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G Macino

Publications and source records attributed to G Macino.

At least 37 records · Page 2Linked to original sources
PubMed1997

Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa.

We report the isolation of 15 Neurospora crassa mutants defective in "quelling" or transgene-induced gene silencing. These quelling-defective mutants (qde) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in N. crassa. The recessive nature of the qde mutations indicates that the encoded gene products act in trans. We show that when qde genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the qde mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such, qde genes may be involved in sensing aberrant sense RNA and/or targeting/degrading the native mRNA. The qde mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene.

Genes, Fungal↗
PubMed1997

Mutants of Neurospora crassa defective in regulation of blue light perception.

A new selection system was used to isolate mutants deficient in regulation of blue light perception in Neurospora crassa. This selection system has two possible applications and was used for the isolation of either blind or constitutive mutants. We isolated 17 UV-induced mutants that showed the pleiotropic white collar (wc) phenotype and were completely blocked in transduction of the light signal. From the segregation pattern in sexual crosses, we tentatively assigned the 17 mutants to either the wc-1 or wc-2 gene. Furthermore, two Neurospora mutants, ccb-1 and ccb-2, were isolated that showed constitutive carotenoid biosynthesis in the dark. Analysis of these mutants for transcripts of the carotenoid biosynthesis genes al-3 and al-1 revealed no higher steady-state levels in the dark than in the control strain. The mutant cch-2 showed major differences in mRNA levels only for conidiation and developmental genes. The lack of a specific change in mRNA levels in response to light, and the mutant phenotype, which seems to reflect a step in conidiation, indicate a role for the cch-2 product in a developmental process, such as conidiation. In contrast, the mutant cch-1 showed a threefold overinduction of carotenoid biosynthesis genes in response to light. This effect was not observed for the conidiation genes examined. The recessive nature of the ccb-1 mutation, together with its specific effect on light induction of the carotenoid biosynthesis genes, indicate that the gene product of ccb-1 acts as a repressor of transcription in some light-regulated processes, but not others.

Carotenoids↗
PubMed1997

White collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa.

A saturating genetic dissection of 'blind' mutants in Neurospora crassa has identified a total of two non-redundant loci (wc-1 and wc-2) each of which is required for blue-light perception/signal transduction. Previously, we demonstrated that WC1 is a putative zinc finger transcription factor able to bind specifically to a light-regulated promoter. Here, we present the cloning and characterization of the wc-2 gene. We demonstrate using mutation analysis and in vitro DNA-binding assays that WC2, the second partner of this light signal transduction system, encodes a functional zinc finger DNA-binding protein with putative PAS dimerization and transcription activation domains. This molecular genetic dissection of the second of two components of this light signal transduction system has enabled us to devise a model whereby WC1 and WC2 are proposed to interact via homologous PAS domains, bind to promoters of light-regulated genes and activate transcription. As such, this study provides the first insight into two co-operating partners in blue-light signal transduction in any organism and describes the molecular tools with which to dissect this enigmatic process.

Amino Acid Sequence↗
PubMed1997

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.

Color↗
PubMed1997

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.

Circadian Rhythm↗
PubMed1997

Homology modeling of Neurospora crassa geranylgeranyl pyrophosphate synthase: structural interpretation of mutant phenotypes.

A model of the tertiary structure of the Neurospora crassa carotenogenic prenyltransferase, geranylgeranyl pyrophosphate synthase (GGPPS), is presented, based on structural homology with other prenyltransferases and on the crystal structure of recombinant avian farnesyl pyrophosphate synthase (FPPS). The conserved aspartate-rich motifs DDxx(xx)D and associated basic residues, considered to be the active sites for binding and catalysis in all prenyltransferases, are highly conserved in the N. crassa GGPPS protein, while other regions display a lower degree of sequence homology; thus the GGPPS model structure is predicted to be highly reliable in the active site region. A number of carotene-deficient mutants have been generated utilizing the repeat-induced point mutation (RIP) mechanism: mutant al-3RIP1 carries a Ser-to-Asn mutation in position 336 which falls within the predicted active site of the enzyme. Analysis of the model structure of this mutant indicates that Ser336 may be involved in substrate uptake. Two other mutants, al-3RIP3 and al-3RIP6, carry mutations in positions in the GGPPS protein, homologous to regions of the avian FPPS enzyme proposed to be involved in enzyme dimerization and substrate uptake, respectively, suggesting an explanation for the reduced carotene content of these mutants.

Alkyl and Aryl Transferases↗
PubMed1996

Mild RIP-an alternative method for in vivo mutagenesis of the albino-3 gene in Neurospora crassa.

