA genes. Cells were relieved from this inhibition of transcription when Rapa was removed from the medium. Under similar conditions, transcriptions of U3 snRNA genes remained unaffected. Cytoplasmic extracts prepared from P1798 cells treated with 1 microgram/ml Rapa for 24 h failed to support transcription from cloned mouse rRNA promoter. This treatment does not affect the RNA polymerase I activity of P1798 cells. Addition of a highly purified murine transcription initiation factor specific for RNA polymerase I reconstitutes the extracts from Rapa-treated P1798 cells. Our data indicate that this new immunosuppressive agent modulates transcription of rRNA genes via regulation of specific transcription factor function.

Cyclosporine A inhibits the activity of a TATA box-binding protein that is required for transcription from the adenovirus major late promoter.

Nuclear extracts from P1798 lymphoma cells support transcription from the adenovirus major late promotor (AdMLP) and the human histone H4 promoter. Nuclear extracts prepared from P1798 cells treated with 1 microgram/ml cyclosporine A for 24 h fail to support transcription from AdMLP, whereas transcription from the histone H4 promoter is unimpaired. Both control and cyclosporine-treated extracts contain proteins that interact with synthetic deoxyoligonucleotides that correspond to the CAAT box, TATA box, and upstream stimulatory element of AdMLP. Cyclosporine had no discernible qualitative or quantitative effect upon such DNA-protein interactions, as observed by gel mobility shift assays. Analysis of 5' deletion mutants of AdMLP indicates that deletion of sequences upstream of the TATA box reduces AdMLP transcription by only 50%. This observation suggests that cyclosporine A, which inhibits AdMLP transcription by > 90%, is unlikely to act through changes in the amount or activity of upstream activators such as upstream stimulatory factor- or CAAT box-binding proteins. On the other hand, deletion of TATA box sequences between -50 and -11 base pairs virtually eliminates transcription from AdMLP in vitro. A partially purified TFIID fraction was obtained from control P1798 nuclear extracts. The TFIID fraction reconstitutes transcription from AdMLP when added to extracts from cyclosporine A-treated cells. Recombinant TATA box-binding protein also reconstitutes transcription from AdMLP in cyclosporine A-treated extracts. These results are consistent with the hypothesis that cyclosporine A regulates the activity of a subset of general transcription factors which are required for initiation from some promoters (such as AdMLP) but not from others (such as histone H4).

Copurification of RNA polymerase I and the glucocorticoid-regulated transcription factor IC.

A simple and rapid method to copurify RNA polymerase I and the glucocorticoid-regulated transcription factor, TFIC, is described. This protocol results in 1262-fold purification and 15% total recovery of the enzyme and factors needed to support faithful transcription in vitro from cloned mouse rRNA gene (rDNA). Using this method, proteins involved in rDNA transcription were purified from exponentially growing lymphosarcoma P1798 cells as well as cells treated with 0.1 microM dexamethasone. A combination of transcription and reconstitution assays using G-free cassette-containing constructs and polyacrylamide gel electrophoretic analysis upon silver staining were used to detect TFIC activity as well as the characteristic TFIC polypeptides in control and dexamethasone-treated cell extracts. Treatment of P1798 cells with 0.1 microM dexamethasone for 24 h results in an over 95% reduction of TFIC activity, but no significant differences in the amount of TFIC polypeptides in the final product purified from control and glucocorticoid-treated cells could be detected. Our data indicate that glucocorticoid regulation of transcription of rDNA is mediated via post-translational modulation of the activity of TFIC.

Glucocorticoid regulation of c-myc promoter utilization in P1798 T-lymphoma cells.

Glucocorticoids rapidly inhibit the expression of c-myc mRNA in P1798 lymphoma cells. Statistically significant decreases can be observed within 5-10 min after the addition of glucocorticoids. Although transcription of c-myc decreases within a few hours after dexamethasone is added to P1798 cell cultures, nuclear run-on transcription cannot be used to demonstrate that the very early changes in mRNA abundance reflect corresponding changes in transcriptional activity. An RNase protection assay has been used to measure the abundance and rates of turnover of the two major c-myc transcripts arising from the P1 and P2 initiation sites. The relative rates of synthesis of the c-myc mRNAs (i.e. transcription) can be calculated from such data. The abundance of the P2 transcript exceeds that of P1 mRNA by 3- to 4-fold in midlog phase cells. The turnover rates of the two c-myc mRNAs are essentially identical (0.02 min-1), indicating that the P2 promoter is 3-4 times stronger than P1. This was confirmed by measuring the relative transcriptional activities of templates containing the individual c-myc promoters in P1798 extracts in vitro. The expression of P1 and P2 mRNAs decreases at different rates in glucocorticoid-treated cells. A 50% decrease in the abundance of P1 mRNA occurs within 1 h after the addition of dexamethasone. Expression of P2 mRNA is reduced by 50% within 4 h. However, the turnover rates of the major c-myc transcripts do not change in glucocorticoid-treated cells. The t1/2 values of P1 and P2 mRNAs are about 25-30 min and not different from the turnover rates measured in control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid regulation of rRNA synthesis.

