Identification of a Gene Encoding GMP Synthetase from a Neurospora
crassa cDNA library by bacterial complementation
Jeffrey A. Smiley1 and David K. Asch2. Departments of Chemistry1 and Biological Sciences2, Youngstown State University, Youngstown, OH 44555
We report the isolation and identification of a gene encoding GMP synthetase from a Neurospora crassa cDNA library. Phage infection of the purine-requiring Escherichia coli strain SØ3834 using the NO3- induced cDNA phage library from the Fungal Genetics Stock Center resulted in colonies able to grow on minimal media with no added purine source. A plasmid, termed pGMPS1, was isolated from one of these colonies and shown to reproducibly support growth of strain SØ3834 in the absence of purines in the media. Identification of this gene as one encoding GMP synthetase is confirmed by DNA sequencing and comparison to the known guaA gene from yeast.
To extend the collection of GMP synthetase genes from different organisms, we attempted isolation of the gene from Neurospora crassa using bacterial complementation. The auxotrophic Escherichia coli strain, SØ3834 (Chang et al.1991, Biochemistry 30: 2273-2280), lacks both genes for GMP synthetase (guaA) and adenosine deaminase (add), and requires a purine source that can be metabolized to GMP for growth. In guaA- add+ strains, the purine source can be 2,6-diaminopurine, since this compound can be enzymatically ribosylated and subsequently deaminated by adenosine deaminase to guanosine, but in SØ3834, 2,6-diaminopurine is incapable of providing the purine requirement. Thus, colonies in the complementation screen that arise on media containing 2,6-diaminopurine could contain either the guaA or add genes, but those that arise on purine-free media could only contain guaA.
From colonies arising in a complementation screen of about 105 phage [NO3- induced cDNA phage library, Fungal Genetics Stock Center; Fungal Genetics Newsletter, 45 (Supplement), 8], one colony yielded plasmid DNA from an alkaline lysis preparation that, when reinserted into the host strain, provided the ability to grow on purine-free media. This plasmid was presumed to contain the GMP synthetase gene and was designated pGMPS1. Sequencing of the inserted region within the vector was carried out at the Automated DNA Sequencing Facility, University of North Carolina, Chapel Hill, using the M13 (-20) primer and primers designed from newly acquired sequence information. An open reading frame of 1629 nucleotides was identified; when translated into the amino acid sequence, a high degree of similarity was found with the yeast GMP synthetase gene (duJardin et al. 1994, Gene 139:127-132). Of the 543 amino acids from the deduced sequence, 354 (65%) have identical counterparts in the yeast sequence (Figure 1). The high degree of sequence similarity with the yeast GMP synthetase gene and the ability of the gene to support growth of E. coli strain SØ3834 in the absence of purines confirm the identification of a GMP synthetase gene from an N. crassa library.
We thank Dr. Per Nygaard, Department of Biological Chemistry, University of Copenhagen, for providing E. coli strain SØ3834. Contributions to this work from Maria T. Kovach and Jennifer R. Shook are gratefully acknowledged.
MAA GEQV SNMFDTILVL DFGSQYSHLI TRRLREFNIY AEMLPCTQKI ||| || ||||| | |||||| ||| ||| || || |||||| || MAAATLGEVP TKAFDTILTL DFGSQYTHLI TRRMRELNIL SEMLPCTTKI SELGWTPKGV ILSGGPYSVY AEDAPHVDHA IFDLNVPILG ICYGMQELAW 97 | |||| |||||||||| ||||| | | | ||||| ||||||| || ADLDYKPKGV ILSGGPYSVY EDGAPHVDPA VFELGVPILG ICYGMQEIAW 100 INGKQVGRG DKREYGPATL KVI DDSNS LFKGMNDS TVWMSHGDK | | |||| | | || || | || |||||||| RASPENVIAG VHREYGHSNL KALKGDDAHV DRLFAGLEDS MRVWMSHGDK LHGLPTGYKT IATSDNSPYC GIVHETKPIY GIQFHPEVTH STQGKTLLKN 191 | || | | | |||| | | | ||||| | |||||||| | | |||| LGALPEGFHT VAVSDNSEYA AIAHKTKPIY GLQFHPEVTH SQNGTQLLKN 200 FAVDLCHAKQ NWTMENFIDT EINRIRKLVG PTAEVIGAVS GGVDSTVASK |||| | | |||| | | || ||| ||| | | |||| |||||||| | FAVDICGCAQ NWTMARFLDQ EIARIRDLVG PEGQVLGAVS GGVDSTVAAK LMTEAIGDRF HAILVDNGVL RLNEAANVKK TLVEGLGINL MVVDASEEFL 291 || ||||||| | || ||| || | | | ||||| | ||| || LMKEAIGDRF WAVLVNNGVM RLDECEQVER DLKQHLGINL TVIDASKDFL 300 SKLKGVTDPE KKRKIIGNTF IHVFEREAEK IKP KDGK EIQFLLQG ||| ||| ||| || | | ||| || | | | || | | ||| EGLKGLHDPE QKRKFIGGKF IDVFEAEAQK IEEAAAKSGK GTKIGFFLQG TLYPDVIESI SFKGPSQTIK THHNVGGLLE NMK LKL IEPLRELFKD 382 ||||||||| |||||| ||| |||||||| | | | | ||||| | || TLYPDVIESL SFKGPSATIK THHNVGGLPE RMTNGQGLQL IEPLRSLYKD 400 EVRHLGELLG IPHDLVWRHP FPGPGIAIRV LGEVTKEQVE IARKADNIYI ||| || || | || ||| ||||||| | ||||| | | ||| || | | EVRELGRTLG IHEELVMRHP FPGPGIAVRI LGEVTEEKVR IARQADHIFI EEIKKAGLYN QISQAFACLL PVKSVGVMGD QRTYDQVIAL RAIETTDFMT 482 || ||||| ||||| | | |||||| | | | | || |||||| SEIRKAGLYD QISQAYAAVD PSRAVGVMGD KRVYGYIIIL RAVTTTDFMT 500 ADWFPFEHSF LKKVASRIVN EVDGVARVTY DITSKPPATV EWE 525 | | | | | | |||| || || |||| ||||||| | | | AEAFNFPWDF LQRVMNRIVN EVNGVCRVTY DITSKPPGTI ELE 543
Figure 1. Amino acid sequence comparison of GMP synthetases. Yeast sequence is shown in light type, sequence from N. crassa is bold. Vertical dashes between sequences indicate identical amino acids.
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