Wheat (Triticum aestivum L. cv. Oasis) seedlings were grown on commercial potting soil (Hoffman, Landisville, PA) in a growth chamber under 14 h photoperiod at 23C (night temperature, 19C) with a minimum relative humidity of 70% for 7 days. Primary leaves were cut above the coleoptile and immediately frozen in liquid nitrogen. Total RNA was isolated by the guanidine thiocyanate/CsCI centrifugation method and poly (A)⁺ RNA was purified by 2 cycles of oligo-dT cellulose chromatography by following the standard protocols (Sambrook et al. 1989). The RT-PCR amplification using the degenerate oligonucleotides which are synthesized based on the yeast ERG6 Amino acids sequence, GARTCNATHAARCGNCAYGARCAYTTYCT and CCADATNACYTCRAANCCNGCYTGYTT (where N=A,C,G or T; H=A,T or C; D=G,A or T; R= A or G; Y=T or C; K=A or T; S=G or C), was carried out as described previously (Subramaniam. et al. 1996).

Double-stranded cDNA was synthesized by Gubler and Hoffman method using a commercially available kit (Promega, Madison, WI), ligated to lambda ZAP Express vector (Stratagene, La Jolla, CA) using an EcoRI adapter and in vitro packaged in Gigapack 11 Gold packaging extract (Stratagene) by following the manufacturer's protocols. This library, which contained about 2 x 10⁶ pfu, was screened using the RT-PCR product as described earlier (Subramaniam et al. 1996).

Chromosomal location of TA-MT was determined by aneuploid analysis using the ditelosomic (DT) and nullisomic-tetrasomic (NT) lines of the standard wheat cultivar Chinese Spring (CS) (Sharp et al. 1989). This method essentially involves the hybridization of the probe (radio-labeled pWMP to a Southern blot containing the genomic DNA extracted from the euploid CS plants and its aneuploids that were digested by an enzyme that yields the characteristic '3-band' pattern (in this case HindIII). The hybridization conditions were as described earlier (Subramaniam et al. 1996).

The isolation of wheat genomic DNA, Southern hybridization, genomic library construction, library screening and DNA sequencing were carried out as described in detail earlier (Subramaniam et al. 1996).


Results and discussion

Cloning of a sterol C-24 methyltransferase cDNA from wheat.
Degenerate oligonucleotide primers were synthesized based on the amino acid sequence of the yeast sterol methyltransferase ERG6 (Hardwick and Pelham. 1994; Welihinda et al. 1994), and used in RT-PCR reactions with wheat cDNA as template. The nucleotide sequence of one of the DNA fragments (500 bp) obtained by RT- PCR, when compared with the GenBank database, showed high similarity to that of the yeast ERG6 gene which codes for sterol C-24 methyltransferase (Hardwick and Pelham 1994; Welihinda et al. 1994). A wheat cDNA library was screened using the RT-PCR product as probe and a full-length cDNA clone (pWMT; 1392 bp) was obtained. The nucleotide sequence of both the strands of the pWMT clone was determined.

Sequence comparison with other sterol C-24 methyltransferases.
The plant SMTs were classified into two families, smtl and smt2 (Grebenok et al. 1997; Bouvier-Nave et al. 1998). Sixteen other plant and yeast SMT genes that have high homology with TA-MT were retrieved from the GenBank and analyzed. The amino acid sequence alignment and the phylogenetic analysis clearly show that there are three distinct groups among these SMTs (Fig. 1). Two maize SMTs that belong to the Smt 1 class have the highest percentage (88.1%) similarity with TA-MT. An alignment of these SMT genes is shown in Fig. 2. There are three conserved motifs among these plant and yeast SMTs.

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