A Network of HMG-box Transcription Factors Regulates Sexual Cycle in the Fungus

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High-mobility group (HMG) B proteins are eukaryotic DNA-binding proteins characterized by the HMG-box functional motif. These transcription factors play a pivotal role in global genomic functions and in the control of genes involved in specific developmental or metabolic pathways. The filamentous ascomycete Podospora anserina contains 12 HMG-box genes. Of these, four have been previously characterized; three are mating-type genes that control fertilization and development of the fruit-body, whereas the last one encodes a factor involved in mitochondrial DNA stability. Systematic deletion analysis of the eight remaining uncharacterized HMG-box genes indicated that none were essential for viability, but that seven were involved in the sexual cycle. Two HMG-box genes display striking features. PaHMG5, an ortholog of SpSte11 from Schizosaccharomyces pombe, is a pivotal activator of mating-type genes in P. anserina, whereas PaHMG9 is a repressor of several phenomena specific to the stationary phase, most notably hyphal anastomoses. Transcriptional analyses of HMG-box genes in HMG-box deletion strains indicated that PaHMG5 is at the hub of a network of several HMG-box factors that regulate mating-type genes and mating-type target genes. Genetic analyses revealed that this network also controls fertility genes that are not regulated by mating-type transcription factors. This study points to the critical role of HMG-box members in sexual reproduction in fungi, as 11 out of 12 members were involved in the sexual cycle in P. anserina. PaHMG5 and SpSte11 are conserved transcriptional regulators of mating-type genes, although P. anserina and S. pombe diverged 550 million years ago. Two HMG-box genes, SOX9 and its upstream regulator SRY, also play an important role in sex determination in mammals. The P. anserina and S. pombe mating-type genes and their upstream regulatory factor form a module of HMG-box genes analogous to the SRY/SOX9 module, revealing a commonality of sex regulation in animals and fungi.

Vyšlo v časopise: A Network of HMG-box Transcription Factors Regulates Sexual Cycle in the Fungus. PLoS Genet 9(7): e32767. doi:10.1371/journal.pgen.1003642
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003642

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Zdroje

1. BustinM (2001) Revised nomenclature for high mobility group (HMG) chromosomal proteins. Trends Biochem Sci 26: 152–153.

2. Koopman, P. 2010. HMG Domain Superfamily of DNA-bending Proteins: HMG, UBF, TCF, LEF, SOX, SRY and Related Proteins. eLS. DOI: 10.1002/9780470015902.a0002325.pub2

3. BianchiME, ManfrediAA (2007) High-mobility group box 1 (HMGB1) protein at the crossroads between innate and adaptive immunity. Immunol Rev 220: 35–46.

4. YanaiH, BanT, WangZ, ChoiMK, KawamuraT, et al. (2009) HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature 462: 99–103.

5. ReadCM, CaryPD, Crane-RobinsonC, DriscollPC, NormanDG (1993) Solution structure of a DNA-binding domain from HMG1. Nucleic Acids Res 21: 3427–3436.

6. WeirHM, KraulisPJ, HillCS, RaineAR, LaueED, et al. (1993) Structure of the HMG box motif in the B-domain of HMG1. EMBO J 12: 1311–1319.

7. GieseK, CoxJ, GrosschedlR (1992) The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell 69: 185–195.

8. StrosM (2010) HMGB proteins: interactions with DNA and chromatin. Biochim Biophys Acta 1799: 101–113.

9. SoullierS, JayP, PoulatF, VanackerJM, BertaP, et al. (1999) Diversification pattern of the HMG and SOX family members during evolution. J Mol Evol 48: 517–527.

10. JantzenHM, AdmonA, BellSP, TjianR (1990) Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins. Nature 344: 830–836.

11. GubbayJ, CollignonJ, KoopmanP, CapelB, EconomouA, et al. (1990) A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 346: 245–250.

12. van de WeteringM, OosterwegelM, DooijesD, CleversH (1991) Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. EMBO J 10: 123–132.

13. StabenC, YanofskyC (1990) Neurospora crassa a mating-type region. Proc Natl Acad Sci U S A 87: 4917–4921.

14. O'SullivanAC, SullivanGJ, McStayB (2002) UBF binding in vivo is not restricted to regulatory sequences within the vertebrate ribosomal DNA repeat. Mol Cell Biol 22: 657–668.

15. GadalO, LabarreS, BoschieroC, ThuriauxP (2002) Hmo1, an HMG-box protein, belongs to the yeast ribosomal DNA transcription system. Embo J 21: 5498–5507.

