Although nearly sixty temperature-sensitive lesions have been mapped in Neurospora crassa, most of their functions have not been identified. These loci are called unknown (un). As part of an effort to identify the open reading frame associated with one of these, we undertook to walk to un-16 using the complementation of temperature-sensitivity as a selection. Cosmids complementing un-16 were identified and the un-16 gene was subcloned. DNA sequence analysis of un-16 revealed that it encodes the highly conserved S9 protein of the 40S ribosomal subunit. This gene has proven useful as a selectable marker and may provide a simple mechanism for the controlled alteration of protein synthesis in N. crassa

Madole, David

McCluskey, Kevin

Plamann, Michael

Walker, Sheera A

Yedlin, Rachel L

English

Kansas State University

Fungal Genetics Reports Volume 54 Article 3 Complementation of un-16 and the development of a selectable marker for transformation of Neurospora crassa Kevin McCluskey University of Missouri- Kansas City Sheera A. Walker University of Missouri- Kansas City Rachel L. Yedlin University of Missouri- Kansas City See next page for additional authors Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation McCluskey, K., S.A. Walker, R.L. Yedlin, D. Madole, and M. Plamann (2007) "Complementation of un-16 and the development of a selectable marker for transformation of Neurospora crassa," Fungal Genetics Reports: Vol. 54, Article 3. https://doi.org/10.4148/1941-4765.1097 This Regular Paper is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Complementation of un-16 and the development of a selectable marker for transformation of Neurospora crassa Abstract Although nearly sixty temperature-sensitive lesions have been mapped in Neurospora crassa, most of their functions have not been identified. These loci are called unknown (un). As part of an effort to identify the open reading frame associated with one of these, we undertook to walk to un-16 using the complementation of temperature-sensitivity as a selection. Cosmids complementing un-16 were identified and the un-16 gene was subcloned. DNA sequence analysis of un-16 revealed that it encodes the highly conserved S9 protein of the 40S ribosomal subunit. This gene has proven useful as a selectable marker and may provide a simple mechanism for the controlled alteration of protein synthesis in N. crassa. Authors Kevin McCluskey, Sheera A. Walker, Rachel L. Yedlin, David Madole, and Michael Plamann This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol54/iss1/3 Number 54, 2007 9Complementation of un-16 and the development of a selectable marker  for transformation of Neurospora crassaKevin McCluskey, Sheera A. Walker, Rachel L. Yedlin, David Madole and Michael PlamannSchool of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110Fungal Genetics Newsletter 54: 9-11Although nearly sixty temperature-sensitive lesions have been mapped in Neurospora crassa, most of their functions have not beenidentified. These loci are called unknown (un). As part of an effort to identify the open reading frame associated with one of these,we undertook to walk to un-16 using the complementation of temperature-sensitivity as a selection. Cosmids complementing un-16were identified and the un-16 gene was subcloned. DNA sequence analysis of un-16 revealed that it encodes the highly conservedS9 protein of the 40S ribosomal subunit. This gene has proven useful as a selectable marker and may provide a simple mechanismfor the controlled alteration of protein synthesis in N. crassa.Temperature-sensitive mutants can be very useful tools for various studies, because they allow temperature-controlled inactivationof essential cellular processes such as DNA, RNA and protein syntheses. An extensive set of temperature-sensitive N. crassamutants is available from the Fungal Genetics Stock Center (FGSC); however, only a few of these mutants have been studied indepth (Perkins et. al., 2001). To enhance the usefulness of this potentially valuable collection of mutants, we have begun to cloneand define the respective mutations in some of these strains. Direct selection of complementing cosmids offers a straight forwardapproach to identifying the underlying lesion associated with temperature-sensitive mutants in a number of organisms (for example,see Osherov et al, 2000). This is made simpler when the mutation being studied has been mapped on the genome. We haveundertaken to complement temperature-sensitive mutants using cosmids mapped on the genome by the Neurospora genome program(Galagan et al. 2003).un-16 was known to be near mating type on linkage group I (Inoue and Ishikawa, 1970, Ishikawa and Perkins, 1983); therefore,we endeavored to walk to un-16 