White collar 2,a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa
1997; Springer Nature; Volume: 16; Issue: 1 Linguagem: Inglês
10.1093/emboj/16.1.98
ISSN1460-2075
Autores Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle1 January 1997free access White collar 2, a partner in blue-light signal transduction, controlling expression of light–regulated genes in Neurospora crassa H. Linden H. Linden Dipartimento di Biopatologia Umana, Sezione Biologia Cellulare, Università di Roma La Sapienza, Viale Regina Elena, 324, 00161 Roma, Italy Search for more papers by this author G. Macino Corresponding Author G. Macino Dipartimento di Biopatologia Umana, Sezione Biologia Cellulare, Università di Roma La Sapienza, Viale Regina Elena, 324, 00161 Roma, Italy Search for more papers by this author H. Linden H. Linden Dipartimento di Biopatologia Umana, Sezione Biologia Cellulare, Università di Roma La Sapienza, Viale Regina Elena, 324, 00161 Roma, Italy Search for more papers by this author G. Macino Corresponding Author G. Macino Dipartimento di Biopatologia Umana, Sezione Biologia Cellulare, Università di Roma La Sapienza, Viale Regina Elena, 324, 00161 Roma, Italy Search for more papers by this author Author Information H. Linden1 and G. Macino 1 1Dipartimento di Biopatologia Umana, Sezione Biologia Cellulare, Università di Roma La Sapienza, Viale Regina Elena, 324, 00161 Roma, Italy The EMBO Journal (1997)16:98-109https://doi.org/10.1093/emboj/16.1.98 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The capacity of sensing and responding to environmental light signals is widespread throughout the biological kingdom. In higher plants, there are at least three different families of photoreceptors, the phytochromes (red and far-red light absorption), blue-light receptor(s) and ultraviolet receptor(s) (Deng, 1994). Although the best studied signalling pathway in plants involves phytochrome, considerable research has been carried out in the past decades in order to unravel the blue-light perception and signal transduction pathway (Kaufman, 1993; Short and Briggs, 1994). Research on blue-light signal transduction in plants has been complicated by the fact that phytochrome absorbs blue and UV light to a certain degree in addition to red/far-red light. Furthermore, the light signalling pathways of higher plants appear to be extremely complex. They include general signal transduction components such as G proteins, cGMP and Ca2+ and seem to be regulated by cross-talk and feedback regulation (Kaufmann, 1993; Bowler and Chua, 1994). Only recently has the first candidate for a blue-light photoreceptor in plants been cloned (CRY1; Ahmad and Cashmore, 1993). The CRY1 protein reveals a close homology to bacterial DNA photolyase and was also shown to bind flavin, although its mode of action and interacting partners are as yet unknown (Lin et al., 1995). Although putative mutants affected in blue-light signal transduction events have been identified in plants, nothing is yet known about the genes coding for the protein components of the blue-light signalling system (Liscum and Hangarter, 1994). Furthermore, numerous genes coding for DNA-binding proteins have been cloned in plants, but none of these proteins has been assigned unequivocally a function in light-induced transcription (Terzaghi and Cashmore, 1995). The fungus Neurospora crassa remains a paradigm for molecular genetic studies on blue-light signal transduction. In contrast to plants, in Neurospora only blue light is perceived and has been shown genetically and physiologically to regulate many different developmental processes, e.g. mycelial carotenogenesis (Harding and Turner, 1981), formation of conidia (Lauter and Russo, 1991) and phototropism of peritecial beaks (Harding and Melles, 1984). Blue-light-regulated transcription has also been observed for many genes in Neurospora such as the carotenoid biosynthesis genes al-1, al-2 and al-3 (Nelson et al., 1989; Schmidhauser et al., 1990, 1994), the conidiation genes con-5 and con-10(Lauter and Russo, 1991), the circadian clock gene frequency and the clock-controlled genes ccg-1 and ccg-2 (Loros, 1995). Several classes of Neurospora mutants have been isolated and characterized which appear to be affected in blue-light signal transduction (Harding and Shropshire, 1980; Paietta and Sargent, 1981, 1983; Carattoli et al., 1995). In the two riboflavin mutants rib-1 and rib-2, flavin deficiency is correlated with a reduced sensitivity to blue light, implicating a role for flavin in light perception (Paietta and Sargent, 1981). By contrast, the wc-1 and wc-2 mutants are completely blind to blue light based on their dark-grown phenotypes observed even after light induction (Harding and Turner, 1981). In spite