White collar proteins: PASsing the light signal in Neurospora crassa.

The filamentous fungus Neurospora crassa is an excellent paradigm for the study of blue light signal transduction. The isolation and characterization of the genes for two central regulators of the blue light response, white collar-1 and white collar-2, have begun to shed light on the mechanism of blue light signal transduction in fungi. These proteins are not only proposed to encode blue-light-activated transcription factors but also to be elements of the blue light signal transduction pathway.

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs.

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.

The bromodomain of Gcn5p interacts in vitro with specific residues in the N terminus of histone H4.

Whereas the histone acetyltransferase activity of yeast Gcn5p has been widely studied, its structural interactions with the histones and the role of the carboxy-terminal bromodomain are still unclear. Using a glutathione S-transferase pull down assay we show that Gcn5p binds the amino-terminal tails of histones H3 and H4, but not H2A and H2B. The deletion of bromodomain abolishes this interaction and bromodomain alone is able to interact with the H3 and H4 N termini. The amino acid residues of the H4 N terminus involved in the binding with Gcn5p have been studied by site-directed mutagenesis. The substitution of amino acid residues R19 or R23 of the H4 N terminus with a glutamine (Q) abolishes the interaction with Gcn5p and the bromodomain. These residues differ from those known to be acetylated or to be involved in binding the SIR proteins. This evidence and the known dispensability of the bromodomain for Gcn5p acetyltransferase activity suggest a new structural role for the highly evolutionary conserved bromodomain.

Ty1 extrachromosomal circular copies in Saccharomyces cerevisiae.

The eukaryotic transposable element Ty1 is present in about 20-30 integrated copies per yeast aploid genome, variably localized in different strains. Here, we report the presence in yeast of circular extrachromosomal molecules homologous to Ty1, 6 kilobases in size (the same as integrated copies) present in about 1 circular copy/250-300 cells. This finding shows another analogy between eukaryotic-transposable elements and the pro-viral integrative form of retroviruses.

The bromodomain: a chromatin browser?

Reversible modification of histone tails is a regulatory step in chromatin remodeling. The N-terminal tails of histones are signaling platforms that carry amino acid residues for post-translational modification and contribute to chromosomal higher order structure. These modifications are performed by a number of chromatin modulators such as histone (h) acetyltransferase, h-deacetylase, h-methyltransferase and h-kinase. Large numbers of these enzymes as well as other chromatin-associated proteins share the bromodomain, a signature protein motif. Structural studies reveal not only wide structural conservation of bromodomains but also envision a possible role of this domain in the recognition of specific modified residues in the histone tails. The widespread presence of bromodomains in leukemogenic and cancer genes has provided a fundamental tool for studies of the role of epigenetic and chromatin remodeling in malignant diseases.

Circular extrachromosomal copia-like transposable elements in Drosophila tissue culture cells.

We find that in the circular extrachromosomal DNA from Drosophila tissue culture cells the transposable elements copia, 412, 297, and mdg 1 are present in variable amounts. There is no detectable circular DNA homologous to B104 . From the relationship between the intra- and extrachromosomal forms it appears that the amount of different circular elements is not related to the amount of the respective chromosomal elements.

Stimulation of yeast RNA polymerase II transcription by critical values of supercoiling.

RNA chains of discrete length were obtained in vitro by yeast RNA polymerase II directed transcription of a supercoiled plasmid. On the basis of the amount and the molecular weight of the RNA chains synthesized in the absence of reinitiation events, the number of actively transcribing RNA polymerase molecules has been calculated. A stimulation of transcriptional activity was found to be related to the torsional strength of negative supercoiling of the template. The DNA unwinding angle measured in the complexes formed with the enzyme in the presence of three ribonucleoside triphosphates equals 485 +/- 30 degrees, marking a melting effect of 14 base pairs per bound enzyme molecule.

Blue light regulation in Neurospora crassa.

The fungus Neurospora crassa has been shown to be a paradigm for photobiological, biochemical, and genetic studies of blue light perception and signal transduction. Several different developmental and morphological processes of Neurospora are regulated by blue light and can be divided into early and late blue light responses. The characterization of two central regulator proteins of blue light signal transduction in Neurospora crassa, WC1 and WC2, and the isolation of light-regulated genes, indicate transcriptional control as a central step in blue light signalling.

Rapid alkaline preparation for yeast circular covalently closed DNA molecules.

The alkaline preparation of prokaryotic plasmids (Birnboim and Doly, 1979) has been here adapted to yeast. By simple denaturation and renaturation steps we recovered, from Saccharomyces cerevisiae and Schizosaccharomyces pombe, a population of nucleic acid molecules highly enriched in circular forms. In S. cerevisiae killer strains it is possible to copurify double stranded RNA molecules. The overall recovery was estimated to be 10-30% of the total circular molecules.

Yeast RNA polymerase II transcription of circular DNA at different degrees of supercoiling.

Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling. Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates. For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription. P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation. However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero. P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity. Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density. Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription.

Escherichia coli ribonucleic acid polymerase binding to the deoxyribonucleic acid of the echinoid Paracentrotus lividus: properties of the complexes and distribution of stable binding sites.