from the mat locus. Strain 4306 (un-16 A) was transformed using the protoplast protocol describedby Vollmer and Yanofsky (1986) using cosmid DNA prepared by the Qiagen midi prep procedure. Selection was carried out byholding the plates at room temperature for several hours prior to incubating plates at 37C. Because the recipient strain does nothave any secondary markers, we included a negative, no-DNA control. In eight transformation experiments, no revertants were seenon these controls. Cosmid pLorist6Xh 54B6 (Kelkar, et al. 2001) was one of the first chosen for the walk as it includes the matingtype locus on its right end. This cosmid was found to complement un-16. Additional cosmids that contain genomic insertsoverlapping the insert contained in cosmid 54B6 were used in transformation experiments to define the region of the genomecontaining the complementing gene(Table 1). Table 1. Cosmids assayed for complementation of un-16Cosmid total transformants ReplicatespLorist6Xh 54B6 465 5pLorist6Xh 67D4 131 6pMOcosX* X5D12 1 2pMOcosX X9B4 0 1#pMOcosX X14D1 0 1pLorist6Xh 63H10* 1 1*Orbach and Sachs, 1991from assembly 3#The high efficiency of complementation by cosmids 67D4 and 54B6 encouraged us to look carefully at the 29 Kb overlap betweenthese two cosmids (Figure 1). This area included five open reading frames, NCU01948.3, NCU01949.3, NCU01950.3,NCU01951.3 and NCU01952.3. Sakai et al. (2002) had previously complemented un-16 with NCU01949.3 as part of theirinvestigation of upr-1.   Our results, plus those of Sakai et al. lead to the conclusion that NCU01949.3 encodes the un-16phenotype.1McCluskey et al.: Complementation of un-16 and the development of a selectable markPublished by New Prairie Press, 201710 Fungal Genetics NewsletterFigure 1. Overlapping cosmids complement un-16. Cosmids shown as solid arrows complement the un-16 mutation while thoseshown as open arrows do not.Based upon its homology to other identified genes including one in Podospora, un-16 encodes the 40S ribosomal protein S9(Dequard-Chablat, 1985). This protein is highly conserved among eukaryotes and archaea (Figure 2). The 40S ribosomal S9 proteinis distantly related to the bacterial S4 ribosomal protein and has been found to function in maintaining the accuracy of proteinsynthesis (Vincent and Liebman, 1992). Sacchromyces cerevisiae is unusual in that it has two genes encoding the S9 proteins, S9-Aand S9-B. Two mutations have been identified in yeast S9-B (SUP46-1 and SUP46-2) that result in increased misreading of mRNAby translating ribosomes (Vincent and Liebman, 1992). The respective positions of these mutations are listed in Figure 2.                                                     *Nc un-16        ----MAPRSYSKTAKVPRRPFEAARLDSELKLVGEYGLRNKREVWRVLLTLSKIRRAARQ 56Sc 40S rp S9-B  --MPRAPRTYSKTYSTPKRPYESSRLDAELKLAGEFGLKNKREIYRISFQLSKIRRAARD 58At 40S rp S9    MVHVCYYRNYGKTFKGPRRPYEKERLDSELKLVGEYGLRNKRELWRVQYSLSRIRNAARD 60Hs 40S rp S9    -MPVARSWVCRKTYVTPRRPFEKSRLDQELKLIGEYGLRNKREVWRVKFTLAKIRKAARE 59Mt 30S rp S4    ---MGYPGKSHKTYDRPRKPWEADRMANEVELIKTYGLRNKRELWKAESILRKYRRVGRM 57                                                               *Nc un-16        LLTLDEK-DPKRLFEGNALIRRLVRVGVLDESRMKLDYVLALKAEDFLERRLQTCVYKLG 115Sc 40S rp S9-B  LLTRDEK-DPKRLFEGNALIRRLVRVGVLSEDKKKLDYVLALKVEDFLERRLQTQVYKLG 117At 40S rp S9    LLTLDEK-SPRRIFEGEALLRRMNRYGLLDESQNKLDYVLALTVENFLERRLQTIVFKSG 119Hs 40S rp S9    LLTLDEK-DPRRLFEGNALLRRLVRIGVLDEGKMKLDYILGLKIEDFLERRLQTQVFKLG 118Mt 30S rp S4    LLASKARGEARADIEAAAVINRLSRFGILKDG-ADLDAILSLKITDILERRLQTQVYRQG 116Nc un-16        LAKSIHHARVLIRQRHIRVGKQIVNVPSFVVRLDSQKHIDFALTSPFG-GGRPGRVRRKK 174Sc 40S rp S9-B  LAKSVHHARVLITQRHIAVGKQIVNIPSFMVRLDSEKHIDFAPTSPFG-GARPGRVARRN 176At 40S rp S9    MAKSIHHSRVLIRQRHIRVGKQLVNIPSFMVRLDSQKHIDFALTSPFG-GGRPGRVKRRN 178Hs 40S rp S9    LAKSIHHARVLIRQRHIRVRKQVVNIPSFIVRLDSQKHIDFSLRSPYG-GGRPGRVKRKN 177Mt 30S rp S4    LANTIRQARQFITHGHIQVAGQRVTVPSYLVKRGDEMTIDYYAGSPLAREGHPERSSKIV 176Nc un-16        A---KAAEGGEAAEE-EDEE 190Sc 40S rp S9-B  AA-RKAEASGEAAEEAEDEE 195At 40S rp S9    EKSASKKASGGGDADGDDEE 198Hs 40S rp S9    AK---KGQGGAGAGDDEEED 194Mt 30S rp S4    AR-----TGGSA-------- 183Figure 2. Alignment of N. crassa un-16 with the 40S ribosomal S9 subunit.  Nc, Neurospora crassa; Sc, Saccharomyces cerevisiae40S ribosomal protein S9-B (accession number NP_009748); At, Arabidopsis thaliana 40S ribosomal protein S9 (accession numberNP_197024); Hs, Homo sapiens 40S ribosomal protein S9 (accession number NP_001004); Mt, Methanosaeta thermophila PT,40S ribosomal protein S4 (accession number YP_844026).  Asterisks indicate the positions of the L34R and L103R mutations,respectively.  