of a saturating screen, no additional wc loci other than wc-1 and wc-2 could be isolated, suggesting that these are the two central and non-redundant components of blue-light signal transduction in N.crassa(Degli-Innocenti and Russo, 1984; Linden et al., 1997). The white collar mutants each have pigmented conidia whereas the mycelia are white due to a specific deficiency in light-induced carotenoid synthesis in mycelia. In addition, most blue-light-regulated genes are unable to respond to light in a wc-1 or wc-2 mutant background. As the wc-1 and wc-2 mutants are defective only in blue-light-induced processes but not generally affected in their growth behaviour or in the expression of non-light-regulated genes, their gene products are proposed to be dedicated to blue-light signal transduction. Furthermore, it has been suggested that the wc mutants correspond to two components of a common signal transduction pathway (Ballario et al., 1996). Using a molecular genetic approach, we have begun to examine the role of WC1 and WC2 in blue-light signal transduction of N.crassa. The wc-1gene has been cloned and characterized to encode a putative transcription factor containing a zinc finger DNA-binding domain and a putative transcription activation domain (Ballario et al., 1996). As the WC1 fusion protein was shown to bind in vitro to the promoter of blue-light-regulated genes, it is proposed that WC1 is a transcription factor that affects the induction of light-regulated genes. Here, we present the isolation and characterization of the wc-2gene coding for the second central regulator of blue-light responses. The wc-2 gene represents the first example of a second component of a blue-light signal transduction pathway that has been characterized in any organism. The wc-2 gene, which was isolated by use of an integrational mutagenesis approach, encodes characteristic features of transcription factors such as a putative zinc finger domain, putative transcription activation domains and a putative dimerization domain. Therefore, both WC proteins are putative transcription factors specifically involved in blue-light regulation. Due to a novel PAS dimerization domain present in both WC polypeptides, an interaction of WC1 and WC2 is proposed. A model is put forward in which a light-induced heterodimerization of WC1 and WC2 results in binding and transcriptional activation of light-induced genes. Results Isolation of a wc-2 mutant by integrational mutagenesis A selection system was used to isolate regulatory mutants which are either hampered or completely blocked in the transduction pathway of blue-light perception in N.crassa(Carattoli et al., 1995). In this selection system, the light-induced al-3 promoter was fused to the coding region of the mtr gene. The mtr protein is responsible for the uptake of neutral, aliphatic and aromatic amino acids and their toxic analogues in Neurospora (Stuart et al., 1988). After transformation of an mtr−/trp− strain with this construct, the resulting strain 13-1 grows on a medium supplemented with the toxic amino acid analogue p-fluoro-phenylalanine (FPA) only in darkness as the al-3::mtr gene construct is not expressed. In the light, however, the al-3::mtr promoter is induced, causing mtrexpression and the uptake of the drug which inhibits cell growth (Linden et al., 1997). Therefore, only mutants impaired in blue-light perception or signal transduction will grow in the light in the presence of FPA. This selection system was applied successfully to the isolation of wc-1 and wc-2 mutants after UV mutagenesis. Thus, this selection system was also used on insertion mutants to identify tagged wc-2 mutants and to isolate the gene. In N.crassa, transformation occurs by random non-homologous integration of the transforming DNA into the genome (Paietta and Marzluf, 1985). Therefore, a large pool of independent N.crassa transformants should include mutants with the foreign DNA integrated into a specific target gene. The powerful al-3::mtrscreening system was used to identify the wc-2 mutant alleles in the DNA-tagged N.crassa lines, allowing cloning of the wc-2 gene. Strain 13-1 was transformed with plasmid pES200 carrying markers selectable in both Escherichia coli (ampicillin) and Neurospora (hygromycin). The transformed sphaeroplasts were grown on solid medium supplemented with hygromycin to allow the formation of homocaryotic conidia. After 8 days' growth, the conidia of transformants were harvested and grown in the presence of FPA to select for light-impaired mutants. Out of ∼2×105 independent transformants tested, one mutant (T13) was isolated which showed a white collar phenotype and was resistant to hygromycin. In genetic crossing experiments, it was shown that the tagged T13 