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A UV-induced mutation in neurospora that affects translational regulation in response to arginine

The Neurospora crassa arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. The levels of arg-2 mRNA and mRNA translation are negatively regulated by arginine. An upstream open reading frame (uORF) in the
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  Copyright zyxwvutsrqpo   996 zyxwvutsrqpon y the Genetics Society of America A UV-Induced Mutation zyx n Neurospora That Affects Translational Regulation in Response to Arginine Michael Freitag, Nelima Dighde and Matthew zyx . Sachs Department zyxwvutsrqpo f Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science zyx   Technology, Portland, Oregon 97291-1000 Manuscript received August 7, 1995 Accepted for publication October 3, 1995 ABSTRACT The Neurospora crmsu arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. The levels of arg-2 mRNA and mRNA translation are negatively regulated by arginine. zy n upstream open reading frame (uORF) in the transcript’s 5’ region has been implicated in arginine- specific control. zyxwvuts n arg-2-hph fusion gene encoding hygromycin phosphotransferase conferred arginine- regulated resistance to hygromycin when introduced into N. crassa. We used an arg-2-hph strain to select for UV-induced mutants that grew in the presence of hygromycin and arginine, and we isolated 46 mutants that had either of two phenotypes. One phenotype indicated altered expression of both arg-2- hph and urg-2 genes; the other, altered xpression of urg-2-hph but not arg-2. One of the latter mutations, which was genetically closely linked to arg-2-hph, was recovered from the 5’ region of the arg-2-hph gene using PCR. Sequence analyses and transformation experiments revealed a mutation at uORF codon 12 (Asp to Asn) that abrogated negative regulation. Examination of the distribution of ribosomes on arg- 2-hph transcripts showed that loss of regulation had a translational component, indicating the uORF sequence was important for Arg-specific translational control. Comparisons with other uORFS suggest common elements in translational control mechanisms. C RBAMOYL phosphate is a key intermediate in the biosynthesis of arginine (Arg), yrimidine nucleo- tides and urea. Most prokaryotes contain a single car- bamoyl phosphate synthetase (CPS), whereas eukary- otes, exemplified here by Neurospora zyxwvut rassa, contain two CPSs (HONG t al. 1994). Neurospora CPSA is a two sub- unit enzyme located in the mitochondrial matrix and functions in Arg biosynthesis, whereas CPSP is located in the nucleus and functions in pyrimidine biosynthesis (DAVIS 986). The level of N. crassa CPSA activity is generally the rate-determining component of flux through the Arg pathway (DAVIS nd STOW 1987). CPSA activity is de- termined by the level of the holoenzyme’s small gluta- mine amidotransferase subunit (DAVIS 1986), which is encoded by the nuclear arg-2gene (ORBACH t al. 1990). arg-2 is the only gene encoding an N. massa Arg biosyn- thetic enzyme that is subject to negative regulation by Arg (DAVIS 1986). Arg negatively regulates both levels of arg-2 transcript and arg-2 translation (ORBACH t al. 1990; SACHS nd YANOFSKY 1991 Luo et al. 1995). This phenomenon is referred to as kg-specific regulation. The arg-2 transcript contains a 24codon upstream open eading rame (uORF) (ORBACH t al. 1990) whose sequence is important n Arg-specific transla- tional regulation (LUO et al. 1995; Z. LUO and M. S. Corresponding authoc Matthew S. Sachs, Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science