Cloning, sequence, and photoregulation of al-1, a carotenoid biosynthetic gene of Neurospora crassa.

Carotenoid biosynthesis is regulated by blue light during growth of Neurospora crassa mycelia. We have cloned the al-1 gene of N. crassa encoding the carotenoid-biosynthetic enzyme phytoene dehydrogenase and present an analysis of its structure and regulation. The gene encodes a 595-residue polypeptide that shows homology to two procaryotic carotenoid dehydrogenases. RNA measurements showed that the level of al-1 mRNA increased over 70-fold in photoinduced mycelia. Transcription run-on studies indicated that the al-1 gene was regulated at the level of initiation of transcription in response to photoinduction. The photoinduced increase of al-1 mRNA levels was not observed in two Neurospora mutants defective in all physiological photoresponses. Analysis of cosmid containing al-1 and of a translocation strain with a breakpoint within al-1 indicated that al-1 transcription proceeds towards the centromere of linkage group I of N. crassa.

Light-induced dephosphorylation of a 33 kDa protein in the wild-type strain of Neurospora crassa: the regulatory mutants wc-1 and wc-2 are abnormal.

Light induces the dephosphorylation of a 33 kdalton protein within 8 min in the wild-type strain of Neurospora crassa. The regulatory mutants, wc-1 and wc-2, have an altered pattern of phosphoproteins in darkness and also after irradiation. Because the wc genes have previously been implicated in photodifferentiation (F. Degli Innocenti and V. E. A. Russo, Genetic analysis of blue light-induced responses in Neurospora crassa, in H. Senger (ed.), Blue Light Effects in Biological Systems, Springer-Verlag, Berlin, Heidelberg, 1984, pp. 213-219), we suggest that protein dephosphorylation may constitute a necessary step in the light-transduction chain of Neurospora crassa.

Alternative microbial testing: a novel DNA-based detection system for specified microorganisms in pharmaceutical preparations.

Fluorescence-coupled PCR technology was employed to quantify DNA segments specific for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. The PCR procedure is put forward as an alternative method for detecting microbial contaminations in pharmaceutical preparations and is compared to the tests for specified microorganisms described in European Pharmacopoeia (EP) 2, 2.6.13 and the USP, chapter 61. Data presented here describe the validation of this analytical method when used for proof of absence of specified microorganisms. The detection systems were specific for the microorganisms analyzed, and led to linear results over a wide range (more than 6-7 log intervals). The correlation coefficients lay above 0.99. The precision of replicate determinations within a single test was observed to be high, the relative standard deviation being between 0.39% and 1.53%. The precision between different tests was also high, with a relative standard deviation between 0.76% and 1.91%. The sensitivity without pre-enrichment amounted to 1-10 CFU. Since determination of the specified bacteria was performed following pre-enrichment, the limit of detection amounted to 1 CFU. Equivalent results were obtained in a study on nine batches of a milky hydrophilic cream (SH-No. M 440 A) with the conventional test for microbial contamination and the PCR procedure. The data presented here strongly indicate that the use of fluorescence-coupled PCR techniques can prove the absence of specified bacteria faster and more efficiently than conventional methods.

Root-specific expression of the LeRse-1 gene in tomato is induced by exposure of the shoot to light.

The tomato gene LeRse-1 was isolated from a root hair-specific cDNA library. The amino acid sequence of the LeRse-1 product displayed a high degree of identity to that of AgMtd, a mannitol dehydrogenase-encoding gene from celery. Expression of LeRse-1 was found to be restricted to the root organs of tomato, with no detectable transcripts in stems and leaves. Transcripts of LeRse-1 were detected in root hairs, as well as other root tissues of mature plants and seedlings. Root-specific LeRse-1 expression increased upon exposure of the shoot to light. Since roots were kept in darkness, shoot-to-root communication is required for light-induced LeRse-1 RNA accumulation.

Blue light induction of conidiation-specific genes in Neurospora crassa.

The con genes of Neurospora crassa are preferentially expressed during a developmental process known as conidiation. We present evidence indicating that transcription of con-5 and con-10 is also stimulated by blue light. Transcription of these genes was not photoinducible in wc-1 and wc-2 mutant strains. The response of con-5 and con-10 to blue light was similar to that of al-1 and al-2, genes involved in carotenoid biosynthesis, and bli-3 and bli-4, blue light inducible genes.

Day/night and circadian rhythm control of con gene expression in Neurospora.