mutant does not recombine with the authentic wc-2 mutants (alleles ER33 and 234w) which identifies it as a newly created wc-2 mutant. In backcrosses to Neurospora wild-type, the wc-2 phenotype of T13 co-segregated with the hygromycin resistance conferred by the transforming DNA. It was concluded, therefore, that the insertion of plasmid pES200 had occurred either in or close to the wc-2 gene. As previously observed, wc-2 mutants are impaired in blue-light induction not only of the carotenoid biosynthesis genes al-1, al-2 and al-3 (Nelson et al., 1989; Schmidhauser et al., 1990, 1994) but also in the induction of other blue-light-regulated genes such as the conidiation genes con-10 and con-8(Lauter and Russo, 1991). To confirm that the T13 allele of wc-2 was also similarly impaired, the steady-state levels of al-1, al-3 and con-10RNA were examined in the T13 mutant grown in darkness and also following light induction (Figure 1). In contrast to the control strains wild-type and 13-1, none of the above genes were inducible by light in the T13 mutant (Figure 1). The region flanking the integrated plasmid was amplified by inverse PCR using oligonucleotide primers complementary to the plasmid sequence and a 0.6 kb fragment containing only Neurospora DNA was cloned (indicated in Figure 2). This DNA fragment, presumed to contain a portion of the wc-2 gene, was used subsequently as a probe in a Southern blot of genomic DNA from the wild-type, strains 13-1 and T13. The results confirmed that the fragment is in fact Neurospora DNA flanking the integrated plasmid in the wc-2 mutant, T13 (data not shown). Figure 1.DNA-tagged wc-2 mutant T13 shows a specific defect in light-regulated gene expression. Northern blot analysis of wild-type, strain 13-1 and wc-2 mutant T13. Mycelia were isolated after growth in the dark (D) and after a light induction of 20 min (L). For hybridization, specific probes of al-1, al-3 and con-10 were used. For normalization, the filter was hybridized with sod-1. Download figure Download PowerPoint Figure 2.The wc-2gene structure. Restriction sites and length of the genomic DNA sequenced are shown in kb. The black box indicates the predicted WC2 protein composed of 530 amino acids interrupted by the two introns. The horizontal arrow above shows the direction of transcription and the putative wc-2 transcript of ∼4 kb. The integration of pES200 in mutant T13 and the deletion are indicated by vertical arrows (DEL). The ClaI–SalI flanking region which was used for the isolation of the wc-2 genomic clones is shown in bold. Download figure Download PowerPoint Isolation of genomic DNA clones, subcloning of the wc-2 gene and complementation of the wc-2 mutant phenotype The cloned DNA insert proposed to contain a portion of the wc-2 gene was used as a hybridization probe for the screening of a Neurospora genomic DNA cosmid library, and four cosmid clones were isolated. Transformation of the wc-2 mutant alleles ER33, 234w and T13 with each of these four cosmids resulted in the complementation of the wcphenotype in each mutant. Using the same flanking DNA fragment, a 4.5 kb PstI–PstI fragment of Neurospora DNA was subcloned (Figure 2). This was used as a probe for subcloning of several restriction fragments from one of the cosmid clones. The subclones subsequently were used in complementation experiments. After transformation of a wc-2 mutant with plasmid pCBWC2A containing a SmaI–SmaI 6.75 kb restriction fragment (Figure 2), complementation of the wc-2 mutant phenotype was observed. Northern blot analysis indicated that the transformed wc-2 mutants had regained the capacity of blue-light-induced transcriptional activation (data not shown). Thus, it was concluded by complementation that a functional wc-2 gene was present on this plasmid. Homology-induced gene inactivation independently confirms the identity of the wc-2 gene In the above section, we showed that plasmid pCBWC2A contained the wc-2 gene by using functional complementation of a wc-2 mutant. To confirm this designation independently, we showed that the pCBWC2A plasmid could be used to create a wc-2 mutant by homology-induced gene inactivation.In N.crassa, two different phenomena of transgene-induced gene silencing have been described: 'quelling' and 'RIPing' which occur during the vegetative and sexual cycle, respectively (Selker, 1990; Romano and Macino, 1992). Both 'quelling' and 'RIPing' affect not only the exogenous but also the endogenous copy of a duplicated gene, and have, therefore, been used to confirm the identity of cloned genes by the ability of the cloned gene to induce a mutant phenotype (Ballario et al., 1996). Plasmid pCBWC2A was transformed in Neurospora wild-type and ∼30% of all transformants were shown to have