Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process.

BACKGROUND: In fungi, light is primarily known to influence general morphogenesis and both sexual and asexual sporulation. In order to expand the knowledge on the effect of light in fungi and to determine the role of the light regulatory protein ENVOY in the implementation of this effect, we performed a global screen for genes, which are specifically effected by light in the fungus Hypocrea jecorina (anamorph Trichoderma reesei) using Rapid Subtraction Hybridization (RaSH). Based on these data, we analyzed whether these genes are influenced by ENVOY and if overexpression of ENVOY in darkness would be sufficient to execute its function. RESULTS: The cellular functions of the detected light responsive genes comprised a variety of roles in transcription, translation, signal transduction, metabolism, and transport. Their response to light with respect to the involvement of ENVOY could be classified as follows: (i) ENVOY-mediated upregulation by light; (ii) ENVOY-independent upregulation by light; (iii) ENVOY-antagonized upregulation by light; ENVOY-dependent repression by light; (iv) ENVOY-independent repression by light; and (v) both positive and negative regulation by ENVOY of genes not responsive to light in the wild-type. ENVOY was found to be crucial for normal growth in light on various carbon sources and is not able to execute its regulatory function if overexpressed in the darkness. CONCLUSION: The different responses indicate that light impacts fungi like H. jecorina at several cellular processes, and that it has both positive and negative effects. The data also emphasize that ENVOY has an apparently more widespread cellular role in this process than only in modulating the response to light.

Involvement of the Oct-1 regulatory element of the gadd45 promoter in the p53-independent response to ultraviolet irradiation.

The gadd45 gene, a growth arrest and DNA damage (gadd)-induced gene, is transcriptionally activated by UV irradiation through two distinct pathways. One requires the sequence-specific binding of the p53 tumor suppressor protein to a responsive element within the third intron of the gadd45 gene, and the other is p53-independent activation of the gadd45 promoter region, although the UV-response element that mediates this has yet to be defined. To investigate the sequences involved in induction of gadd45 by UV irradiation in a p53-independent pathway, we performed mutation analyses of the human gadd45 promoter fused to the luciferase reporter gene in cell lines in which p53 was inactivated. We found that the UV-responsive element was involved in the Oct-1 binding site at -99 bp relative to the transcription start site. Electrophoretic mobility shift assays showed that Oct-1, a transcription factor, bound this element on the gadd45 gene, although the intensity and mobility pattern of the retarded bands were not altered by UV irradiation. These results suggest that the Oct-1 regulatory element might be one of the essential elements involved in the activation of the gadd45 promoter by UV irradiation in a p53-independent pathway.

Radiation induction of immediate early genes: effectors of the radiation-stress response.

Recent studies have demonstrated that the early response genes c-jun, Egr-1, c-fos, and NF kappa B are induced following exposure of mammalian cells to ionizing radiation. We propose that the products of these early response genes regulate downstream genes that are important in the adaptation of cells and tissues to radiation-induced stress. Potential downstream targets include cytokine and growth factor genes as well as deoxyribonucleic acid (DNA) repair genes. Early response gene products may also regulate cell cycle progression following cellular x-irradiation. Signal transduction pathways that allow cells to adapt to radiation may provide molecular targets to modify tumor and normal responses to radiotherapy.

[Induction of transposon Tn1 translocations as affected by different mutagens]. / Induktsiia translokatsii transpozona Tn1 pri deistvii razlichnykh mutagenov.

Transposition of ampicillin Tn1 transposon is repressed under normal conditions and occurs with low frequency (10(-4) per cell). Treatment of Escherichia coli cells with 22 c+mytomycin C, nitrosoguanidine and UV light induced transposition of Tn1 into different replicons. The degree of induction depended upon the strain of bacteria and the replicon which the transposon was inserted into. Mutation recA did not influence spontaneous translocation of the transposon but fully repressed induction of transposition during the period of mutagen treatment. In the present paper, the connection of inducible transposition process with the recA and lexA inducible functions of E. coli is discussed.