In the filamentous fungus Neurospora crassa, several events in the process of conidiation are influenced by light. Two genes, con-6 and con-10, which were previously shown to be transcriptionally activated during conidiation and by exposure to light, were found to be unexpressed in mycelium maintained in constant darkness or in constant light. However, when mycelium was shifted from darkness to light, transcripts of both genes appeared and were abundant. Upon further illumination both transcripts disappeared--i.e., their continued production was light repressed. When dark-grown mycelium was exposed to a light pulse and reincubated in the dark, expression of con-6 and con-10 exhibited a 20-hr circadian periodicity. Both genes were photoinducible throughout the stages of the circadian cycle. In the mutant strains bd and bd;frq9, con-6 and con-10 were light inducible but were not normally light repressible. Mutant genes such as acon-2, acon-3, and fl that block developmental expression of con-6 and/or con-10 did not prevent their photoinduction.

Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora.

The surface of many fungal spores is covered by a hydrophobic sheath termed the rodlet layer. We have determined that the rodlet protein of Neurospora crassa is encoded by a cloned gene designated bli-7, and that bli-7 is identical to the known gene eas (easily wettable). Using eas DNA as a probe we show that eas mRNA is abundant in illuminated mycelia and conidiophores but is not detectable or is barely detectable in dark-grown mycelia, mature macroconidia, microconidia, and ascospores. Mutations in the genes acon-2, acon-3, and fl block early conidiophore development; of these, only fl prevents normal eas transcription. The EAS protein is homologous to the rodlet protein (RodA) of Aspergillus nidulans, and the hydrophobins of Schizophyllum commune. eas is the first cloned conidiation (con) gene of N. crassa that is associated with a phenotypic alteration.

Light and developmental regulation of the gene con-10 of Neurospora crassa.

The gene con-10 of Neurospora crassa is expressed preferentially during conidiation and following illumination of vegetative mycelia with blue light. In this study we have examined the segmental locations of the genetic elements associated with con-10 that are responsible for light and developmental expression. A translational fusion was prepared between the initial segment of con-10 and Escherichia coli lacZ. Deletions were then introduced into the con-10 upstream region associated with this translational fusion. Each construct was integrated at the his-3 locus of N. crassa by transformation and homologous recombination. Photoinduction of mycelia containing the translational fusion with the intact upstream region revealed a two phase stimulus-response curve. Exposure to light for as little as 5 sec induced a transcriptional response. Following this initial induction, a period of 15 min in the dark or light was required for appearance of a second phase response. Only a brief light treatment was necessary for induction of the second phase response. Deletions within the upstream region altered normal light and developmental expression of constructs containing the con-10-lacZ translational fusion. The deleted segments appear to contain a mycelial repression site, two conidiation activation sites, and two dark repression sites. A repeated 17-bp sequence acted as a transcriptional enhancer. One copy of this enhancer is in the upstream region. The second copy, with the opposite orientation, is located in the first con-10 intron. The enhancer was required for proper mycelial and conidial expression of the con-10-lacZ fusion. The initial 10 bp of this enhancer sequence were sufficient to restore conidial expression to a deletion construct lacking both copies of the 17-bp repeat. Proteins were detected in extracts of mycelia and conidia that specifically bound to the enhancer sequence in vitro. Our findings suggest that conidiation-specific and mycelial-specific expression of con-10 requires the action of several factors acting independently and/or in concert at distinct sites located in the regulatory regions for con-10.

Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato.

Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment. They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake. To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library. Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves. Expression was root cell type-specific and regulated by nitrogen availability. LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate. In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied. Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium. Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport. Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen.

Fast light-regulated genes of Neurospora crassa.

Several physiological reactions including the sexual differentiation of the ascomycete Neurospora crassa are triggered by blue light. Mutants in the white-collar genes wc-1 and wc-2 are blind for all the blue light effects tested so far. We have previously shown that blue light induces some translatable mRNAs at different times after beginning the illumination. Here we report the cDNA cloning of four genes that are induced by blue light. Induction of these transcripts is temporally ordered (lag times from 2 to 45 min). Analysis of run-on transcripts show that the increases in mRNA levels are due to de novo transcription. None of these transcripts is inducible in white-collar mutants.

Characterization of al-2, the phytoene synthase gene of Neurospora crassa. Cloning, sequence analysis, and photoregulation.

We have cloned the al-2 gene of Neurospora crassa and have analyzed its structure and regulation. The gene encodes a 603-residue polypeptide with a segment homologous to prokaryotic and other eukaryotic phytoene synthases. RNA measurements showed that the level of al-2 mRNA increased over 30-fold in photoinduced mycelia compared with dark-grown mycelia. This observation is consistent with the fact that carotenoid biosynthesis is induced by blue light during growth of N. crassa mycelia. The photoinduced increase in al-2 mRNA levels was not observed in two Neurospora mutants, wc-1 and wc-2, that are defective in all physiological photoresponses.

