DNA sequence of histidine-3 from two Neurospora wild-types.
P.J. Yeadon, Arnis Petersen and David E.A. Catcheside - School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia.
We report sequence differences between laboratory strains of Neurospora within his-3,and corrections to published sequence for this locus for ST74A strains, of possible significance to experiments using histidine mutants for gene replacement and gene targeting.
In order to investigate polymorphism within the his-3 gene, PCR primers were designed using his-3 sequence for the St. Lawrence strain ST74-OR23-IVA (Legerton and Yanofsky 1985 Gene 39:129-140). The complete sequence was obtained from T391, a his-3 K26 mutant derived in Lindegren Y8743 (Angel et al. 1970 Aust. J. Biol. Sci. 23:1229-1240), and partial sequence from Lindegren a (FGSC 541). A single base-pair difference between sequences of the two Lindegren strains defined the position of the K26 mutant site. There was substantial difference between the St. Lawrence and Lindegren sequences, with the latter showing greater similarity to the HIS4 sequence of Saccharomyces cerevisiae (Donahue et al. 1982 Gene 18:47-59). We therefore also obtained sequence from G3:6F, a cosmid from the Orbach/Sachs pMOcosX library (Orbach 1994 Gene 150:159-162) made from strain ST74-OR23-IVA. The newly determined his-3 sequence for St. Lawrence 74A was a closer match to that from Lindegren a than was the published sequence and predicted restriction enzyme sites absent in the published sequence but present in the gene. The intron position in his-3 was determined by comparison of sequence from the ST74-OR23-IVA cDNA library (Orbach et al. 1990 J. Biol. Chem. 265:10981-10987) and those from St. Lawrence 74A and T391. Neurospora intron consensus sequences (Bruchez et al.1993 Fungal Genet. Newsl. 40:85-96) did not conflict with this intron position.
Since his-3 sequences are frequently used in targeting and gene replacement vectors (for example: Legerton and Yanofsky 1985 Gene 39:129-140, Ebole 1990 Fungal Genet. Newsl. 37:15-16, Sachs and Ebole 1990 Fungal Genet. Newsl. 37:35-36, Aramayo and Metzenberg 1996 Fungal Genet. Newsl. 43:9-13, Margolin et al.1997 Fungal Genet. Newsl. 44:34-36), we thought it appropriate to make available corrections to the sequence for 74A strains, and note the fact that this gene is substantially polymorphic in Neurospora laboratory strains of different wild-type provenance. There are 14 single base-pair differences between the his-3 sequences of St. Lawrence and Lindegren. Eight of the single base-pair variations are in the third base position including the stop codon which is TAG in St Lawrence but TAA in Lindegren. The gene sequence is 2676 bp in total in both wild types, including a single invariant intron of 63 bp. We have not yet investigated the influence of this degree of heterology on the integration of DNA during gene replacement.
his-3 sequences for St. Lawrence 74A and Lindegren a laboratory wild-types are available from GenBank and have accession numbers of AF045455 and AF045456 respectively.