[Time of the action of genes controlling the activity of aldolase in embryonal development of groudling]. / Vremia deistviia genov, kontroliruiushchikh aktivnost' al'dolazy v embrional'nom razvitii v'iuna

The time of action of the genes controlling the decrease of aldolase activity (21-23 hrs of development) and its subsequent increase (23-36 hrs) was determined by means of inactivation of the nuclei by actinomycin or heavy doses of irradiation at succesive developmental stages. There exist two distinct periods of gene activity; the former (15-18 hrs) determines the rapid fall of maternal aldolase activity and the latter (21-27 hrs) its subsequent replacement by embryonic aldolase. This result is confirmed by the data concerning the changes in aldolase heat resistance in the hybrids of the loach and tropical cyprinids. The genes controlling the synthesis of the new aldolase and the morphogenesis which takes place at the same developmental stages are functioning at different times, i.e. the biochemical and morphological differentiations may occur relatively independently.

[Synergistic mechanisms in the combined carcinogenic action of radiation and other factors: the role of mutation and the activation of oncogenes and genes controlling growth factor synthesis]. / O mekhanizmakh sinergizma pri kombinirovannom kantserogennom deistvii izluchenii i drugikh faktorov: rol' mutatsii i aktivatsii onkogenov i genov, kontroliruiushchikh sintez rostovykh faktorov.

A hypothesis is proposed on the role in the radiation carcinogenesis of mutation and (or) activation of combination of the several prooncogenes, some of which are homologous on the growth factors or probably to the actin. The antibodies to the products of oncogenes may be used to study mechanisms of carcinogenic action of radiation and others factors or even as diagnostic tool or remedy.

UV-induced reversion patterns of constitutive and repressed Salmonella histidine auxotrophs.

An unlinked regulatory mutation hisT1504, causes an approximate 11-fold derepression of the histidine (his) operon and a linked constitutive mutation hisO1242 causes an approximate 15-fold derepression. In this study we demonstrate that hisT1504 provokes a significant increase in the UV-induced reversion frequency of his ochre and frameshift mutations. Analysis of revertants derived from frameshift mutants show that this increment in derepressed strains compared to the repressed strains is due to better growth of suppressed revertants by weak frameshift suppressors. The frequency of revertants suppressed by strong frameshift suppressors appears to be the same in repressed and derepressed strains. In contrast, intragenic revertants appear at two-fold decreased frequency in derepressed strains carrying either of the histidine constitutive mutations, hisT1504 or hisO1242. A possible competition is indicated between frequently transcribing RNA polymerase and error-promoting recombinational repair within the histidine operon.

recA+-dependent inactivation of the lambda repressor in Escherichia coli lysogens by gamma-radiation and by tif expression.

When gamma lysogens of E. coli are induced by gamma-radiation the gamma repressor, as measured by its specific binding to gamma DNA, is rapidly inactivated by a recA+-dependent process which does not require new protein synthesis. This rapid inactivation is similar to inactivation of repressor by expression of the temperature sensitive E. coli mutation tif. In contrast, induction by UV irradiation or mitomycin C treatment requires new protein synthesis and there is a lag before the repressor is inactivated (Tomizawa and Ogawa, 1967; Shinagawa and Itoh, 1973).

Correlation between UV dose requirement for lambda bacteriophage induction and lambda repressor concentration.

Escherichia coli K-12 wild type and a uvrA mutant derivative were used to construct isogenic strains bearing one, two, three, or more phage lambda cI genomes and containing increasing concentration of lambda repressor as measured by in vitro operator DNA-binding assays. The survival and phage induction in response to UV irradiation were determined. In both strains, dose-response relationships were obtained as a function of the cellular repressor concentration. The uvrA lysogens required one-tenth the UV fluence of the wild-type counterparts for induction. Lysogenic strains containing plasmids that overproduce the lambdaind+ repressor and the same lysogens with plasmids overproducing the lambdaind- repressor displayed the same survival curves as the nonlysogenic parental strain; however, only the former produced infectious centers (at a frequency of 2 x 10(-3) to 5 x 10(-4) in response to radiation.

Genetic and molecular analysis of Sn, a light-inducible, tissue specific regulatory gene in maize.