a wc phenotype ('quelling'). A similar frequency of 'quelling' was also observed for other non-essential genes such as al-1 (Romano and Macino, 1992). Several of the transformants which had a wild-type phenotype subsequently were backcrossed to wild-type and ∼9% of the progeny revealed a wc phenotype ('RIPing'). Therefore, by both 'RIPing' and 'quelling', the pCBWC2A plasmid was able to induce a wc-2 phenotype by DNA-induced gene inactivation, again indicating that the wc-2 gene is present on this cloned fragment of Neurospora DNA. Nucleotide sequencing and isolation of wc-2 cDNAs The entire region of the SmaI–SmaI fragment which encompasses the putative wc-2 gene and adjacent regions was sequenced (Figure 3).Using the same DNA fragment of pCBWC2A, a Neurospora cDNA library was screened. Approximately 20 positive clones were isolated and examined further by restriction analysis and DNA sequencing. All the isolated cDNAs detected the wc-2 transcript depicted in Figure 2. The largest cDNA clone contained a 3 kb insert truncated at the 5′ end. This cDNA revealed an open reading frame (ORF) of 1590 bp coding for a protein of 530 amino acids. Stop codons in all three reading frames upstream of the first start codon (nt 3218 in Figure 3) indicate that the entire ORF is present on this cDNA. The first ATG start codon is followed by several additional ATG codons in-frame. It is not known at present which of these is the actual start codon used, although the third ATG (nt 3270) was found to be in accordance with the consensus AGXXATGG for eukaryotic initiation sites described by Kozak (1981). Comparison with the genomic DNA sequence resulted in the identification of two introns with 156 (intron 1) and 86 nucleotides (intron 2). Intron 5′-donor and 3′-acceptor sites were identified which corresponded very well with the consensus sequences described for N.crassa (Figure 3, Edelmann and Staben, 1994). Two different polyadenylation sites were found by sequencing of several cDNAs, a situation rather common in Neurospora (Bruchez et al., 1993). In comparison with the coding region, a very long mRNA transcript was detected, suggesting that wc-2 contains a long 5′ non-coding region (Figure 3). Reverse transcriptase PCR was used to identify the approximate length of the 5′ non-coding region. When an oligonucleotide at nt 1929 was used, cDNA synthesis was still observed (results not shown), indicating an unusually long 5′ non-coding region of at least 1290 nucleotides. Long 5′ non-coding regions have been identified in other N.crassa genes coding for DNA-binding proteins such as NIT-2 and CPC-1 and have been implicated in the control of RNA translation or stability (Paluh et al., 1988; Fu and Marzluf, 1990). Figure 3.Nucleotide sequence of the 6.7 kb SmaI–SmaI fragment containing the wc-2 gene and flanking sequences. The translated amino acid sequence of the predicted WC2 protein is shown above the nucleotide sequence. Bold letters indicate the putative zinc finger domain. Underlined amino acids indicate the Thr/Gly, Met/Gly and Ser/Gly repeats. Lower case letters correspond to intron 1 (157 bp) and intron 2 (86 bp). Underlined nucleotides in this region show residues in agreement with intron donor and acceptor sites. The largest cDNA in the 5′ direction found by RT–PCR with different oligonucleotides is indicated by an asterisk and an arrow above the sequence (nt 1928). Use of an oligonucleotide further upstream (indicated by an asterisk, nt 1628) did not result in any cDNA synthesis. The two horizontal arrows beneath the sequence show the region which was used for the overexpression of the GST–WC2 fusion protein in E.coli. (E) As above, the sequence indicates two different polyadenylation sites of the wc-2transcript. Download figure Download PowerPoint The WC2 protein reveals features of a transcription factor and contains a putative PAS dimerization domain The WC2 protein deduced from the nucleotide sequence is composed of 530 amino acids. WC2 has a calculated mol. wt of 56 903 and a statistical pI of 7.4. The major amino acids present are glycine (11.9%), proline (8.3%) and serine (8.3%). A hydropathy plot indicates that WC2 is a soluble protein. An acidic region occurring between amino acids 300 and 350 was also identified. In this region, a helical folding structure is predicted. Another region between amino acids 80 and 120 shows a high percentage of prolines (25%). When compared with other proteins of the SwissProt protein sequence database, no overall homology was found, but several notable features were identified in WC2 subdomains: firstly, from residue 467 onwards, a putative zinc-finger-binding domain with the structure C-X2-C-X18-C-X2-C was found (Figure 3). This domain exhibits a high degree of