16. RayS, GroveA (2009) The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends. Nucleic Acids Res 37: 6389–6399.

17. StillmanDJ (2010) Nhp6: a small but powerful effector of chromatin structure in Saccharomyces cerevisiae. Biochim Biophys Acta 1799: 175–180.

18. KaoLR, MegrawTL, ChaeCB (1993) Essential role of the HMG domain in the function of yeast mitochondrial histone HM: functional complementation of HM by the nuclear nonhistone protein NHP6A. Proc Natl Acad Sci U S A 90: 5598–5602.

19. LambertJR, BilanchoneVW, CumskyMG (1994) The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 91: 7345–7349.

20. TsaponinaO, BarsoumE, AstromSU, ChabesA (2011) Ixr1 is required for the expression of the ribonucleotide reductase Rnr1 and maintenance of dNTP pools. PLoS Genet 7: e1002061.

21. LanF, ZaratieguiM, VillenJ, VaughnMW, VerdelA, et al. (2007) S. pombe LSD1 homologs regulate heterochromatin propagation and euchromatic gene transcription. Mol Cell 26: 89–101.

22. BarrionuevoF, Bagheri-FamS, KlattigJ, KistR, TaketoMM, et al. (2006) Homozygous inactivation of Sox9 causes complete XY sex reversal in mice. Biol Reprod 74: 195–201.

23. FosterJW, Dominguez-SteglichMA, GuioliS, KwokC, WellerPA, et al. (1994) Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 372: 525–530.

24. WagnerT, WirthJ, MeyerJ, ZabelB, HeldM, et al. (1994) Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79: 1111–1120.

25. KnowerKC, KellyS, HarleyVR (2003) Turning on the male-SRY, SOX9 and sex determination in mammals. Cytogenet Genome Res 101: 185–198.

26. Debuchy R, Berteaux-Lecellier V, Silar P (2010) Mating systems and sexual morphogenesis in Ascomycetes. In: Borkovich KA, Ebbole DJ, editors. Cellular and Molecular Biology of Filamentous Fungi. Washington, DC: ASM Press. pp. 501–535.

27. MartinT, LuSW, van TilbeurghH, RipollDR, DixeliusC, et al. (2010) Tracing the origin of the fungal alpha1 domain places its ancestor in the HMG-box superfamily: implication for fungal mating-type evolution. PLoS One 5: e15199.

28. ButlerG, KennyC, FaganA, KurischkoC, GaillardinC, et al. (2004) Evolution of the MAT locus and its Ho endonuclease in yeast species. Proc Natl Acad Sci U S A 101: 1632–1637.

29. KwastKE, BurkePV, BrownK, PoytonRO (1997) REO1 and ROX1 are alleles of the same gene which encodes a transcriptional repressor of hypoxic genes in Saccharomyces cerevisiae. Curr Genet 32: 377–383.

30. SugimotoA, IinoY, MaedaT, WatanabeY, YamamotoM (1991) Schizosaccharomyces pombe ste11+ encodes a transcription factor with an HMG motif that is a critical regulator of sexual development. Genes Dev 5: 1990–1999.

31. QinJ, KangW, LeungB, McLeodM (2003) Ste11p, a high-mobility-group box DNA-binding protein, undergoes pheromone- and nutrient-regulated nuclear-cytoplasmic shuttling. Mol Cell Biol 23: 3253–3264.

32. GryganskyiAP, LeeSC, LitvintsevaAP, SmithME, BonitoG, et al. (2010) Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex. PLoS One 5: e15273.

33. IdnurmA, WaltonFJ, FloydA, HeitmanJ (2008) Identification of the sex genes in an early diverged fungus. Nature 451: 193–196.

34. LeeSC, CorradiN, ByrnesEJ3rd, Torres-MartinezS, DietrichFS, et al. (2008) Microsporidia evolved from ancestral sexual fungi. Curr Biol 18: 1675–1679.

35. WetzelJ, BurmesterA, KolbeM, WostemeyerJ (2012) The mating-related loci sexM and sexP of the zygomycetous fungus Mucor mucedo and their transcriptional regulation by trisporoid pheromones. Microbiology 158: 1016–1023.

36. LeeSC, CorradiN, DoanS, DietrichFS, KeelingPJ, et al. (2010) Evolution of the sex-related locus and genomic features shared in microsporidia and fungi. PLoS One 5: e10539.