The Sn locus of maize is functionally similar to the R and B loci, in that Sn differentially controls the tissue-specific deposition of anthocyanin pigments in certain seedling and plant cells. We show that Sn shows molecular similarity to the R gene and have used R DNA probes to characterize several Sn alleles. Northern analysis demonstrates that all Sn alleles encode a 2.5 kb transcript, which is expressed in a tissue-specific fashion consistent with the distribution of anthocyanins. Expression of the Sn gene is light-regulated. However, the Sn: bol3 allele allows Sn mRNA transcription to occur in the dark, leading to pigmentation in dark-grown seedlings and cob integuments. We report the isolation of genomic and cDNA clones of the light-independent Sn: bol3 allele. Using Sn cDNA as a probe, the spatial and temporal expression of Sn has been examined. The cell-specific localization of Sn mRNA has been confirmed by in situ hybridization using labelled antisense RNA probes. According to its proposed regulatory role, expression of Sn precedes and, in turn, causes a coordinate and tissue-specific accumulation of mRNA of structural genes for pigment synthesis and deposition, such as A1 and C2. The functional and structural relationship between R, B, Lc and Sn is discussed in terms of an evolutionary derivation from a single ancestral gene which gave rise this diverse gene family by successive duplication events.

Studies on an unstable phenotype induced by UV irradiation: the lysine excreting (lex(-)) phenotype of the yeast Saccharomycosis lipolytica.

Unstable clones excreting L-lysine into their growth medium are obtained at a very high frequency following UV irradiation in both haploid and diploid strains of Saccharomycopsis lipolytica, provided they carry a mutation affecting the first enzyme of the lysine pathway and confering resistance to end product inhibition. The phenotype can be stabilized in some sublines; it appears as dominant and coupled with a decrease in spore viability. Excretion in batch cultures is confined to the end of the exponential phase, and seems not to consist in a simple release of the lysine pool content.

Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment.

The regulatory photoreceptor phytochrome controls the transcription of its own phy genes in a negative feedback fashion. We have exploited microprojectile-mediated gene transfer to develop a rapid transient expression assay system for the study of DNA sequences involved in the phytochrome-regulated expression of these genes. The 5'-flanking sequence and part of the structural region of an oat phy gene have been fused to a reporter coding sequence (chloramphenicol acetyltransferase, CAT) and introduced into intact darkgrown seedlings by using high-velocity microprojectiles. Expression is assayable in less than 24 hr from bombardment. The introduced oat phy-CAT fusion gene is expressed and down-regulated by white light in barley, rice, and oat, whereas no expression is detected in three dicots tested, tobacco, cucumber, and Arabidopsis thaliana. In bombarded rice shoots, red/far-red light-reversible repression of expression of the heterologous oat phy-CAT gene shows that it is regulated by phytochrome in a manner parallel to that of the endogenous rice phy genes. These data indicate that the transduction pathway components and promoter sequences involved in autoregulation of phy expression have been evolutionarily conserved between oat and rice. The experiments show the feasibility of using high-velocity microprojectile-mediated gene transfer for the rapid analysis of light-controlled monocot gene promoters in monocot tissues that until now have been recalcitrant to such studies.

Photochemical attachment of lac repressor to bromodeoxyuridine-substituted lac operator by ultraviolet radiation.

The transducing phage lambdah80dlac carries the lac operator, whereas wild-type lambdah80 does not. We find that in high salt (0.18 M KCl), ultraviolet radiation causes the formation of a very stable complex between repressor and 5-bromodeoxyuridine (BrdU)-substituted lambdah80dlac but not to BrdU-lambdah80 DNA. Studies with inducers of the lac operon confirm the specificity of attachment. In low slat (0.01 M KCl), ultraviolet radiation will also attach repressor nonspecifically to BrdU-lambdah80 DNA. The stability of the complex suggests that covalent bonds are formed. We also report that another regulatory protein, the catabolite gene activator protein, can be attached similarly to DNA.

Genetic regulation and photocontrol of anthocyanin accumulation in maize seedlings.