homology with the zinc finger motifs of transcription factors such as WC1 (Ballario et al., 1996) as well as with AREA from Aspergillus, GAT1 and SRD1 from yeast and NIT2 from N.crassa, all belonging to the group of GATA factors (Figure 4A, Orkin, 1992). Secondly, a putative dimerization region was identified in WC2 (Figure 4B) which showed a homology with PAS (for PER-ARNT-SIM), a dimerization domain present in the period protein (PER) and the single-mindedgene product of Drosophila melanogaster and in both subunits of the mammalian dioxin receptor complex, the aryl hydrocarbon receptor nuclear translocator (ARNT) and the aryl hydrocarbon receptor (AHR) (Huang et al., 1993). This PAS dimerization domain is normally comprised of two direct repeats called PAS A and PAS B. Only one of these subdomains was identified in WC2, which showed a higher similarity to the PAS A repeat in the case of ARNT (39.0%) and AHR (42.9%) and to PAS B in the case of SIM (46.4%) and PER (44.0%). The other blue-light regulatory protein of N.crassa,WC1 (Ballario et al., 1996), also contains a previously unidentified putative PAS domain, which exhibits a similarity of 41% with the WC2 PAS domain (Figure 4B). There are only two residues (indicated in Figure 4 by asterisks) which are identical in all PAS A and PAS B domains characterized so far (Wang et al., 1995). These amino acids are also conserved in the PAS domains of both WC2 and WC1 (Figure 4B). In contrast to WC2, a second putative PAS domain was identified in WC1 (amino acids 395–426, data not shown). However, the similarity to other PAS domains is comparably low, and the residues strictly conserved in all other PAS domains could not be found in this domain. The WC2 protein showed an additional similarity of 45.2% over 60 amino acids in the putative PAS domain (amino acids 179–241) to the photoactive yellow protein (PYP, Ectothiorhodospira halophila; Baca et al., 1994), although the similarity of the latter protein to the PAS domain of the other proteins was minimal (data not shown). In addition to the homology in the PAS domain, the WC2 protein shares a second region of homology with the PER protein of D.melanogaster. This domain was found close to the WC2 N-terminus consisting of Thr/Gly, Met/Gly and Ser/Gly repeats (Figure 3; Jackson et al., 1986). Such a domain was also described in the other circadian clock regulatory protein FRQ from Neurospora, although the significance of this domain is not known (McClung et al., 1989). Figure 4.Multiple alignment of different WC2 protein domains with other polypeptides from the SwissProt protein sequence database. (A) Comparison of the putative zinc finger domain of WC2 (amino acids 468–495) with the zinc finger motifs of AREA (Aspergillus nidulans, EMBL, x52491), GAT6 (Rattus norvegicus, EMBL l22760), GLN3 (Saccharomyces cerevisiae, EMBL m35267), NIT2 (N.crassa, EMBL m33956), GAT1 (S.cerevisiae, EMBL u27344), WC1 (N.crassa, EMBL x94300), NTL1 (Nicotiana tabacum, EMBL x73111) and SRD1 (S.cerevisiae, EMBL X063322). Major identities are boxed. An asterisk indicates the mutated amino acid of the wc-2 allele ER33. (B) Multiple alignment of the putative WC2 PAS domain with the PAS A domain of ARNT and AHR, the PAS B domain of SIM and PER and the putative PAS domain of WC1. References are given in the text. Similar residues between the different proteins and WC2 are boxed. The only two conserved residues in all PAS A and PAS B domains are indicated by asterisks. A hyphen indicates a gap introduced to maximize alignment. The numbers of the first amino acid of each sequence are given in parentheses on the left. Download figure Download PowerPoint Binding of WC2 to the al-3 promoter region involved in blue-light regulation The features of WC2 suggest a role for the protein in transcriptional activation. Using a GST–WC2 fusion protein which contained the putative zinc-finger-binding domain and the PAS dimerization motif (as indicated in Figure 3), in vitro DNA-binding experiments were carried out. An al-3 promoter fragment was used as a putative target gene for WC2 binding for the following reasons: Carattoli et al. (1994) identified a promoter region in the al-3 gene which contained all necessary regulatory elements for blue-light gene induction. This fragment contained two canonical GATA sequences and has been applied successfully in DNA-binding experiments with a WC1 fusion protein (Ballario et al., 1996). Furthermore, in wc-2 mutants, no light induction of the al-3 mRNA is observed. In electrophoretic mobility shift assay (EMSA), Escherichia coli lysates from cells expressing either the GST–WC2 fusion protein or GST only were incubated with the labelled al-3 promoter fragment (Figure 5). A band shift was obtained only when