Technology, P.O. Box 91000, Portland, OR 97291-1000. E-mail: SACHS, npublished data). CPAl, the Saccharomyces cere- vzsiae homologue of arg-2, is also subject to Arg-specific regulation (DAVIS 1986). Both transcriptional and post- transcriptional components contribute to CPAl regula- tion (WERNER t al. 1987; CRABEEL t al. 1990). Arg- specific regulation of PA1 also depends on the amino acid coding information of a 25-codon uORF in the CPAl 5’ leader WERNER et al. 1987; DELBECQ t al. 1994). The predicted peptide sequences of the arg-2 and CPAl uORFs are similar. An understanding of uORF functions is important because uORFs are present in the 5‘ regions of many genes involved in growth control (GEBALLE nd MORRIS 1994). The best understood example of uORF-medi- ated eukaryotic translational control is the S. z ereuisiae GCN4 gene’s response to amino acid starvation; ribo- some reinitiation is important for control but specific uORF peptide sequences are not mportant (HINNE- BUSCH 1994). A imilar mechanism may regulate the N. crassa homologue cpc-1 (LUO et al. 1995). Conversely, in the fungal CPSA systems the uORF peptide se- quences, and not ribosome reinitiation, appear to be the important factors (DELBECQ t al. 1994; Z. LUO and M. S. SACHS, npublished data). Previous genetic selection schemes to obtain arg-2 mutants in which Arg-specific regulation was affected used an arg-12 pyr-3 strain. Normally, certain N. crussa pyr-3 mutants that lack CPSP activity (WILLIAMS nd DAVIS 1970) require pyrimidine supplementation in minimal medium. However, the bradytrophic arg-12 Genetics 142: 117-127 (January, 1996)  118 M. Freitag, N. Dighde and M. S. Sachs mutation, when combined with such a zyxwvut 9r-3' allele, per- mits growth on minimal medium zyxwvut DAVIS nd WOOD- WARD 1962). The rg-12 mutation causes the arg-2 gene to be overexpressed (DAVIS 1986), and arg-12' zyxwvut ir-? strains grow on minimal medium because overex- pression of CPS-A provides sufficient carbamoyl phos- phate for both rg and pyrimidine synthesis. Since addi- tion of Arg reduces he expression of arg-2, the arg- zyxwv 2 pyr-? strain acts as a pyrimidine auxotroph on Arg medium. Extensive direct selections in arg-I2 pyr-3 backgrounds for mutations at the arg-2 locus that elimi- nated the uridine requirement in Arg medium failed (DAWS 1986), although alleles of pmb, a gene involved in the import of basic amino acids, were obtained by this scheme (THWAITES 965; THWAITES nd PENDYALA 1969). In contrast, a imilar selection scheme in S. cere- uisiae was used to isolate mutants affecting Arg-specific regulation of CPAl (THURIAUX t al. 1972). Here we describe a novel approach for obtaining mu- tants affecting kg-specific translational regulation in N. crassa. We constructed an N. crassa arg-12 pyr-? strain containing an arg-2-hph reporter gene, in which arg-2 sequences were translationally fused to the Escherichia coli hygromycin B phosphotransferase gene, hph. Hygro- mycin phosphotransferase inactivates the antibiotic hy- gromycin B (Hyg) by phosphate group transfer (RAo et al. 1983) and confers a Hyg' phenotype on trans- formed N. crassa strains (STABEN t al. 1989). The arg- 12 pyr-? arg-2-hph strain grew in medium containing Hyg and uridine, but not Hyg, uridine and Arg. Selec- tion of hygromycin-resistant variants in the presence of Arg yielded two classes of mutants. One of the mutants contained an altered uORF in the arg-2-hph gene and Arg-specific regulation of the fusion gene was abol- ished. MATERIALS AND METHODS zyxwvu Strains and growth of N. crm Wild-type strain 74A-OR23- 1VA was obtained from D. PERKINS, tanford University. arg- 12 pyr-? (DFC-3) zyxwvuts   Jufb A and Jufb a strains were from E. SELKER, niversity of Oregon. The his-3 (1-234723) strain was from the Fungal Genetics Stock Center, University of Kansas Medical School (FGSC #6103). Strains were routinely grown in 10 X 75 mm culture ubes containing solid minimal growth medium (lx Vogel's N, 2% sucrose and 2% agar) (VOGEI. 