The flavonoid pathway leading to anthocyanin biosynthesis in maize is controlled by multiple regulatory genes and induced by various developmental and environmental factors. We have investigated the effect of the regulatory loci R, B, and Pl on anthocyanin accumulation and on the expression of four genes (C2, A1, Bz1, and Bz2) in the biosynthetic pathway during an inductive light treatment. The results show that light-mediated anthocyanin biosynthesis is regulated solely by R; the contributions of B and Pl are negligible in young seedlings. Induction of the A1 and Bz2 genes by high fluence-rate white light requires the expression of a dominant R allele, whereas accumulation of C2 and Bz1 mRNA occurs with either a dominant or recessive allele at R. A1 and Bz2 mRNA accumulate only in response to high fluence-rate white light, but Bz1 is fully expressed in dim red light. Some C2 mRNA is induced by dim red light, but accumulation is far greater in high fluence-rate white light. Furthermore, expression from both dominant and recessive alleles of the regulatory gene R is enhanced by high fluence-rate white light. Seedlings with a recessive allele at R produce functional chalcone synthase protein (the C2 gene product) but accumulate no anthocyanins, suggesting that, in contrast to the R-mediated coordinate regulation of C2 and Bz1 observed in the aleurone, C2 expression in seedlings is independent of R and appears to be regulated by a different light-sensitive pathway.

Distal regulatory functions for the uvrC gene of E. coli.

We find that the uvrC gene is preceded by three promoters (P1, P2 and P3), identified by heparin-resistant RNA polymerase-DNA complex formation, P2 and P3 promoters are located proximal to the 5' end of the uvrC gene, while the P1 promoter is separated from the uvrC structural gene by an interposed DNA region of more than 1 kb. We have reported that P2 and P3 are not sufficient to promote uvrC complementation. However, plasmids containing the direct fusion of the P1 promoter to the uvrC gene complements the uvrC defect. Insertion of IS1 downstream from the P1 promoter leads to efficient synthesis of the uvrC protein as measured in maxicells. Fusion of the lac promoter to the uvrC structural gene can substitute for in vivo regulatory functions. We conclude that uvrC protein synthesis is controlled in a complex manner and that a distal promoter, P1, is required.

Role of the regulatory gene pl in the photocontrol of maize anthocyanin pigmentation.

The pl gene encodes a regulatory protein that controls the transcription of a number of structural genes of the anthocyanin biosynthetic pathway in maize. pl alleles have been classified phenotypically into two categories: dominant (Pl) alleles lead to intense, light-independent pigmentation in vegetative and floral organs of the plant; recessive "sun-red" alleles (pl) lead to light-dependent red pigmentation in which only tissues exposed to light become pigmented. Based on these observations, two alternate pathways leading to anthocyanin synthesis in the plant have been proposed: one requiring light and the other bypassing the light requirement through the action of Pl. To evaluate this hypothesis, we have analyzed light-independent and light-dependent alleles of pl. Sequence analysis revealed that the two types of alleles have very distinct promoters but have the capacity to encode very similar proteins. The protein encoded by one recessive allele was shown to be functional in transient assays. Measurements of husk mRNA levels by quantitative polymerase chain reaction showed that sun-red pl alleles are expressed at much lower levels than a Pl allele, but their expression is increased approximately sixfold by exposure to light. These results lead to the conclusion that the sun-red pl alleles are not null; instead, they synthesize functional mRNA and protein. We propose that the light-dependent pigmentation observed in pl plants is the result of a threshold effect in which light exposure boosts pl mRNA expression past a crucial level necessary to generate sufficient PL protein molecules to activate transcription of the anthocyanin structural genes.

Deficiency in plasma protein synthesis caused by x-ray-incuded lethal albino alleles in mouse.