the WC2 fusion protein was present (Figure 5A, lane 2) but not when a control GST extract was used (Figure 5A, lane 1). The amount of labelled al-3 promoter fragment shifted by the GST–WC2 fusion protein was comparatively low. This could be due to incorrect folding of the GST–WC2 fusion protein. To prove that binding of WC2 to the al-3 promoter fragment was sequence specific, different unlabelled competitor DNAs were used. When increasing amounts of an unlabelled 41mer covering the same region of the al-3 promoter were added to the reaction, binding of WC2 to al-3 was competed (Figure 5A, lanes 3–6). By contrast, when mutated GATA motifs were used as a competitor, competition was observed only at higher concentrations tested (25-fold excess of competitor, Figure 5A, lanes 7–10). Addition of an unrelated oligonucleotide revealed no competition at any concentration (Figure 5B, lanes 3–6). When the reaction was performed in the presence of 50 mM EDTA, the binding of the GST–WC2 fusion protein was abolished, indicating that divalent cations such as zinc are required for the binding of WC2 to al-3 DNA (Figure 5C, lanes 1 and 2). After incubation of equimolar amounts of both WC2 and WC1 fusion proteins with the 32P-labelled al-3 promoter fragment, no additional gel-shifted bands were observed when compared with bandshifts with WC1 and WC2 incubated separately (data not shown). Figure 5.Binding of an al-3 promoter fragment containing two GATA elements by a GST–WC2 fusion protein. In an electrophoretic mobility shift experiment, 1 ng of 32P-labelled promoter fragment (78 bp) was incubated with 5 μg of either GST (A, lane 1) or GST–WC2 protein extract (A, lane 2); lanes 3–6 are the same as lane 2, but with increasing amounts of an al-3 41mer oligonucleotide as an unlabelled competitor (0.5, 2.5, 5.0 and 25.0 ng); lanes 7–10 are the same as lane 2, but with an unlabelled al-3 oligonucleotide with mutated GATA motifs. (B) Lanes 1 and 2 are the same as lanes 1 and 2 in (A), lanes 3–6 are the same as lane 2, but with an unlabelled, unrelated oligonucleotide as competitor (0.5, 2.5, 5.0 and 25.0 ng). (C) Lane 1 is the same as lane 1 in (A) and lane 2 the same as lane 1 but in the presence of 50 mM EDTA. Download figure Download PowerPoint Nucleotide sequence analysis of wc-2 mutants reveals that the zinc finger motif is required for WC2 function in vivo In order to confirm the identity of the isolated wc-2 gene and to obtain information about important functional domains in the WC2 protein, the wc-2 coding regions of three different wc-2 mutants alleles were sequenced and compared with the wild-type wc-2 sequence (Table I). Several mutations were identified and confirmed by sequencing in both directions of at least two independent PCR reactions. The wc-2 mutant ER33 obtained from the FGSC showed two independent mutations. One was a nucleotide exchange in intron 2 of the wc-2 gene (Table I). RT–PCR experiments showed that this mutation did not result in a splicing defect and is, therefore, a neutral mutation. The second mutation in ER33 resulted in the conversion of a conserved glycine to a glutamic acid inside the putative zinc finger domain (Table I, Figure 4A), presumably disrupting this functional domain. Mutant 234w also revealed two mutations; one was a neutral mutation which led to an amino acid exchange close to the amino-terminus of WC2. The other mutation of 234w resulted in a truncated protein of 356 amino acids in which the DNA-binding domain of wc-2 is absent (Table I). The DNA-tagged wc-2 mutant T13 also showed a disruption of the zinc-finger-binding domain of WC2 by the integration of the plasmid DNA. It can be presumed, therefore, that the observed wc-2 phenotype is, at least in some cases, due to the absence or non-function of the putative zinc finger DNA-binding domain. Table 1. Mutations in the wc-2 gene of different wc mutants Neurosporawc-2 mutant strains Mutation in the nucleotide sequence Alteration of the wc-2 amino acid sequence Allele ER33 t4808 →a none G4915→A Gly485→Glu Allele 234W G3258→A Gly14→Ser Del T4440 change in rf and stop after Pro356 Allele T13 integration of pES200 at C4892 and deletion of 1619 nt downstream Del of aa sequence after Pro477 The wc-2 mRNA steady-state levels are light regulated and this light regulation occurs also in a wc-1 and wc-2 mutant background Using RNA from Neurospora wild-type and different wc mutants, the expression pattern of the wc-2 gene was examined (Figure 6). A wc-2 transcript of ∼4 kb was detected in the wild-type which showed a small but significant increase in response to light (Figure 6). Mutant T13 revealed a somewhat smaller wc-2 transcript, which was due to the integration of pl
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