37. BrefortT, MullerP, KahmannR (2005) The high-mobility-group domain transcription factor Rop1 is a direct regulator of prf1 in Ustilago maydis. Eukaryot Cell 4: 379–391.

38. HartmannHA, KahmannR, BolkerM (1996) The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis. EMBO J 15: 1632–1641.

39. UrbanM, KahmannR, BolkerM (1996) Identification of the pheromone response element in Ustilago maydis. Mol Gen Genet 251: 31–37.

40. MurataY, FujiiM, ZolanME, KamadaT (1998) Molecular analysis of pcc1, a gene that leads to A-regulated sexual morphogenesis in Coprinus cinereus. Genetics 149: 1753–1761.

41. KruzelEK, GilesSS, HullCM (2012) Analysis of Cryptococcus neoformans sexual development reveals rewiring of the pheromone-response network by a change in transcription factor identity. Genetics 191: 435–449.

42. LinX, JacksonJC, FeretzakiM, XueC, HeitmanJ (2010) Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans. PLoS Genet 6: e1000953.

43. SonH, SeoYS, MinK, ParkAR, LeeJ, et al. (2011) A phenome-based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum. PLoS Pathog 7: e1002310.

44. Dequard-ChablatM, AllandtC (2002) Two copies of mthmg1, encoding a novel mitochondrial HMG-like protein, delay accumulation of mitochondrial DNA deletions in Podospora anserina. Eukaryot Cell 1: 503–513.

45. DebuchyR, CoppinE (1992) The mating types of Podospora anserina: functional analysis and sequence of the fertilization domains. Mol Gen Genet 233: 113–121.

46. DebuchyR, ArnaiseS, LecellierG (1993) The mat- allele of Podospora anserina contains three regulatory genes required for the development of fertilized female organs. Mol Gen Genet 241: 667–673.

47. Turgeon BG, Debuchy R (2007) Cochliobolus and Podospora: mechanism of sex determination and the evolution of reproductive lifestyle. In: Heitman J, Kronstad JW, Taylor JW, Casselton LA, editors. Sex in Fungi, molecular determination and evolutionary implications. Washington, DC: ASM Press. pp. 93–121.

48. AltschulSF, MaddenTL, SchafferAA, ZhangJ, ZhangZ, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.

49. ParkJ, ParkJ, JangS, KimS, KongS, et al. (2008) FTFD: an informatics pipeline supporting phylogenomic analysis of fungal transcription factors. Bioinformatics 24: 1024–1025.

50. GoughJ, KarplusK, HugheyR, ChothiaC (2001) Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. J Mol Biol 313: 903–919.

51. Marchler-BauerA, LuS, AndersonJB, ChitsazF, DerbyshireMK, et al. (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39: D225–229.

52. GrosseteteS, LabedanB, LespinetO (2010) FUNGIpath: a tool to assess fungal metabolic pathways predicted by orthology. BMC Genomics 11: 81.

53. EspagneE, LespinetO, MalagnacF, Da SilvaC, JaillonO, et al. (2008) The genome sequence of the model ascomycete fungus Podospora anserina. Genome Biol 9: R77.

54. JacksonD, LawsonT, VillafaneR, GaryL (2013) Modeling the structure of yeast MATα1: an HMG-box motif with a C-terminal helical extension. Open J Biophys 3: 1–12.

55. IyerSV, RamakrishnanM, KasbekarDP (2009) Neurospora crassa fmf-1 encodes the homologue of the Schizosaccharomyces pombe Ste11p regulator of sexual development. J Genet 88: 33–39.

56. JohnsonTE (1979) A neurospora mutation that arrests perithecial development as either male or female parent. Genetics 92: 1107–1120.

57. BorkovichKA, AlexLA, YardenO, FreitagM, TurnerGE, et al. (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68: 1–108.

58. FangZA, WangGH, ChenAL, LiYF, LiuJP, et al. (2009) Gene responses to oxygen availability in Kluyveromyces lactis: an insight on the evolution of the oxygen-responding system in yeast. PLoS One 4: e7561.

59. KadoshD, JohnsonAD (2001) Rfg1, a protein related to the Saccharomyces cerevisiae hypoxic regulator Rox1, controls filamentous growth and virulence in Candida albicans. Mol Cell Biol 21: 2496–2505.

60. KimCA, BowieJU (2003) SAM domains: uniform structure, diversity of function. Trends Biochem Sci 28: 625–628.

61. CostiganC, KolodrubetzD, SnyderM (1994) NHP6A and NHP6B, which encode HMG1-like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway. Mol Cell Biol 14: 2391–2403.

