Gaertner, F.

Manger, R.

Morse, C. C.

English

Kansas State University

Fungal Genetics Reports Volume 30 Article 5 Neurospora crassa qa-2 gene fragment. C. C. Morse R. Manger F. Gaertner 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 Morse, C. C., R. Manger, and F. Gaertner (1983) "Neurospora crassa qa-2 gene fragment.," Fungal Genetics Reports: Vol. 30, Article 5. https://doi.org/10.4148/1941-4765.1620 This Research Note 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. Neurospora crassa qa-2 gene fragment. Abstract Neurospora crassa qa-2 gene fragment. This research note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol30/iss1/5 In an effort to isolate the aro cluster-"ene (GaertnerMorse, C.C., R. Manger and F. Gaertnerand Cole 1977 Biochem. Biophys.  Res. Commun. 75: 259) fromN. crassa, we prepared a total BamHI digest of chromosomalDNA, ligated into the Saccharomyces shuttle vector pYE13(Broach et at. 1979 Gene 8: 121), and amplified the resultantgenomic library in E. coli (Procedure I). The amplified gene-bank was used to attempt complementation in four mutants ofE. coli (see E. coli strains lacking aro activities by transformation (Procedure II). Only the E. coli  mutantSK288a (aroD),  which lacks DHQase, was complemented. Isolation of plasmid  DNA (Procedure III), and subsequentrestriction of the DNA with BamHI, showed that the complementing activity was present on a 3.2 kbp BamHIfragment. This fragment in pYE13  also complements the qa-2  gene of N. crassa (Case and Giles, personal communi-cation), but does not complement DHQase minus strains of Saccharomyces cerevisiae (our unpublished results).Subcloning of the 3.2 kbp insert was performed to obtain both orientations within the pYE13 shuttle-vector.Complementation was observed in the E. coli, aroD, mutant in both orientations, indicating that promoter activ-ity was inherent to the fragment and independent of plasmid  promoter functions. Enzyme preparations from thecomplemented E. coli aroD mutant also confirmed the existence of the qa-2  gene on the 3.2 kbp fragment. Thus,we concluded that the 3.2 kbp BamHI fragment from Neurospora crassa carried the structural and promoter infor-mation of the qa-2 dehydroquinase gene.Complementing activities for other E. coli aro mutations relevant to the aro cluster-gene have not beenobserved, suggesting either that BamHI cuts and inactivates these genes, or that some incompatibility existsbetween E. coli and N. crassa with respect to these genetic elements.Procedure I - Library Formation and Plasmid AmplificationLigation of 4.8 micrograms of BamHI digested N. crassa genomic DNA with 1.6 micrograms of BamHI digested,alkaline phosphatased, pYE13 DNA was done in a total volume of 800 microliters, also containing T4 DNA ligasebuffer (lx T4 DNA ligase buffer: 50 mM Tris-Cl 7.8, 10 mM MgC12, 2 mM DTT, 1 mM ATP and 50 micrograms per mlof BSA) and 1600 U of T4 DNA ligase. The ligation was carried out for 18 h in a 15°C water bath. The 800microliter ligation was then added to 16 ml of competent E. coli HB101 cells and our usual transformation wasperformed. The entire transformation mixture was added to 500 ml of LB-medium containing 25 micrograms per mlof ampicillin and grown to a stationary phase overnight in an air shake-incubator at 37°C. These cells werethen spun down and resuspended in 10 ml of LB-medium plus 25 micrograms per ml of ampicillin. Enough of thisresuspension was added to 500 mls of M-g medium to bring the initial O.D. 600 to 0.16 using a 1 cm pathlength.The culture was then grown in an air shake-incubator at 37°C until the O.D. 600 reached 0.8, at which timechloramphenicol was added to a final concentration of 200 micrograms per ml and the culture grown for anadditional 18 h. The plasmid DNA was then recovered as described below in Procedure III.Procedure II Bacterial TransformationBacterial strains were made competent according to the following protocol. A single colony isolate wasstarted in 20 ml of LB-medium and grown at 37°C overnight. The following day, a 560 microliter aliquot of theovernight culture was added to 56 ml of LB-medium in a 250 ml side-arm flask with a 1 cm pathlength. Theculture was then grown to an O.D. 600 of 0.16 and the flask placed in ice-water for 10 minutes. The cellswere then spun down in sterile 30 ml oak-ridge tubes, resuspended in 28 ml of C.M.T. buffer (lx C.M.T. buffer:0.1 M CaCl2,  5 mM MgCl2, 10 mM Tris-Cl  7.8), and incubated on ice for 25 minutes. The cells were spun downagain and resuspended in 1.4 ml of C.M.T. The transformation procedure was begun by the addition of DNA to thecompetent cells in a ratio of 5-10 microliters of aqueous DNA per 200 microliters of competent cells, and in-cubated on ice for 15 minutes. After a 5 minute heat shock in a 37°C water-bath, 800 microliters of LB-mediumwas added per 200 microliter transformation aliquot, and the culture vigorously shook in a 37°C water-bath for60 minutes. The transformation mixture was then plated on selectable medium or amplified directly.Procedure III - Plasmid PurificationPlasmid purification was done according to the alkaline lysis protocol in the Cold Spring Harbor laboratorymanual "Molecular Cloning," T. Maniatis, E.F. Fritsch and J. Sambrook (1982),  without modification.  LB-Medium (1000 ml)                    M-9 Medium (1000 ml) 10x M-9 Salts (500 ml)10 g tryptone 100 ml                       10x M-g salts (see below)         54.7 g Na2HPO4,7H2O5 g yeast extract                    5 g casaminoacids                 15.0 g KH2PO45 g NaCl0.5 ml 5 N NaOHE. coli  Strains2.5 g NaCl5.0 g NH4ClThe "SK" strains of E. coli were a generous gift from Sidnev R. Kushner (The Universitv of Georgia, Athens).   SK494: F- , gal- , thi- ,aroE, spcR, hsr-4, endA-, sbcB15, (DH5 reductase)SK890: F-, gal-, thi-, aroB, strR, tonR  hsr-4, endA-, sbcB15, (DHQ synthase)SK2881: F-, gal-, lac-, srl-, leuC, aroD6-,  tonA, hsdR4, recA1, sbcB15, endA-, (DHQase)SK3337:  F-. qal-K2, acvl, mH-1, xyl-5, his-4, pro-A, thi-1, tetS, λ−  aroA2, hsdR4, (ESP synthase)HBlOl: hsdR-, hsdM-, recAB, supE44, 1ac24, leuB6, proA2, thi-1, SmR(Research sponsored by the Office of Health and Enviromnental Research. U.S. Dept. of Energy, contract W-7405.eng-26 with the Union Carbide Corp. C. Morse supported by an NIH training grant GM-7438, and R. Manger supportedby NCI training grant CA-09104.) - - - The University of Tennessee- Oak Ridge Graduate School of BiomedicalSciences and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830.

Neurospora crassa qa-2 gene fragment.

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Neurospora crassa qa-2 gene fragment.

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