Catalina Sanz

Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism

The fungus Phycomyces blakesleeanus reacts to environmental signals, including light, gravity, touch, and the presence of nearby objects, by changing the speed and direction of growth of its fruiting body (sporangiophore). Phototropism, growth toward light, shares many features in fungi and plants but the molecular mechanisms remain to be fully elucidated. Phycomyces mutants with altered phototropism were isolated ≈40 years ago and found to have mutations in the mad genes. All of the responses to light in Phycomyces require the products of the madA and madB genes. We showed that madA encodes a protein similar to the Neurospora blue-light photoreceptor, zinc-finger protein WC-1. We show here that madB encodes a protein similar to the Neurospora zinc-finger protein WC-2. MADA and MADB interact to form a complex in yeast 2-hybrid assays and when coexpressed in E. coli, providing evidence that phototropism and other responses to light are mediated by a photoresponsive transcription factor complex. The Phycomyces genome contains 3 genes similar to wc-1, and 4 genes similar to wc-2, many of which are regulated by light in a madA or madB dependent manner. We did not detect any interactions between additional WC proteins in yeast 2-hybrid assays, which suggest that MADA and MADB form the major photoreceptor complex in Phycomyces. However, the presence of multiple wc genes in Phycomyces may enable perception across a broad range of light intensities, and may provide specialized photoreceptors for distinct photoresponses.

PTGDR gene in asthma: a functional, genetic, and epigenetic study.

To cite this article: Isidoro‐García M, Sanz C, García‐Solaesa V, Pascual M, Pescador DB, Lorente F, Dávila I. PTGDR gene in asthma: a functional, genetic, and epigenetic study. Allergy 2011; 66: 1553–1562.

Promoter genetic variants of prostanoid DP receptor (PTGDR) gene in patients with asthma

Background:  PTGDR gene has been identified as an asthma‐susceptibility gene. Recently, functional genetic variants have been associated with asthma. The objective of this work was to study −549T>C, −441C>T and −197T>C PTGDR promoter polymorphisms in a Spanish population.

927T>C polymorphism of the cysteinyl-leukotriene type-1 receptor (CYSLTR1) gene in children with asthma and atopic dermatitis.

Asthma and atopic dermatitis share several common features and Cysteinyl‐leukotrienes are mediators that participate in the pathogenesis of both diseases. Recently, a new polymorphism (927T>C) has been identified in cysteinyl‐leukotriene type‐1 receptor (CYSLTR1) gene. This gene is found on the X chromosome. The aim of this study was to analyze this SNP in a population of children with asthma and atopic dermatitis. In this study, 166 individuals, 79 adult controls (CTR) and 87 children with asthma (AA) were included. Forty‐one patients with asthma presented atopic dermatitis (AA‐AD). Adults were chosen as controls to confirm lack of development of asthma and allergy during childhood. Standardized history, physical examination, skin prick tests, and lung function measurements were performed in all patients. The 927T>C CYSLTR1 SNP was analyzed by direct sequencing after PCR amplification. In males (53 individuals), the C allele was significantly more common among AA‐AD patients (47%) than in CTR (8%) (Fishers p < 0.005; Monte Carlo p < 0.008; OR:9.78; 95%CI: 1.73–55.30). When comparing AA‐AD vs. AA‐NAD (patients with asthma but not atopic dermatitis), significant differences were observed, (47% vs. 15%, Fishers p = 0.014; Monte Carlo p = 0.022; OR: 4.97; 95%CI: 1.29–19.13). No differences in allele distribution were observed between these disease sub‐groups in females. The 927T>C is a silent SNP; however, it could affect transcription or translation or may be linked to an unidentified, functional polymorphism and thus may pre‐dispose male children to asthma and atopic dermatitis in our population. Further studies are needed to confirm these findings.

Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution

Significance Photolyases repair UV-B–induced DNA lesions. They form a large protein family together with cryptochrome photoreceptors (cryptochrome/photolyase family, CPF). A more recently discovered CPF subclade consists of DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH), present in bacteria, plants, animals, and fungi. Cry-DASH are considered as photoreceptors with residual repair activity for DNA lesions in single-stranded DNA. Canonical photolyases repair such lesions in single-stranded and double-stranded DNA. Here, we show that mucoromycotina fungi except Umbelopsis ramanniana, which is an early diverging lineage within the mucoralean fungi, encode only cry-DASH. They possess the full spectrum of DNA repair activity as canonical photolyases as exemplified for the Phycomyces CryA. This finding is a unique example of CPF evolution where a canonical CPD-photolyase was lost but its function was maintained by cry-DASH. DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B–induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi.