62. DowellNL, SperlingAS, MasonMJ, JohnsonRC (2010) Chromatin-dependent binding of the S. cerevisiae HMGB protein Nhp6A affects nucleosome dynamics and transcription. Genes Dev 24: 2031–2042.

63. GalaganJE, CalvoSE, BorkovichKA, SelkerEU, ReadND, et al. (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422: 859–868.

64. BrunS, MalagnacF, BidardF, LalucqueH, SilarP (2009) Functions and regulation of the Nox family in the filamentous fungus Podospora anserina: a new role in cellulose degradation. Mol Microbiol 74: 480–496.

65. FuC, IyerP, HerkalA, AbdullahJ, StoutA, et al. (2011) Identification and characterization of genes required for cell-to-cell fusion in Neurospora crassa. Eukaryot Cell 10: 1100–1109.

66. SimoninAR, RasmussenCG, YangM, GlassNL (2010) Genes encoding a striatin-like protein (ham-3) and a forkhead associated protein (ham-4) are required for hyphal fusion in Neurospora crassa. Fungal Genet Biol 47: 855–868.

67. LalucqueH, MalagnacF, BrunS, KickaS, SilarP (2012) A non-Mendelian MAPK-generated hereditary unit controlled by a second MAPK pathway in Podospora anserina. Genetics 191: 419–433.

68. SilarP (2011) Grafting as a method for studying development in the filamentous fungus Podospora anserina. Fungal Biol 115: 793–802.

69. Read ND, Fleissner A, Roca GM, Glass L, N. (2010) Hyphal fusion. In: Borkovich KA, Ebbole DJ, editors. Cellular and Molecular Biology of Filamentous Fungi. Washington, DC: ASM Press. pp. 260–273.

70. CoppinE, Berteaux-LecellierV, BidardF, BrunS, Ruprich-RobertG, et al. (2012) Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body. PLoS One 7: e37488.

71. CoppinE, ArnaiseS, ContamineV, PicardM (1993) Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation. Mol Gen Genet 241: 409–414.

72. Jamet-ViernyC, DebuchyR, PrigentM, SilarP (2007) IDC1, a pezizomycotina-specific gene that belongs to the PaMpk1 MAP kinase transduction cascade of the filamentous fungus Podospora anserina. Fungal Genet Biol 44: 1219–1230.

73. BidardF, Ait BenkhaliJ, CoppinE, ImbeaudS, GrognetP, et al. (2011) Genome-wide gene expression profiling of fertilization competent mycelium in opposite mating types in the heterothallic fungus Podospora anserina. PLoS One 6: e21476.

74. RidderR, OsiewaczHD (1992) Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency. Curr Genet 21: 207–213.

75. du PrelJB, HommelG, RohrigB, BlettnerM (2009) Confidence interval or p-value?: part 4 of a series on evaluation of scientific publications. Dtsch Arztebl Int 106: 335–339.

76. NakaiK, HortonP (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24: 34–36.

77. CoppinE, DebuchyR (2000) Co-expression of the mating-type genes involved in internuclear recognition is lethal in Podospora anserina. Genetics 155: 657–669.

78. BaileyTL, ElkanC (1994) Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2: 28–36.

79. van BeestM, DooijesD, van De WeteringM, KjaerulffS, BonvinA, et al. (2000) Sequence-specific high mobility group box factors recognize 10–12-base pair minor groove motifs. J Biol Chem 275: 27266–27273.

80. KunitomoH, HiguchiT, IinoY, YamamotoM (2000) A zinc-finger protein, Rst2p, regulates transcription of the fission yeast ste11(+) gene, which encodes a pivotal transcription factor for sexual development. Mol Biol Cell 11: 3205–3217.

81. AonoT, YanaiH, MikiF, DaveyJ, ShimodaC (1994) Mating pheromone-induced expression of the mat1-Pm gene of Schizosaccharomyces pombe: identification of signalling components and characterization of upstream controlling elements. Yeast 10: 757–770.

82. TsongAE, TuchBB, LiH, JohnsonAD (2006) Evolution of alternative transcriptional circuits with identical logic. Nature 443: 415–420.

83. TuchBB, GalgoczyDJ, HerndayAD, LiH, JohnsonAD (2008) The evolution of combinatorial gene regulation in fungi. PLoS Biol 6: e38.