Glucocorticoids inhibit transcription of the genes encoding rRNA (rDNA) in P1798 lymphoma cells. This is due to a decrease in the amount or activity of a transcription factor, called TFIC. TFIC has been purified to apparent homogeneity and the properties of this protein have been investigated in detail. TFIC is tightly associated with RNA polymerase I. The data indicate that TFIC is a bona fide initiation factor that is required for formation of the first phosphodiester bond of nascent pre-rRNA. Extracts from dexamethasone-treated cells are devoid of this factor and cannot form initiated complexes in vitro.

Glucocorticoid inhibition of transcription from adenovirus major late promoter.

Glucocorticoids inhibit proliferation of lymphosarcoma P1798 cells in culture. This is accompanied by inhibition of expression of class I as well as some class II genes. We have used the technique of transcription in vitro and the adenovirus major late promoter (AdMLP) to investigate molecular mechanisms of glucocorticoid inhibition of transcription of class II genes. Nuclear extracts from exponentially growing P1798 cells support faithful transcription from AdMLP. However, treatment of P1798 cells with 10(-7) M dexamethasone (a synthetic glucocorticoid) for 24 h impairs the ability of nuclear extracts prepared from such cells to support faithful transcription from AdMLP. Transcription from the human histone H4 gene promoter is unaffected by dexamethasone treatment. Gel mobility shift assays using synthetic oligonucleotide probe indicate no difference in the CAAT box binding activity of control and dexamethasone-treated cell extracts. Similarly, dexamethasone treatment does not affect the upstream stimulatory factor activity of P1798 cells. Furthermore, up-stream stimulatory factor purified from control cell extracts is unable to reconstitute the glucocorticoid-treated cell extracts for transcription from AdMLP. Fractionation of the control cell extracts on Sepharose S yields two protein fractions, neither of which supports transcription from AdMLP. However, one of these fractions, S-II, confers upon glucocorticoid-treated cell extracts the ability to support faithful transcription from AdMLP. We conclude that glucocorticoids regulate the transcription from AdMLP by regulating the activity of a trans-acting transcription factor which copurifies with fraction S-II.

Hormonal regulation of transcription of rDNA. Purification and characterization of the hormone-regulated transcription factor IC.

Glucocorticoids reversibly inhibit transcription of ribosomal RNA genes in murine lymphosarcoma P1798 cells in culture. Inhibition of rDNA transcription is due to reduction in the amount or activity of an RNA polymerase I transcription factor called transcription factor IC (TFIC). TFIC has been purified over 100,000-fold. The highly purified preparation contains neither RNA polymerase I activity nor any of the conventional RNA polymerase I subunits. TFIC activity co-purifies with three polypeptides of approximately 55, 50, and 42 kDa molecular mass. These polypeptides are present in a stoichiometric ration of 1:1:1.

Hormonal regulation of transcription of rDNA. Formation of initiated complexes by RNA polymerase I in vitro.

This paper describes studies of initiation of transcription by RNA polymerase I in vitro. The protocols take advantage of the observation that active transcription complexes precipitate when incubated with S100 extracts. The pellets contain less than 5% of the protein present in unfractionated extracts and are stable to centrifugal washing. This permits rapid manipulation of the reaction conditions and facilitates kinetic studies of aspects of the initiation reaction. An initiated complex has been defined which forms rapidly at 30 degrees C and is associated with formation of the first phosphodiester bond of nascent rRNA. Once formed, initiated complexes are capable of elongation in the presence of heparin or KCl in concentrations sufficient to preclude subsequent initiation. One can therefore estimate the number of initiated complexes formed in a given reaction by measuring the number of full length transcripts recovered in a KCl or heparin-start reaction. The number of such complexes formed correlates well with the formation of a presumptive initiator trinucleotide ApCpU.

Hormonal regulation of transcription of rDNA. The role of TFIC in formation of initiation complexes.

Glucocorticoids inhibit transcription of rDNA in P1798 lymphoma cells. This observation can be recapitulated in vitro in that extracts from hormone-treated cells are virtually incapable of transcribing from the cloned mouse rRNA promoter. However, such extracts can be reconstituted by addition of a RNA polymerase I transcription factor, called TFIC. TFIC has been purified to homogeneity. Assays have been developed which facilitate analysis of various aspects of initiation of transcription by RNA polymerase I in vitro. This paper describes a series of experiments designed to test two related hypotheses. It is proposed that TFIC is a bona fide initiation factor and that the inability of hormone-treated cells to synthesize rRNA is due to failure to form initiation complexes on rDNA. The data indicate that extracts from hormone-treated cells cannot form KCl or heparin-resistant initiated complexes upon the rRNA promoter. The ability to form such complexes is dependent upon the addition of TFIC. The lack of TFIC precludes formation of the first phosphodiester bond. At low concentrations of TFIC there is a more or less direct relationship between the amount of the factor and the number of initiated complexes formed. At higher concentrations, the system saturates and addition of TFIC beyond 80 pg/microliters (approximately 0.5 nM) has no effect upon initiation. Addition of TFIC to control extracts does not influence the formation of initiated complexes. This is consistent with the conclusion that control extracts contain excess TFIC, whereas hormone-treated extracts are depleted in this respect. The kinetics of reconstitution have been examined, and the results suggest that association of highly purified TFIC with the transcriptional apparatus is a relatively slow process, with a t1/2 of about 2 min. The data are consistent with the hypothesis that TFIC is an initiation factor and suggest that the active form of RNA polymerase I is associated with TFIC. It is proposed that in the absence of this association, initiation of transcription of rDNA does not occur.