We describe the properties of the complexes that form between Escherichia coli RNA polymerase and Paracentrotus lividus DNA: dissociation kinetics, temperature dependence of the complex formation, resistance to heparin, and range of RNA polymerase-DNA weight/weight ratios that give rise to the stable binding events. The amount and distribution of the sites that form stable binding [class A sites as defined by Hinkle & Chamberlin [Hinkle, D., & Chamberlin, M. J. (1972) J. Mol. Biol. 70, 157]] with E. coli RNA polymerase were determined by the analysis of the dissociation of complexes formed by the enzyme on DNA fragments of various length. The P. lividus appears to form 3.1 X 10(5) stable (t1/2 greater than or equal to 15 min) complexes per haploid genome; the great majority of these complexes shows a short-range distribution (1000-2000 base pairs). The observed attributes of the stable binding sites of P. lividus DNA for E. coli RNA polymerase (amount, distribution, and quantitative ability to start in vitro RNA chains) point to the conclusion that E. coli and sea urchin DNA are nearly indistinguishable by the criteria adopted. The behavior of the sea urchin stable binding sites for the E. coli enzyme is not consistent with the expected behavior of the in vivo promoters.

Purification of sea-urchin RNA polymerase II. Characterization by template requirements and sensitivity to inhibitors.

The purification of RNA polymerase II from gastrulae of Paracentrotus lividus is described. The enzyme obtained is homogeneous as judged by electrophoresis under non-denaturing conditions. It is able to transcribe both native high-Mr P. lividus DNA and Psammechinus miliaris h22 histone DNA, although single-stranded and nicked DNAs are better templates. P. lividus RNA polymerase II forms with homologous native DNA stable binary complexes that are able to initiate RNA chains after exposure to heparin. Heparin-resistant complexes do not form on nicks of DNA molecules. Sensitivity of sea-urchin RNA polymerase II to rifamycin derivatives and alpha-amanitin has been determined.

GCN5, a yeast transcriptional coactivator, induces chromatin reconfiguration of HIS3 promoter in vivo.

Gcn5p, the nuclear histone acetyltransferase (HAT A), is a component of the multiprotein adaptor complex, ADA. Its role as a transcriptional coactivator is required for full induction of most of the genes regulated by GCN4. In this study we present experimental evidence demonstrating that, during gene activation, the nuclease sensitive region of HIS3 promoter, harbouring the poly (dA:dT) and the GCN4 binding site, is invaded by nucleosomes in a gcn5 disrupted strain. These data demonstrate, for the first time, that Gcn5p affects directly the chromatin organization of a chromosomal gene during its transcriptional activation.

Homologous RNA polymerase binding to Escherichia coli DNA: a study of the distribution of stable binding sites.

The number and the distribution of the sites of Escherichia coli DNA that form stable complexes with the homologous RNA polymerase (class A sites according to Hinkle and Chamberlin [3]) have been investigated. Almost all the DNA can bind RNA polymerase, even when fragmented at short (undergenic) size; this general non-promoter-specific binding is highly labile and is not temperature-dependent. The range of RNA polymerase/DNA ratios that give rise to the stable temperature-dependent complexes was examined. The amount and the distribution of class A complexes were studied analysing the dissociation of complexes formed by RNA polymerase on DNA fragments of various length. The E. coli genome appears to form 3.8 X 10(3) stable complexes; the majority of these complexes shows a short-range distribution (800-1200 base pairs). The rest is more widely spaced (1200-6000 base pairs).

The Neurospora crassa carotenoid biosynthetic gene (albino 3) reveals highly conserved regions among prenyltransferases.

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al-3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophosphate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNA clones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in dark-grown and light-induced mycelia. The analysis revealed that the al-3 gene is not expressed in the dark and that its transcription is induced by blue light (Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276). The al-3 gene encodes a polypeptide of 428 amino acids. Comparison of the deduced amino acid sequence of al-3 with the sequences of prenyltransferases of other species, from bacteria to humans, showed three highly conserved homologous regions. These homologous regions may be involved in the formation of the catalytic site of the prenyltransferases.

Role of a white collar-1-white collar-2 complex in blue-light signal transduction.

Mutations in either white collar-1 (wc-1) or white collar-2 (wc-2) lead to a loss of most blue-light-induced phenomena in Neurospora crassa. Sequence analysis and in vitro experiments show that WC-1 and WC-2 are transcription factors regulating the expression of light-induced genes. The WC proteins form homo- and heterodimers in vitro; this interaction could represent a fundamental step in the control of their activity. We demonstrate in vivo that the WC proteins are assembled in a white collar complex (WCC) and that WC-1 undergoes a change in mobility due to light-induced phosphorylation events. The phosphorylation level increases progressively upon light exposure, producing a hyperphosphorylated form that is degraded and apparently replaced in the complex by a newly synthesized WC-1. WC-2 is unmodified and also does not change quantitatively in the time frame examined. Light-dependent phosphorylation of WC-1 also occurs in a wc-2 mutant, suggesting that a functional WC-2 is dispensable for this light-specific event. These results suggest that light-induced phosphorylation and degradation of WC-1 could play a role in the transient expression of blue-light-regulated genes. Our findings suggest a mechanism by which WC-1 and WC-2 mediate light responses in Neurospora.

Selective in vitro transcription by purified yeast RNA polymerase II on cloned 2 micron DNA.

The in vitro transcription properties of purified yeast RNA polymerase II have been analyzed on prokaryotic plasmids (pBR322 and pBR313) and chimaeric plasmids bearing yeast 2 micron sequences (BTYP 1, BTYH 2 and BTYH 3). Conditions for selective transcription of the 2 micron DNA sequences in chimaeric plasmids have been determined. pBR322 and pBR313 are not transcribed by the purified RNA polymerase II when not bearing eukaryotic inserts. We show that the agarose gel electrophoretic analysis of ternary transcription complexes allows the localization of nascent RNA chains. The RNA produced has been visualized by electron microscopy (nascent RNA hybridization loops) and by gel electrophoretic analysis. All the observed properties are shared by RNA polymerase II purified by a conventional method (1) and by a rapid alternative procedure described herein. The peculiar properties of a partially purified form of RNA polymerase II are reported.

White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein.

The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs.

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.