The positions of the S. cerevisiae SUP46-1 (D94N) and SUP46-2 (L97W) are underlined in the Sc 40S rp S9-Bsequence. The colors represent categories of amino acids: blue (acidic) - aspartate and glutamate; pink (basic) - arginine, histidine,and lysine; green (polar) - asparagine, cysteine, glutamine, serine, threonine, and tyrosine; red (non-polar) - alanine, glycine,isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine.2Fungal Genetics Reports, Vol. 54 [2007], Art. 3https://newprairiepress.org/fgr/vol54/iss1/3DOI: 10.4148/1941-4765.1097Number 54, 2007 11To further characterize N. crassa un-16, DNA sequence analysis was used to define the respective mutations in two independentun-16 mutants, allele T42M38(t) in strain FGSC#4306 and allele T42M69 in strain FGSC#7558. Allele T42M38(t) of un-16 wasfound to contain a single nucleotide difference changing leucine codon 34 to an arginine codon, and the un-16 allele T42M69contained a single nucleotide difference changing leucine codon 103 to an arginine codon (Figure 2).  The L34R mutation is locatedin the N-terminal domain of the S4/S9 protein, whereas the L103R mutation is located within the S4 domainN. crassa un-16 mutants were easily complemented by the wild-type allele suggesting that the un-16 mutations are recessive.However, because un-16 encodes a mutant ribosomal protein S9, it is possible that it could disrupt protein synthesis inheterokaryons with strains carrying a wild type un-16 gene. To address this issue, we attempted to make heterokaryons of un-16and a strain with a wild type un-16 locus. Strains FGSC#4306 and FGSC#7558 were paired individually with strain FGSC#8964(a  arg-3 ad-3A) and with each other on minimal medium and incubated at 37C. In two separate replicates, both un-16 strainsm33grew vigorously with FGSC#8964 at 37C while they did not grow at 37C when paired with each other or when alone. StrainFGSC#8964 did not grow alone on minimal medium.The ease of use of un-16 as a selectable marker led us to clone the un-16 fragment by amplifying a DNA fragment containing theun-16 gene. Primers were selected to amplify a 1.5 Kb fragment and the fragment was cloned into the pSC-A cloning vector usingtopoisomerase I-mediated ligation followed by Cre-mediated recombination (Stratagene). DNA from eight of the resulting cloneswere prepared using a Wizard prep (Promega) and transformed into prepared un-16 protoplasts.  Seven of the resulting clonescomplemented the un-16 lesion. One clone, pUN16-6, gave the best transformation and it is  available through the FGSC.Acknowledgements  This work was supported by NSF grants 0235887 and 0235871.ReferencesDequard-Chablat, M. 1985. Identification of the structural gene for the S9 ribosomal protein in the fungus Podospora anserina:a new protein involved in the control of translational accuracy. Mol. Gen. Genet. 200:343-5. Galagan J.E., et al. 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature. 422:859-68. Inoue, H., and T. Ishikawa.  1970. Macromolecule synthesis and germination of conidia in temperature-sensitive mutants ofNeurospora crassa. Jpn. J. Genet. 45: 357-369.Ishikawa, T. and D. D. Perkins. 1983. Additional irreparable temperature-sensitive mutants. Neurospora Newl. 30:9Kelkar, H.S., J. Griffith, M.E. Case, S.F. Covert, R.D. Hall, C.H. Keith, J.S. Oliver, M.J. Orbach, M.S. Sachs, J.R. Wagner, M.J.Weise, J.K. Wunderlich, and J. Arnold. 2001. The Neurospora crassa genome: cosmid libraries sorted by chromosome. Genetics157: 979-990.Osherov N, J. Mathew and G.S. May. 2000. Polarity-defective mutants of Aspergillus nidulans. Fungal Genet. Biol. 31:181-8.Orbach, M.J. and M.S. Sachs. 1991. The Orbach/Sachs cosmid library of N. crassa DNA sequences (pMOcosX). Fungal Genet.Newsl. 38: 97.Perkins, D. D., A. Radford, and M. Sachs. 2001. The Neurospora compendium: chromosomal loci.  Academic Press, San Diego.Sakai, W.,  C. Ishii, and H. Inoue. 2002. The upr-1 gene encodes a catalytic subunit of the DNA polymerase . which is involvedin damage-induced mutagenesis in Neurospora crassa. Mol Genet Genomics 267: 401-408Vincent, A. and S.W. Liebman. 1992. The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functionaland structural homolog of the Escherichia coli S4 ram  protein. Genetics 132: 375-386.Vollmer, S. J. and C. Yanofsky.  1986. Efficient cloning of genes of Neurospora crassa. Proc. Natl. Acad. Sci. USA. 83: 4869-4873.3McCluskey et al.: Complementation of un-16 and the development of a selectable markPublished by New Prairie Press, 2017

Complementation of un-16 and the development of a selectable marker for transformation of Neurospora crassa

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Complementation of un-16 and the development of a selectable marker for transformation of Neurospora crassa

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