We have used a biological phenomenon that occurs in Neurospora crassa, termed Repeat-Induced Point mutation (RIP), to create partially functional mutant alleles of the albino-3 (al-3) gene encoding geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids and diverse prenylated compounds. A total of 70 RIP-induced al-3 mutants were identified by their pale albino phenotype, resulting from inactivation of carotenoid bio-synthesis. Nucleotide sequence analysis of the al-3 gene in five of the RIP-induced mutants revealed that in each case RIP had introduced no more than six point mutations. The low frequency of RIP mutants (0.42%) and the isolation of only leaky mutants with very few mutations suggest that ascospores containing a heavily mutated al-3 gene do not survive. These results are evidence that the RIP phenomenon, used to inactivate and silence duplicated genes in N. crassa, may be exploited in its mild version as a method of sequence-specific in vivo mutagenesis to obtain functional mutant alleles of Neurospora genes. This mild form of mutagenesis may be particularly advantageous in selecting for leaky mutations in essential Neurospora genes.

Alkyl and Aryl Transferases↗
PubMed1996

Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation.

The molecular mechanisms involved in transgene-induced gene silencing ('quelling') in Neurospora crassa were investigated using the carotenoid biosynthetic gene albino-1 (al-1) as a visual marker. Deletion derivatives of the al-1 gene showed that a transgene must contain at least approximately 132 bp of sequences homologous to the transcribed region of the native gene in order to induce quelling. Transgenes containing only al-1 promoter sequences do not cause quelling. Specific sequences are not required for gene silencing, as different regions of the al-1 gene produced quelling. A mutant defective in cytosine methylation (dim-2) exhibited normal frequencies and degrees of silencing, indicating that cytosine methylation is not responsible for quelling, despite the fact that methylation of transgene sequences frequently is correlated with silencing. Silencing was shown to be a dominant trait, operative in heterokaryotic strains containing a mixture of transgenic and non-transgenic nuclei. This result indicates that a diffusable, trans-acting molecule is involved in quelling. A transgene-derived, sense RNA was detected in quelled strains and was found to be absent in their revertants. These data are consistent with a model in which an RNA-DNA or RNA-RNA interaction is involved in transgene-induced gene silencing in Neurospora.

Base Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

The isoprenoid pathway: cloning and characterization of fungal FPPS genes.

Farnesylpyrophosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis. Several classes of essential metabolites, including sterols, quinones, carotenoids and gibberellins, are terpenoids with high biological activity. The structural gene for FPP synthase was isolated from two ascomycete fungi, Neurospora crassa and Gibberella fujikuroi. A comparative analysis of the nucleotide sequences of both FPPS genes revealed the presence of introns at the same positions at the 5' end of the coding regions. Furthermore, the most conserved region of the gene was isolated from two other plant pathogenic fungi, Sphaceloma manihoticola and Claviceps purpurea, by PCR. Sequence analysis showed a high degree of similarity between the deduced proteins of all known FPP synthase genes. In contrast to animals, all analyzed fungi contain a single copy of the gene, although FPP is the precursor for essential sterol and quinone biosynthesis and secondary metabolites, such as gibberellins, as well. Transcription analysis in different light regimes has shown that the FPPS genes in G. fujikuroi and N. crassa are not regulated by light induction.

Alkyl and Aryl Transferases↗
PubMed1995

A chimeric light-regulated amino acid transport system allows the isolation of blue light regulator (blr) mutants of Neurospora crassa.

We have developed a system for the isolation of Neurospora crassa mutants that shows altered responses to blue light. To this end we have used the light-regulated promoter of the albino-3 gene fused to the neutral amino acid permease gene mtr. The product of the mtr gene is required for the uptake of neutral aliphatic and aromatic amino acids, as well as toxic analogs such as p-flurophenylalanine or 4-methyltryptophan. mtr trp-2-carrying cells were transformed with the al-3 promoter-mtr wild-type gene (al-3p-mtr+) to obtain a strain with a light-regulated tryptophan uptake. This strain is sensitive to p-fluorophenylalanine when grown under illumination and resistant when grown in the dark. UV mutagenesis of the al-3p-mtr(+)-carrying strain allowed us to isolate two mutant strains, BLR-1 and BLR-2 (blue light regulator), that are light-resistant to p-fluorophenylalanine and have lost the ability to grow on tryptophan. These two strains have a pale-orange phenotype and show down-regulation of all the photoregulated genes tested (al-3, al-1, con-8, and con-10). Mutations in the BLR strains are not allelic with white collar 1 or white collar 2, regulatory genes that are also involved in the response to blue light.