Cyclosporin A inhibits rDNA transcription in lymphosarcoma P1798 cells.

Effects of cyclosporin A (CsA) on rRNA synthesis in vivo and in vitro were studied using lymphosarcoma P1798 in culture. Pulse labeling with [3H]uridine indicated that treatment of P1798 cells with 1 microgram/ml of CsA for 24-h reduced rRNA levels by 50-60%, whereas rRNA levels of cells rescued from CsA and grown for 24 h were similar to those of controls. Transcription experiments using nuclei from control, treated, and rescued cells indicated that the reduction in rRNA synthesis in treated cells was due to reversible inhibition of transcription of rDNA. Transcription studies in vitro indicated that S100 extracts from CsA-treated cells were unable to carry out faithful transcription of cloned mouse rDNA, even though RNA polymerase I levels of control and treated cell extracts were similar. Mixing experiments indicated that the inability of the CsA-treated cell extract to transcribe cloned rDNA in vitro was not due to the presence of inhibitor(s) or nuclease(s) in such extracts. Supplementation of CsA-treated cell extract with partially or highly purified preparations of a transcription initiation factor for RNA polymerase I, obtained from control cell extracts, conferred transcriptional ability on the CsA-treated cell extract. Extracts from cells treated with cyclosporin H, an inactive analogue of CsA, faithfully transcribed rDNA, indicating the specificity of CsA action. These data indicate that CsA-treated cells lack the ability to initiate rDNA transcription in vivo and in vitro, due to specific, reversible reduction in the amount or activity of transcription factor IC. Significance of these results in understanding the mechanisms of the lymphostatic activity of CsA is discussed.

Effect of oxidizing agents on rabbit mammary prolactin receptors.

Treatment of rabbit mammary membrane-bound and solubilized prolactin receptors with the oxidizing agents N-chlorobenzene sulfonamide and N-bromosuccinimide resulted in total inactivation of the ability of the receptor to bind prolactin. A similar inactivation was obtained with 2-hydroxy-5-nitrobenzylbromide. The results suggest the possible importance of tryptophan in maintaining the activity of the prolactin receptor.

Inactivation of rabbit mammary prolactin receptor by N-acetylimidazole.

Treatment of rabbit mammary prolactin receptor with N-acetylimidazole resulted in loss of prolactin binding activity. Loss of activity of the particulate receptor was time and concentration dependent with 100 mM reagent causing total inactivation in 10 min. Similar results were obtained with solubilized receptor preparations, but at lower reagent concentrations. The loss of binding activity was due to loss in the number of binding sites. Incubation of the reagent inactivated membranes or soluble receptor with hydroxylamine for 3 hr resulted in 80-90% reactivation of the prolactin binding activity. These results indicate the possible involvement of tyrosyl residue(s) on the receptor in the prolactin-receptor interaction.

Cyclosporin A and rabbit mammary prolactin receptors.

The binding of cyclosporin A and ovine prolactin to rabbit mammary gland membranes was determined. CsA bound with a Kd of 2.2 X 10(-6)M whereas prolactin bound with a Kd of 2 X 10(-10)M. The binding of each ligand was an independent event and neither ligand influenced the binding of the other ligand showing that CsA does not inhibit the binding of prolactin to its specific receptor in this system.

Novel approaches to the purification of penicillin acylase.

Penicillin acylase of E. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0-7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purified enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.

Purification of rabbit mammary prolactin receptor by acidic elution from a prolactin affinity column.

Membrane-bound prolactin receptors from the mammary gland of 6-7-day postpartum lactating rabbits were solubilized using the zwitterionic detergent, Zwittergent 3-12 (3- dodecyldimethylammonio )-1-propanesulfonate). The solubilized receptor from one rabbit was bound to an ovine prolactin-agarose affinity gel and eluted at pH 4.2. The receptor appeared as a single band upon staining sodium dodecyl sulfate-polyacrylamide gels with Coomassie blue. The protein yield from one rabbit was about 8 micrograms and the overall yield of receptor was over 50%. The apparent molecular weight was 42,000 on sodium dodecyl sulfate gels but varied on molecular weight columns due to the type of detergent. Receptor inactivated by iodination had an apparent molecular weight of 21,000 on sodium dodecyl sulfate gels. The purified receptor did not bind to concanavalin A-agarose or Lens culinaris-agarose.

Purification of amyloglucosidase.

Amyloglucosidase (glucoamylase; EC 3.2.1.3) has been purified from the culture filtrates of Aspergillus candidus Link var. aureus using hydrophobic interaction chromatography and DEAE-cellulose treatment. The enzyme thus obtained has a specific activity of 329 units/mg protein with the overall recoveries between 70 and 75%. The process appears to be of industrial promise.