1956) with supplements as required. The arg-12 pyr-? arg-2- hph transformant and mutants derived from it were grown on the following: minimal medium (Min), minimal medium to which was added 0.5 mg/ml uridine (Uri), 0.5 mg/ml Arg (Arg), both Uri and Arg (Uri zyxwvutsr   Arg), hygromycin B (Hyg (Calbiochem, La Jolla, CA), Hyg Uri, Hyg Arg, and Hyg + Uri Arg. Media containing different concentrations of Hyg were used where indicated. Min was also supplemented with 2 mg/ml thialysine (Sigma, St. Louis, MO) where appro- priate. The his-? auxotroph was grown on Min supplemented with 0.5 mg/ml histidine (His). Procedures described previously (LUO t al. 1995) were used to grow N. crussa in liquid media. In typical experiments, 30 ml of medium in 125 ml Erlenmeyer flasks were inoculated with 2 X IO conidia per ml; cultures were incubated at 34 with orbital shaking (200 rpm) for 6.5 hr and harvested by vacuum filtration onto Whatman #541 filter paper. The mycelial mat was divided and either used immediately in enzyme assays or quickly frozen in liquid nitrogen for analyses of total RNA. Plasmids: Plasmids were maintained in E. coli strain DH5a. Plasmid DNA was isolated by equilibrium centrifugation as described (SAMBROOK t al. 1989). Plasmid pCV4 contained a translational fusion of the 5' regulatory region of the arg-2 gene to the E. coli hph gene. To construct pGV4, pAUl (OK- BACH et al. 1990) was digested with Sty1 and the large 4.4-kb fragment (containing vector sequences and he 5 and 3' regions of arg-2) made blunt by incubation with the Klenow fragment of E. coli DNA polymerase I and dNTPs. The 1.0- kb BamHI hph fragment from pConlO/AKCHyg (D. ERBOI.~: Texas A M University) was also made blunt by this proce- dure and then ligated to the large Sty1 fragment of pAUl to yield pGV4 (Figure 1A). This ligation translationally fused the hph gene to the arg-2 coding region at codon 10 of the pre- dicted CPS-A small subunit open reading frame. Plasmid pMFl 1-wt and pMFl1-D12N contained PCR-ampli- fied, 638-nt EcoRI 5' arg-2-hph fragments from the single copy nrg-12 py~? rg-Z-hph transformant (strain MF13-3) and the arg-12 pyr-3 arg-Bhph (Dl2N) class I mutant (strain MF13-3- 40), described below, inserted at he unique EcoRI site in plasmid pMF2. Plasmid pMF2 was designed to target integra- tion of nrg-2-hph constructs to the N. crassa chromosomal his- 3 locus. A truncated his-3 fragment can be used to transform certain N. crmm hi. -3 mutants to His prototrophy by restoring a functional copy ofthe hi-?gene equence after homologous recombination at the hk-3 locus (SACHS nd EBBOLE 1990). pMF2 (6.7 kb) was constructed by ligating the 2.5-kb EmRL ClaI his-? fragment from pH303 (K. HAGEK, ale University) to the unique EcoRI and QaI sites of pGV4. Plasmids for sequencing were constructed by cloning EroRI- digested products obtained from PCR amplification into the unique EcoRI site of pBS SUI+ (Stratagene, La Jolla, CA). Plasmid tgCMV/HyTK, which contained an in-frame fusion of the E. coli hph gene to the herpes simplex virus type 1 thymidine kinase gene, tk, driven by the human cytomegalovi- rus immediate early promoter I94 (LLIPTON t 11. 