Amino Acid Transport Systems↗
PubMed1995

Light induction of the clock-controlled gene ccg-1 is not transduced through the circadian clock in Neurospora crassa.

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungus Neurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genes ccg-1 and ccg-2. In this paper we report studies on the regulation of ccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes in Neurospora, we report that ccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression of ccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction of ccg-1 requires the products of the Neurospora wc-1 and wc-2 genes, but surprisingly the requirement for wc-2 is suppressed in conditional mutants of cot-1, a gene that encodes, a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression of ccg-1.

Blotting, Northern↗
PubMed1995

Light and development regulate the expression of the albino-3 gene in Neurospora crassa.

The albino-3 gene of Neurospora crassa codes for geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids. The albino-3 locus encodes two overlapping transcription units that give rise to two mRNAs of 2.2 kb (al-3(c)) and 1>6 kb (al-3(m)), with the promoter of the latter residing in the transcribed region of the former. The 1.6-kb transcript was transiently expressed in the mycelium after light induction, while the 2.2-kb mRNA appeared in conidiating cultures only, independently of light. Al-3(c) mRNA was enhanced in the conidiophores and was impaired in mutants blocked at the early stages of conidiation; al-3(c) therefore represents a conidiation-specific transcript. The al-3(c) mRNA level increased upon illumination with blue light and fluctuated according to the circadian cycle.

Base Sequence↗
PubMed1995

Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide.

Purification of the glutamate synthase (GOGAT) enzyme from Saccharomyces cerevisiae showed that it is an oligomeric enzyme composed of three identical 199-kDa subunits. The GOGAT structural gene was isolated by screening a yeast genomic library with a yeast PCR probe. This probe was obtained by amplification with degenerate oligonucleotides designed from conserved regions of known GOGAT genes. The derived amino-terminal sequence of the GOGAT gene was confirmed by direct amino-terminal sequence analysis of the purified protein of 199 kDa. Northern (RNA) analysis allowed the identification of an mRNA of about 7 or 8 kb. An internal fragment of the GOGAT gene was used to obtain null GOGAT mutants completely devoid of GOGAT activity. The results show that S. cerevisiae has a single NADH-GOGAT enzyme, consisting of three 199-kDa monomers, that differs from the one found in prokaryotic microorganisms but is similar to those found in other eukaryotic organisms such as alfalfa.

Amino Acid Sequence↗
PubMed1994

Internal translational initiation in the mRNA from the Neurospora crassa albino-3 gene.

The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA.

Alkyl and Aryl Transferases↗
PubMed1994

Suppression of gene expression by homologous transgenes.

When a wild-type strain of Neurospora crassa is transformed with different portions of the carotenogenic albino 1 or albino 3 genes, up to 30-35% of the transformants show an albino phenotype. The albino transformants presented a variety of phenotypes ranging from white or yellow to dark yellow colour. The ectopically integrated sequences provoke a severe impairment of the expression of the endogenous al-1 or al-3 genes. This phenomenon, that has been termed quelling, is found to be spontaneously and progressively reversible. In fact, all of the albino transformants have an unstable phenotype and revert progressively to wild type or intermediate phenotypes over a prolonged culturing time. The phenotypic reversion is characterised by a progressive release of the transcriptional inhibition and seems to correlate with the reduction of the number of the ectopic integrated sequences. However, there is no strict correlation between the copy number of the ectopic sequences and the intensity of quelling, as indicated by the existence of albino transformants containing only 1-2 ectopic sequences. The nature of the molecular events determining the onset of quelling is unclear, in any event, these are likely to involve some kind of interaction between the resident genes and ectopically integrated exogenous sequences. Recent evidences on a possible mechanism are presented.

Carotenoids↗
PubMed1994

Molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino-3 gene of Neurospora crassa.

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light, principally through transcriptional activation of some key genes in the carotenogenic enzymatic pathway. Here we report the characterization of the photoinducible promoter of the albino-3 (al-3) gene, encoding GGPP synthase. We have modified the 5' non-coding sequence of the cloned al-3 gene by deletion and site-directed mutagenesis, and we have tested the residual photoinducibility of the different constructs by transformation and subsequent analysis of gene expression in dark-grown and light-induced mycelia. The results indicate that a promoter region between positions -226 and -55 contains all the necessary information for blue light photoinduction. Multiple regulatory elements are involved in the regulated expression of the al-3 gene. One (termed the APE element) is important for the light-induction switch on of the gene and a second belongs to the CCAAT boxes family. The putative APE element is also found in the promoter of other N. crassa photoinducible genes.

Alkyl and Aryl Transferases↗