A new PTGDR promoter polymorphism in a population of children with asthma

Recently, functional genetic variants of the PTGDR gene have been associated with asthma. The objective of this work was to study polymorphisms of the promoter region of PTGDR and their haplotype and diplotype combinations in a Spanish population of children with asthma. In this study, 200 Caucasian individuals were included. Asthma was specialist–physician diagnosed according to the ATS criteria. The polymorphisms were analyzed by direct sequencing. In the study, the new polymorphism (‐613C > T) in the promoter region of PTGDR was analyzed. The CT genotype was more common in controls (17%) than in patients with asthma (1%) (p‐value = 0.0003; OR, 0.057; 95% CI, 0.007–0.441). The CCCT CCCC diplotype (promoter positions ‐613, ‐549, ‐441, and ‐197) was more frequent in the group of patients with asthma [Fisher’s p‐value = 0.012; OR, 10.24; 95% CI (1.25–83.68)]; this diplotype is unambiguous. To our knowledge, this is the first study of ‐613C > T PTGDR polymorphism in patients. This analysis provides more complete information on influence of diplotype combinations of PTGDR polymorphisms in asthma.

Analysis of 927T > C CYSLTR1 and -444A > C LTC4S polymorphisms in children with asthma

INTRODUCTION The cysteinyl leukotrienes (Cys-LTs) are potent inflammatory mediators in asthma. It has been suggested that the different response of patients to Cys-LTs inhibitors could be due to the presence of polymorphisms in the genes implicated in this pathway. METHODS In this study, polymorphisms 927T > C CYSLTR1 and -444A > C LTC4S were analysed in a Spanish population of 188 individuals (109 asthmatic children and 79 controls). Standardised history, skin prick tests and lung function measurements were performed in all patients. Genotypes were determined by sequencing after PCR amplification. RESULTS Differences were observed in 927T > C CYSLTR1, regarding the severity of asthma in males. A greater presence of allele C in the population with persistent asthma versus the control group (Fishers p-value = 0.001; Monte Carlo p-value = 0.003; OR: 12.35; 95 %CI: 2.18-70.00) was observed. Differences were also detected in the combined study of both polymorphisms, among controls and asthmatic patients (Monte Carlo p-value = 0.0002). In the group of males with asthma, an increase of AC variant (-444A LTC4S and 927C CYSLTR1) and a reduction in the AT genetic combination were detected. CONCLUSIONS The combined study of polymorphisms in genes of the leukotriene pathway could explain the differences observed in the studies reported on polymorphism -444A < C LTC4S individually analysed.

Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

Protein-DNA interactions in the promoter region of the Phycomyces carB and carRA genes correlate with the kinetics of their mRNA accumulation in response to light.

Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type.

A Ras GTPase associated protein is involved in the phototropic and circadian photobiology responses in fungi

Light is an environmental signal perceived by most eukaryotic organisms and that can have major impacts on their growth and development. The MadC protein in the fungus Phycomyces blakesleeanus (Mucoromycotina) has been postulated to form part of the photosensory input for phototropism of the fruiting body sporangiophores, but the madC gene has remained unidentified since the 1960s when madC mutants were first isolated. In this study the madC gene was identified by positional cloning. All madC mutant strains contain loss-of-function point mutations within a gene predicted to encode a GTPase activating protein (GAP) for Ras. The madC gene complements the Saccharomyces cerevisiae Ras-GAP ira1 mutant and the encoded MadC protein interacts with P. blakesleeanus Ras homologs in yeast two-hybrid assays, indicating that MadC is a regulator of Ras signaling. Deletion of the homolog in the filamentous ascomycete Neurospora crassa affects the circadian clock output, yielding a pattern of asexual conidiation similar to a ras-1 mutant that is used in circadian studies in N. crassa. Thus, MadC is unlikely to be a photosensor, yet is a fundamental link in the photoresponses from blue light perceived by the conserved White Collar complex with Ras signaling in two distantly-related filamentous fungal species.