84. DyerPS (2008) Evolutionary biology: genomic clues to original sex in fungi. Curr Biol 18: R207–R209.

85. Tsong AE, Brian BT, Johnson AD (2007) Rewiring transcriptional circuitry: mating-type regulation in Saccharomyces cerevisiae and Candida albicans as a model for evolution. In: Heitman J, Kronstad JW, Taylor JW, Casselton LA, editors. Sex in Fungi, molecular determination and evolutionary implications. Washington, DC: ASM Press. pp. 75–89.

86. Wong Sak HoiJ, DumasB (2010) Ste12 and Ste12-like proteins, fungal transcription factors regulating development and pathogenicity. Eukaryot Cell 9: 480–485.

87. DesjardinsAE, BrownDW, YunSH, ProctorRH, LeeT, et al. (2004) Deletion and complementation of the mating type (MAT) locus of the wheat head blight pathogen Gibberella zeae. Appl Environ Microbiol 70: 2437–2444.

88. FerreiraAV, AnZ, MetzenbergRL, GlassNL (1998) Characterization of mat A-2, mat A-3 and ΔmatA mating-type mutants of Neurospora crassa. Genetics 148: 1069–1079.

89. CoppinE, de RentyC, DebuchyR (2005) The function of the coding sequences for the putative pheromone precursors in Podospora anserina is restricted to fertilization. Eukaryot Cell 4: 407–420.

90. KickaS, SilarP (2004) PaASK1, a mitogen-activated protein kinase kinase kinase that controls cell degeneration and cell differentiation in Podospora anserina. Genetics 166: 1241–1252.

91. KickaS, BonnetC, SoberingAK, GanesanLP, SilarP (2006) A mitotically inheritable unit containing a MAP kinase module. Proc Natl Acad Sci U S A 103: 13445–13450.

92. KjaerulffS, Lautrup-LarsenI, TruelsenS, PedersenM, NielsenO (2005) Constitutive activation of the fission yeast pheromone-responsive pathway induces ectopic meiosis and reveals ste11 as a mitogen-activated protein kinase target. Mol Cell Biol 25: 2045–2059.

93. BerbeeML, TaylorJW (2010) Dating the molecular clock in fungi - how close are we? Fungal Biol Rev 24: 1–16.

94. RizetG, EngelmannC (1949) Contribution à l'étude génétique d'un ascomycète tatrasporé: Podospora anserina. Rhem Rv Cytol Biol Veg 11: 201–304.

95. RizetG (1952) Les phénomènes de barrage chez Podospora anserina. I. Analyse génétique des barrages entre souches S and s. Rev Cytol Biol Veg 13: 51–92.

96. PuntaM, CoggillPC, EberhardtRY, MistryJ, TateJ, et al. (2012) The Pfam protein families database. Nucleic Acids Res 40: D290–D301.

97. ColotHV, ParkG, TurnerGE, RingelbergC, CrewCM, et al. (2006) A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci U S A 103: 10352–10357.

98. El-KhouryR, SellemCH, CoppinE, BoivinA, MaasMF, et al. (2008) Gene deletion and allelic replacement in the filamentous fungus Podospora anserina. Curr Genet 53: 249–258.

99. SilarP (1995) Two new easy to use vectors for transformations . Fungal Genet Newslett 42: 73.

100. VandesompeleJ, De PreterK, PattynF, PoppeB, Van RoyN, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034.

101. AndersenCL, JensenJL, OrntoftTF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64: 5245–5250.

102. PfafflMW, HorganGW, DempfleL (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30: e36.

103. van HoutenV, DenkersF, van DijkM, van den BrekelM, BrakenhoffR (1998) Labeling efficiency of oligonucleotides by T4 polynucleotide kinase depends on 5′-nucleotide. Anal Biochem 265: 386–389.

104. HoldenNS, TaconCE (2011) Principles and problems of the electrophoretic mobility shift assay. J Pharmacol Toxicol Methods 63: 7–14.

105. LarkinMA, BlackshieldsG, BrownNP, ChennaR, McGettiganPA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.

106. WaterhouseAM, ProcterJB, MartinDM, ClampM, BartonGJ (2009) Jalview Version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189–1191.

107. LeSQ, GascuelO (2008) An improved general amino acid replacement matrix. Mol Biol Evol 25: 1307–1320.

108. PavlidisP, NobleWS (2003) Matrix2png: a utility for visualizing matrix data. Bioinformatics 19: 295–296.

109. PhilleyML, StabenC (1994) Functional analyses of the Neurospora crassa MT a-1 mating type polypeptide. Genetics 137: 715–722.