Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase

Science

Volumn 286, Issue 5448, 1999, Pages 2342-2344

  Cogoni, Carlo ^a   Macino, Giuseppe ^a  

^a SAPIENZA UNIVERSITY OF ROME   (Italy)

Author keywords

[No Author keywords available]

Indexed keywords

HELICASE;

ARTICLE; DNA BINDING; GENE SILENCING; NEUROSPORA CRASSA; NONHUMAN; PRIORITY JOURNAL; SEQUENCE ANALYSIS; TRANSGENE;

ADENOSINE TRIPHOSPHATASES; AMINO ACID SEQUENCE; BLOOM SYNDROME; CAMPTOTHECIN; DNA HELICASES; DNA, FUNGAL; ENZYME INHIBITORS; ETOPOSIDE; FUNGAL PROTEINS; GENE SILENCING; GENES, FUNGAL; GENETIC COMPLEMENTATION TEST; HUMANS; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; MUTAGENESIS, INSERTIONAL; NEUROSPORA CRASSA; RECQ HELICASES; SEQUENCE ALIGNMENT; TRANSCRIPTION, GENETIC; TRANSGENES; WERNER SYNDROME;

ANIMALIA; FUNGI; NEUROSPORA; NEUROSPORA CRASSA;

EID: 0033579344     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5448.2342     Document Type: Article

References (31)

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14. The N. crassa al-1 silenced strain 6XW, used in insertional mutagenesis, and qde mutant strains were described previously (11). The general growth medium was composed of Vogel's minimal medium with 2% sucrose in agar plates; heterokaryon analysis and maintenance of stock cultures were performed as described (11). Plasmid pMXY2 containing the Neurospora selectable marker benomyl was used for insertional mutagenesis. Insertional transformants were selected on minimal medium containing benomyl (1 mg/ml). Release or restoration of al-1 gene silencing after transformation or heterokaryon formation was evaluated by visual inspection of conidia color: wild-type conidia were orange, whereas white to yellow conidia were indicative of al-1 gene silencing.
15. Cloning of the DNA region flanking the integrated plasmid was achieved with a plasmid rescue approach. Chromosomal DNA (100 ng) extracted from strain 627 was digested with BgI II restriction enzyme, and the derived restriction fragments were ligated in a reaction volume of 0.2 ml in order to favor intramolecular ligation events. The DNA was ethanol precipitated and used to transform E. coli. Chromosomal DNA flanking the integration site was isolated with the enzymes BgI II or Sal I, and the resulting restriction fragment was used as a probe on a N. crassa genomic cosmid library. Two positively hybridizing cosmids (6E8 and 54D7), containing 30-kb inserts, were introduced by transformation into insertional mutant qde-3 strain 627 and into an UV irradiation-induced qde-3 mutant strain. The subcloning of restriction fragments from genomic library clones was performed in pBluescript SK plasmid (Stratagene).
16. To map the intron positions, we used total RNA as the template in RT reactions containing, as primers, an oligonucleotide 5′-ATGGTTGACTTGATCCAG-3′ to map the first intron or an oligonucleotide 5′-TGGAACTTCGTTTCTTGG-3′ for the second intron. The two cDNA reactions were subsequently amplified by PCR with the oligonudeotide pair 5′-GACTACAGCCGGCAACTG-3′ and 5′-GATGTGAGGAAGGCTCTC-3′ or the oligonudeotide pair CGAGCACGGTGGCGTGTG-3′ and 5′-CAGGGTGGAAAGTTCTTG-3′, respectively. The resulting PCR fragments were inserted in TA cloning vector (Invitrogen) and sequenced.
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27. A. Pickford, C. Cogoni, G. Macino, unpublished data.
28. The sensitivity of qde-3 mutants to increasing doses of methyl methansulfonate (MMS), hydroxyurea (HU), mitomycin C (MC), and UV irradiation was tested. MMS, HU, and MC were added directly to conidia suspensions as described [C. Ishii, K. Natamuran, H. Inoue, Mol. Gen. Genet. 288, 33 (1991)], and UV irradiation was carried out as described [H. Inoue, T. M. Hong, F. J. deSerres, Mutat. Res. 80, 27 (1981)].
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30. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
31. A special thanks to G. Azzalin for skillful technical assistance. We also thank G. Coruzzi and A. Pickford for revising the manuscript. Supported in part by grants from the Istituto Pasteur Fondazione Cenci Bolognetti, from the Ministero dell' Università e della Ricerca Scientifica e Tecnologica, and from the European Union BIOTECH program.