1991), was obtained from S. LUPTON, argeted Genetics Co., Seattle, WA. Transformation of E. coli and N. zyx rmsa: Competent E. coli strain DH5a cells were prepared and transformed by electro- poration in an Electro Cell Manipulator 600 using the manu- facturer's protocol (BTX Inc., San Diego, CA). Transformants were selected on LB medium containing 100 pg/ml ampicil- lin (SAMBROOK I al. 1989). N. crassa protoplasts were prepared and transformed as de- scribed (SELITRENNIKOFF nd SACHS 1991). Transformants were selected on minimal medium supplemented with Uri and 0.2 mg/ml Hyg. Plates were incubated for 2-5 days at 34 . Plasmids pMF2, pMFll-wt and pMFll-Dl2N were targeted to the N. crassa his-3 locus (SACHS nd EBBOLK 990). Trans- formants were selected on Min or Min supplemented with 0.2 mg/ml Hyg. Isolation and Southern analyses of genomic N. crmsa DNA Genomic zyx   ram DNA was isolated and Southern analyses were performed as described (OAKLEY t at. 1987; LLIO et nl. 1995). The arg-2 probe was the 1.4kb PvuII-Ncol fragment from pARl (LUO et nl. 1995). The cox-5 probe was the 0.8-kb EcoRI fragment from pSRCOX5 (SA(:HS rt al. 1989). The rpc-1 probe was the 1.2-kb BglII-BamHI fragment from pCPC-1-2 (PALUH et al. 1988). The hph probe was the I-kb BamHI fragment from pGV4. UV mutagenesis: Conidia (1 X 10') of transformant MF13- 3 were suspended in IO ml of water in an uncovered 100-mm glass Petri dish and irradiated using a Stratagene Stratalinker 1800 with 254 nm UV light at 700 or 900 J/m', which yielded  Translational Control in eurospora 119 25 or 10% spore survival, respectively. Then, under dim light to minimize photoreactivation repair, conidia (2 zyxwvut   zyxwvuts o6 UV- mutagenized conidia per 100-mm Petri plate) were spread onto medium containing Hyg Uri + Arg. Colonies were isolated after 2-5 days of incubation at 34 . The growth phe- notypes of putative mutants were analyzed in 10 X 75-mm culture tubes containing Min, Uri, Arg, Uri + Arg, Hyg, Hyg + Uri, Hyg Arg or Hyg + Uri + Arg. Homokaryotic cultures of putative mutants were obtained by serial isolation of single asexual spores (DAVIS nd DE SERRES 970) or by isolation of microconidia (EBBOLE nd SACHS 990). Genetic analyses: Genetic crosses were performed and na- lyzed as previously described (DAVIS nd DE SERRES 970). MFl3-3 zyxwvutsrqpo arg-12 zyxwvutsrqp yr-3 arg-2-hph a) or UV-induced mutants de- rived from this strain were backcrossed to wild type. Asco- spores were heat-shocked in water for 45 min at 62 and spread onto X Vogel's N medium containing .05% fructose, 0.05% glucose and 1% sorbose (FGS) supplemented with Uri, or FGS supplemented with Uri + Arg. Spread ascospores were incubated for 8-12 hr at 34 , and single germinated spores were picked to small culture tubes containing Uri or Uri Arg. Alternatively, 50 or 100 random ungerminated asco- spores per cross were picked to 10 X 75 mm culture tubes containing Uri. The segregation of pyr-3 (LGIVR), arg-12 (LGIIR) and arg- 2-hph (LG unknown) were examined in progeny obtained from wild type X arg-12 pyr-3 arg-2-hph. The arg-12 pyr-? arg- 2-hph a parent (MF13-3) and eight class I mutants were crossed to wild type. The progeny obtained from all of the crosses were tested on Min, Arg, Uri, Uri + Arg, Hyg, Hyg + Uri, Hyg + Arg and Hyg + Uri Arg to score for arg-12, Pyr- zyxwvu   and arg-2-hph. Recovery zyxwvuts f zyxwvutsr he a7 2hph reporter gene by PCR: PCR con- tained either 1,2.5 or ng of plasmid DNA or 50, 100 or 200 ng of N. crussa genomic DNA as template. Primer ARG2-Eco (5 -CGGAATTCTACCAGATCCAATCAA-3 ) as identical to nucleotides 654-674 of the published arg-2 sequence (OR- BACH et al. 1990); an EcoRI site was created by adding a CGG clamp at the 5' end. Primer