Plasma protein synthesis was studied in mice bearing x-ray induced lethal mutations at the albino locus. Newborn albino mutants showed a decrease in each of the three principal plasma proteins, albumin, alpha-fetoprotein, and transferrin, when compared with colored littermate controls. Incorporation of [14C]leucine into plasma proteins of the newborn albinos 30 min after injection was only 1/5 that of the controls, but incorporation into total liver protein was only slightly diminished. Incorporation of [14C]leucine into an albumin fraction obtained by immunoprecipitation from livers incubated in vitro in an amino acid mixture was also strongly diminished. Thus, the liver of 18-day-old albino fetuses incorporated into this fraction 1/3 and that of newborn albinos 1/8 as much as the controls, but in both cases the incorporation into total liver protein was only 25% less than in the respective controls. These results indicate that the rather severe structural abnormalities observed in the mutants in the endoplasmic reticulum and the Golgi apparatus are not associated with a general deficiency of hepatic protein synthesis. Instead the data from this and previous work show that the progressive deficiency from fetal life to birth involves certain specific proteins represented by several perinatally developing enzymes and by plasma proteins. It is suggested that the mutational effects observed in these mice are due to deletions involving regulatory rather than structural genes at or near the albino locus.

Phytochrome-mediated transcriptional up-regulation of ALLENE OXIDE SYNTHASE in rice seedlings.

Allene oxide synthase (AOS) is a key enzyme for the biosynthesis of jasmonic acid (JA). We identified four AOS gene homologs, named OsAOS1-4, in the database of a japonica rice genome and cloned a full-length cDNA of OsAOS1. The analysis of deduced amino acid sequences indicated that only OsAOS1 has a chloroplast transit peptide among all the identified monocot AOSs including OsAOSs. We found that the transcripts of OsAOS1 and OsAOS4 are up-regulated by red and far-red light in seedling shoots. The response in OsAOS1 transcripts occurred rapidly and transiently, while the response in OsAOS4 transcripts was slower and more sustainable; the maximal enhancement was greater in OsAOS1 transcripts than in OsAOS4 transcripts. The transcript of OsAOS1 was also up-regulated transiently in response to wounding, as reported for dicot AOSs. No wound-induced enhancement occurred, however, in OsAOS4 transcripts. Our results also indicated that OsAOS1, responding to both light and wounding, is the most highly expressed of all the OsAOSs in seedling shoots. By using phyA mutants of rice, it was demonstrated that the photoregulation of the AOS transcript level is mediated by phytochrome. It is suggested that this transcriptional photoregulation participates in the phytochrome-mediated inhibition of rice coleoptile growth.

Transcription of Leishmania major Friedlin chromosome 1 initiates in both directions within a single region.

Almost nothing is known about the sequences involved in transcription initiation of protein-coding genes in the parasite Leishmania. We describe here the transcriptional analysis of chromosome 1 (chr1) from Leishmania major Friedlin (LmjF) which encodes the first 29 genes on one DNA strand, and the remaining 50 on the opposite strand. Strand-specific nuclear run-on assays showed that a low level of nonspecific transcription probably takes place over the entire chromosome, but an approximately 10-fold higher level of coding strand-specific RNA polymerase II (Pol II)-mediated transcription initiates within the strand-switch region. 5' RACE studies localized the initiation sites to a <100 bp region. Transfection studies support the presence of a bidirectional promoter within the strand-switch region, but suggest that other factors are also involved in Pol II transcription. Thus, while in most eukaryotes each gene possesses its own promoter, a single region seems to drive the expression of the entire chr1 in LmjF.

Ordered cooperative functions of PRMT1, p300, and CARM1 in transcriptional activation by p53.

Transcriptional coactivators that modify histones represent an increasingly important group of regulatory factors, although their ability to modify other factors as well precludes common assumptions that they necessarily act by histone modification. In an extension of previous studies showing a role for acetyltransferase p300/CBP in p53 function, we have used systems reconstituted with recombinant chromatin templates and (co)activators to demonstrate (1) the additional involvement of protein arginine methyltransferases PRMT1 and CARM1 in p53 function; (2) both independent and ordered cooperative functions of p300, PRMT1, and CARM1; and (3) mechanisms that involve direct interactions with p53 and, most importantly, obligatory modifications of corresponding histone substrates. ChIP analyses have confirmed the ordered accumulation of these (and other) coactivators and cognate histone modifications on the GADD45 gene following ectopic p53 expression and/or UV irradiation. These studies thus define diverse cofactor functions, as well as underlying mechanisms involving distinct histone modifications, in p53-dependent gene activation.