Photoregulation of cot-1, a kinase-encoding gene involved in hyphal growth in Neurospora crassa.

Blue light plays a key role as an environmental signal in the regulation of growth and development of fungi and plants. Here we demonstrate that in Neurospora crassa hyphae branch more frequently in cultures grown in light. Previous studies had identified cot-1 as a gene that controls apical hyphal cell elongation. In the cot-1 mutant, cessation of elongation is accompanied by hyperbranching. Here we demonstrate that the cot-1 gene encodes two transcript species of about 2100 nt (cot-1 (s)) and about 2400 nt (cot-1 (l)) in length and that the ratio of both transcript species abundance is photoregulated. The origin of the difference between cot-1 (l) and cot-1 (s) was localized to the 5' end of the cot-1 transcripts, suggesting that two COT1 isoforms with different activities may be formed. Both light effects, on branching and on cot-1 expression, were dependent on functional wc-1 and wc-2 gene products. In addition to light, L-sorbose comprises another environmental cue that controls hyphal branching in N. crassa. In the presence of L-sorbose, photoregulation of cot-1 was blocked, suggesting the involvement of alternative and potentially interdependent signaling pathways for the regulation of hyphal elongation/branching.

Detection of BRCA1 and BRCA2 mutations in breast cancer families by a comprehensive two-stage screening procedure.

We have developed a 2-stage protocol for BRCA1 and BRCA2 mutation screening from blood spot paper. Stage 1 screening was aimed to analyze patients at highest risk for the most common disease-associated sequence variants listed in the BIC database. Accordingly, stage1 testing implied detection of 18 disease- associated BRCA1 and 9 BRCA2 mutations by adapting the 5' nuclease assay to heterozygote screening. For stage 2 screening, we applied the conformation sensitive gel electrophoresis (CSGE) method by adapting this technique to automated heteroduplex analysis of BRCA1 and BRCA2 using fragment scanning on an ABI 377 sequencing device. Of the 120 patients with a family history of breast and ovarian cancer who took part in this study so far, 45 entered stage 1 testing. Disease-associated mutations were detected in 6 patients by stage 1 testing (13%). For these patients, the final result was available within 10 days. Mutation 300T-->G was found in 2 patients. One patient with mutation 3036delACAA in BRCA2 reported only 1 sister with a multifocal bilateral breast cancer. New disease-associated mutations were detected in 2 of the 114 patients who entered the stage 2 test (1.7%). Of particular interest was 1 patient who was diagnosed with a medullary breast carcinoma at age 39 and who had no family history of breast cancer. We conclude that pre-screening by 5' nuclease assay for the mutations most frequently seen in a given population represents a relatively effective first line of analysis. Subsequent detailed analysis by fluorescence conformation sensitive gel electrophoresis (F-CSGE) and fragment sequencing is a sensitive alternative to full nucleotide sequencing.

Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato.

To elucidate the role of NH4+ transporters in N nutrition of tomato, two new NH4+ transporter genes were isolated from cDNA libraries of root hairs or leaves of tomato. While LeAMT1;2 is closely related to LeAMT1;1 (75.6% amino acid identity), LeAMT1;3 is more distantly related (62.8% identity) and possesses two short upstream open reading frames in the 5' end of the mRNA and a particularly short N-terminus of the protein as unique features. When expressed in yeast mutants defective in NH4+ uptake, all three genes complemented NH4+ uptake. In roots of hydroponically grown plants, transcript levels of LeAMT1;2 increased after NH4+ or NO3- supply, while LeAMT1;1 was induced by N deficiency coinciding with low glutamine concentrations, and LeAMT1;3 was not detected. In aeroponic culture, expression of LeAMT1;1 and LeAMT1;2 was higher in root hairs than in the remaining root fraction. Growth of plants at elevated CO2 slightly decreased expression of LeAMT1;2 and LeAMT1;3 in leaves, but strongly repressed transcript levels of chloroplast glutamine synthetase and photorespiratory serine hydroxymethyl-transferase. Expression of LeAMT1;2 and LeAMT1;3 showed a reciprocal diurnal regulation with highest transcript levels of LeAMT1;3 in darkness and highest levels of LeAMT1;2 after onset of light. These results indicate that in tomato at least two high-affinity NH4+ transporters, LeAMT1;1 and LeAMT1;2, are differentially regulated by N and contribute to root hair-mediated NH4+ acquisition from the rhizosphere. In leaves, the reciprocally expressed transporters LeAMT1;2 and LeAMT1;3 are supposed to play different roles in N metabolism, NH4+ uptake and/or NH3 retrieval during photorespiration.