HPH-Eco (5 GATGCAATAGGT- CAGGCTCTC-3') was complementary to nucleotides 463- 483 of the E. coli hph sequence ( GMTZ nd DAVIES 983). This primer sequence is located near an EcoRI site within hph that was subsequently used for subcloning. In a typical reaction (50 pl), 5 ng plasmid DNA or 100 ng genomic DNA template, 0.5 pM primers, 400 pM of each dNTP, 1 X Vent Polymerase buffer [lo mM KCI, 20 mM Tris- HCI (pH 8.8), 10 mM [NH4]$04, 0.1% Triton X-1001 (New England Biolabs, Beverly, MA) and Vent Polymerase (1 unit) were overlaid with 25 pl of light mineral oil (Aldrich, St. Louis, MO) and cycled 35 times through the following temperature profile: 90 sec at 94 , 30 sec at 50°, 60 sec at 72 . The last extension step was carried out for 5 min. Reactions were cooled on ice, extracted with 60 p1 chloroform and vortexed, and phases were separated by centrifugation for 5 min at 16,000 X g. One-tenth of the reactions were run on an nalyti- cal 0.8% agarose gel. Reactions that yielded discrete bands (662 nt) were directly digested with EcoRI and used for sub cloning following standard procedures (SAMBROOK zyxwv t at. 1989). The arg-2-hph region from plasmid pGV4 or from N. crassa genomic DNA were each amplified in three indepen- dent PCR reactions and subcloned into the unique coRI site of pBS SKII+ or pMF2. DNA sequencing by dideoxynucleotide chain termination: The SequenaseTM Version 2.0 DNA sequencing kit (USB, Cleveland, OH) was used to sequence double-stranded DNA templates using synthetic oligonucleotide primers according to the manufacturer's protocols. Doublestranded DNA tem- plates were purified by equilibrium density gradient centrifu- gation ( SAMBROOK t al. 1989). Hygromycin B phosphotransferase activity assay: Whole cell extracts of N. crussa were prepared (LUO et al. 1995), frozen in liquid nitrogen and stored at 80 . Protein concen- trations were determined by the Bradford assay (BRADFORD 1976) with bovine serum albumin as the standard. The ygro- mycin phosphotransferase activity filter assay using N. crassa cell extracts (FREITAG nd SACHS 1995) was adapted from a previously published protocol (SBRENSON t al. 1992). Serial dilutions of 5, 2.5, 1.25 and 0.63 pg of total protein from whole cell extracts in a 10-pl total volume were assayed for hygromycin phosphotransferase activity in wells of 96well mi- crotiter dishes. Relative amounts of radioactivity in dots on phosphocellulose paper were determined using a Phospori- mager (Molecular Dynamics, Sunnyvale, CA) running IPLab Gel software (Signal Analytics Corporation, Vienna, VA) , Isolation of total RNA, polysomal RNA and Northern analy- ses: Small-scale total RNA samples were prepared by phenol- chloroform extraction of frozen mycelium (LUO et al. 1995). The procedures for polysome gradient preparation and pro- file analyses were essentially as described (LUO et al. 1995), except that heparin was omitted and 12.5 Axe,) nits of homog- enate, in a maximum volume of 400 pl were layered onto gradients. Twelve 1-ml ractions were collected, 1 ml of isopro- panol was added to each, and fractions were stored at -80 until further processing. To extract polysomal RNA, fractions were centrifuged at 4 for 15 min and the supernatant aspi- rated. Pellets were resuspended in 300 pl lysis buffer [20 mM Tris-HC1 (pH 7.4), 100 mM NaCl, 2.5 mM EDTA, 1 sodium dodecyl sulfate in pyrocarbonic acid diethyl ester (DEPC)- treated water] (CICAN t al. 1991) and extracted once in 300 pl of pheno1:chloroform (l:l), followed by one extraction in chloroform. RNA was precipitated twice with NaOAc (pH 5.5) and isopropanol, washed with 70 ethanol, resuspended in 42 pl of sterile DEPGtreated water, frozen in liquid nitrogen and stored at -80 until further analyses by Northern blotting. For the experiments shown in Figure 5, fractions 1 + 2 and 11 + 12 were pooled. Methods for gel electrophoresis of RNA, membrane blot- ting, probe preparation and probe hybridization were as de- scribed (SACHS nd YANOFSKY 991), except that RNA immo- bilized on nylon membranes was visualized by staining with methylene blue (SAMBROOK t al. 1989) and dextran sulfate was omitted from the hybridization solution. Probes were as described for Southern analyses. Northern and polysome analyses were performed orl two independent growth experiments; each experiment was ana- lyzed in duplicate. Only fractions from similarly shaped poly- some profiles were analyzed. The relative levels of mRNA detected by RNA blotting were determined by direct analysis using a phosphorimager running IPLab Gel software. RESULTS Isolation and phenotypic analyses of mutants: Plas- mid pGV4, which contained a translational fusion of the 5 regulatory sequences of N. cram arg-2 to hph (Figure lA), was introduced into the arg-12 fir-3 strain by DNA-mediated transformation, and Hyg' trans- formants were selected. Transformants that showed growth on Hyg + Uri, but not on Hyg + Uri kg, were examined further. Strain MF13-3, which contained a singlecopy ectopic integration of the arg-2-hph fusion gene as determined by Southern blot analysis (Figure lB), was chosen for mutagenesis.  120 zyxwvutsrqpo . Freitag, N. Dighde and M. zyxwv . Sachs A B uORF zyxwvutsr : .._ *<.. .. ., : ..., zyxwvuts  . .. i? .. .. :: '..,'.,'..'; .. Arg2p v zyxwvutsr 4v -i ' . _ --- - 3.8 kb arg-2 .2 kb 1 1 I B Sa P St St St SVN Ps Sa UORF . .. i . . . . . . . HPh zyxwvuts .5 kb hPh .2 kb Ill I I II Sa P SVB E BISVN Ps Sa FIGLIRE .-Structures of the zyxwvutsrq . un.wz nrg-2 and the recombinant nrg-2-hph genes. (A) Partial restriction maps of arg-2 (top) and nrg-2-hj~h bottom). W, the uORF and the nrg-2 coding regions; 0 ntrons; Ed the hph coding region. Introns have been removed from the arg-2-lt/)h gene. * * * delimit the uOKF peptide coding sequences. *, the major nrg-2 transcription start sites; V, redicted translation initiation codons; +, predicted translation termination sites. Restriction enzyme recognition sites are as follows: €3, BnmHI; E, KcoRI; N, NroI; P, RmII; Ps, A/I; Sa, Snd; St, S/yL (R) Southern analyses of the arg-2 and nrg-2-hph genes showed that a single copy of the nrg-2-hph gene is present in the genome of transformant MF133. DNA from wild type 74A), nrg-12 pyr-3 and MF13-3 strains are shown as reference. Genomic DNA 1 pg) was digested with Sad and probed with ' P-labeled nrg-2 or hph DNA as indicated. MF13-3 conidia were UV-irradiated and plated on Hyg Uri + Arg, and 46 independent colonies were isolated. No colonies grew on selective plates noculated with mock-irradiated conidia. Putative mutants and ref- erence comparison strains were grown on eight differ- ent media (Figure 2), and mutants were sorted nto two classes with respect o he new phenotypes they exhibited on Arg-containing media. N. zyxwvutsr anu wild type grew on all media lacking Hyg but, as expected, was sensitive to Hyg at concentrations as low as 0.15 mg/ ml (Figure 2 and data not shown). The nrg-12 fyr-3 strain grew on Min, Uri and Uri + Arg, but not on Arg alone, due to the reduced ctivity of nrg-2 and reduced cross-feeding of carbamoyl phosphate to the pyrimidine pathway in Arg medium. The arg-12 pyr-3 strain was sensitive to Hyg, as expected. The nrg-12' fy-3 rg-2-hph transformant MF13-3 grew on Min, Uri and Uri + Arg, but not on Arg alone, as expected. This transformant also grew on medium containing Hyg or Hyg + Uri, at Hyg concentrations as high as 4 mg/ml but did not grow on medium containing Hyg + Arg or Hyg + Uri + Arg (Figure 2 and data not shown). This was consis- tent with Arg-specific negative regulation of both arg-2 and arg-2-hph (see below). Mutants differed from MF133 with respect to their growth phenotypes. Class I mutants (33 strains) grew on Min, Uri, Uri + Arg, Hyg and Hyg Uri, but not on Arg and Hyg + Arg, as observed for the unmutagenized MFl3-3 parent; but n contrast to MF13-3, class I mutants also grew on Hyg + Uri + Arg (Figure 2), indicating that expression of the q-2-hph gene was altered. Class I mutants (13 strains) grew on all media (Figure 2). I wild type i arg 14 yr 3 arg 2 hph Mutants arg 4 yr 3 MF13-3 Class Class II MIN URI ARG URI ARG HYG HYG URI HYG ARG HYG URI ARG FIGURE .-Growth f wild- type, arg-12 pyr-3 and arg-12 pyr-3 arg-2-hph strains in different me- dia. Minimal medium (Min) was supplemented with Uri, Arg and/ or 2 mg/ml Hyg as indicated. The strains examined were wild type, arg-12 pyr-3 MF133 (the arg-12 pyr-3 arg-2 hph strain used for mutagenesis), the class I mutant MFl3-3-12 and the class I1 mutant MF13.347.  Translational ontrol in Neurospora zyx 121 Thus it appeared that class 1 mutations affected the ex- pression of the zyxwvutsrq rg-2-hph gene, whereas class zyxwv 1 mutations affected the expression of both arg-2-hph and arg-2. Genetic analyses of mutants: The genotypic basis for the mutant phenotypes was examined by comparing progeny from crosses of MF13-3 the nonmutant strain) to wild type with crosses of 0 class 1 and class I1 mutants to wild ype. arg-2-hph segregated as a single genetic locus in a Mendelian manner: in a total of 1059 progeny examined from the 21 Hyg' zyxwvut   Hyg' crosses, Hyg':Hyg progeny segregated 525:534. No linkage of arg-2-hph to arg-12 (LG IIR), zyxwvutsr yr-3 (LG IVR) or mating type (LG IL) was observed in 516 progeny from crosses of the transformed parent or class I mutants to wild type; all loci were unlinked in these crosses (Pfrom zyxwv   > 0.5). The observed percentage of recombination between loci were as follow: pyr-3 - arg-12, 50%; pyr-3 - arg-2- hph, 50%; arg-12 - arg-2-hph, 47%. Since arg-2 and pyr- 3 are separated by three map units on linkage group lVR (PERKINS t al. 1982), the ectopically integrated arg- 2-hph gene was therefore also unlinked to arg-2. The inheritance of genotypes that permitted growth on Hyg + Uri Arg was examined in the progeny of second generation backcrosses to wild type of MF13-3, the class 1 mutant MF13-3-40 and the class I1 mutants MF13-3-37 and MF13-3-39. zyxwvu s expected, none of the 27 Hyg' progeny from the cross of MF13-3 o wild type that grew on Hyg Uri could grow on Hyg Uri + Arg (0/27). In contrast, all of the Hyg' progeny from the cross of the class 1 mutant MF13-3-40 to wild type that grew on Hyg + Uri could also grow on Hyg + Uri + Arg (25/25). Thus the mutation that permitted rowth on Hyg + Uri + Arg was tightly linked to the arg-2-hph gene. Crosses of two class I1 mutants to wild ype re- vealed that half of the progeny that grew on Hyg Uri grew on Hyg + Uri + Arg (14/29 and 13/26). This indicated that these mutations were not linked to the arg-2-hph gene. A premeiotic phenomenon in N, crassa, known as repeat-induced point mutation (RIP), ecognizes dupli- cated sequences and can result in gene inactivation (SELKER 990). The arg-2-hph and arg-2 genes share common sequences. If RIP had recognized the similari- ties between the arg-2-hph and arg-2 genes, mutations might have rendered either of these genes nonfunc- tional. We would have expected a decreased percentage of ascospore germination among the progeny of the crosses of wild type to parent and mutant strains, be- cause of the presence of kg-requiring ascospores among the rogeny of all crosses. This was not observed. In all of the crosses we examined, 85-90% of ascospores tested germinated on Uri and formed colonies after 2- 4 days of incubation at 34 . In crosses of the arg-12 py- 3 arg-2-hph a strain to an arg-12 Pyr-3 A strain, a imilarly high percentage of ascospores formed colonies on both Uri Arg and Min (data not shown). Thus there was no evidence for extensive RIP in the progeny of Hyg' X Hyg' crosses affecting either arg-2-hph or arg-2. Recovery and characterization of the av2-hph re- porter gene: A 662-nt genomic DNA fragment repre- senting he 5' region of the arg-2-hph reporter gene from MF13-3 and the class I mutant MF13-340 were recovered using PCR no discrete fragment was ampli- fied from plasmids or N. crassa strains that lacked arg- 2-hph sequences (Figure 3A and data not shown). The sequences of the subcloned PCR products from the transformant MF13-3 were consistent with the corre- sponding previously published arg-2 (ORBACH t al. 1990) and hph (GRITZ nd DAVIES 983) sequences (Fig- ure 3B and data not shown). In contrast, sequences obtained from the subcloned PCR products of the class I mutant MFl3-3-40 carried a single G to A transition mutation in the uORF coding sequence (Figure 3C and data not shown). This single G to A ransition changes the aspartic acid to an asparagine residue at codon 12 (D12N) of the predicted uORF peptide. Several mutants that exhibited a class 1 phenotype, and that were genetically linked to arg-2-hph, contained no mutations in the 5' regions of the arg-2-hph genes that were recovered by PCR (data not shown). It is possible that mutations in other regions of these genes, for example, in the hygromycin phosphotransferase se- quence, resulted in increased enzyme activity in Hyg + Uri Arg; this remains to be determined. Effect of the D12N mutation on hygromycin phos- photransferase activity: The activity of the product of the arg-2-hph gene, hygromycin phosphotransferase, was assayed in whole cell extracts prepared from strains grown in Uri and Uri + Arg media. As expected, wild- type N. crassa, which lacked the hph gene, produced no detectable enzyme activity, while a strain transformed with gCMV/HyTK, which was Hyg', showed constitu- tive enzyme activity (Figure 4). Hygromycin phospho- transferase activity in MF13-3, which contains the arg-2- hph reporter with the wild-type uORF, was regulated approximately fourfold by Arg; this response was abol- ished in the MFl3-3-40 mutant carrymg the D12N muta- tion (Figure 4). We confirmed that the D12N mutation was sufficient to eliminate Arg-specific regulation by reconstructing arg-2-hph genes using the PCR-amplified, ully e- quenced 5' regions of the wild-type and mutant genes. We analyzed growth phenotypes and measured hygro- mycin phosphotransferase activity in strains that con- tained reconstructed arg-2-hph genes integrated at the his-3 locus. Plasmid pMFll-wt contained an arg-2-hph gene with the wild-type arg-2 5 sequence, and plasmid pMFll-Dl2N was identical except for the single G to A transition mutation at codon 12 of the uORF. Indepen- dent transformants containing these plasmids inte- grated at the chromosomal his-3 locus in single copy were identified by Southern analyses (data not shown). Strains transformed with pMFll-wt grew on Hyg, but
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