Genetic mapping of two acetyl-CoA carboxylase genes
--James J. Caffrey, Eve S. Wurtele and Basil J. Nikolau

The biotin-containing enzyme acetyl-CoA carboxylase (ACCase) catalyses the formation of malonyl-CoA from acetyl-CoA. This reaction occurs in at least two spatially separate compartments. In the plastids, the reaction is the first committed step in de novo fatty-acid biosynthesis; in the cytosol, malonyl-CoA is required for the synthesis of a number of secondary metabolites, including flavonoids. In some non-Gramineae, two structurally distinct forms of the enzyme exist: a "multisubunit type", resembling the E. coli ACCase, which occurs in plastids, and a "multifunctional type" cytosolic enzyme, resembling yeast and mammalian ACCase (Sasaki et al., J. Biol. Chem. 268:25118, 1993; Alban et al., Biochem. J. 300:557, 1994). These isoforms of ACCase can be distinguished by the molecular masses of their biotin-containing subunits and by their susceptibility to the aryloxyphenoxypropionate and cyclohexanedione herbicides: the multisubunit ACCase is resistant to the herbicides, while the multifunctional ACCase is susceptible. In Gramineae such as maize, wheat, and rice, the multisubunit ACCase appears to be absent, which accounts for the susceptibility of these species to graminicides (Konishi and Sasaki, Proc. Nat'l. Acad. Sci. USA 91:3598, 1994).

In maize, two types of multifunctional ACCase can be resolved by ion-exchange chromatography, ACCase I and ACCase II. ACCase I occurs in the plastids of mesophyll cells, and is susceptible to inhibition by graminicides, while ACCase II is not in plastids and is resistant to these herbicides (Egli et al., Plant Physiol. 101:499, 1993). Furthermore, two very similar partial cDNAs coding for the multifunctional ACCase (pA3 and pA4) have been sequenced (Ashton et al., Plant Mol. Biol. 24:35, 1994). Based on the sequence of the 4.3 kb partial cDNA clone (pA3), PCR primers were designed to amplify the 873 bp fragment between positions 3035 and 3907 of the maize ACCase cDNA. The amplified DNA product was gel-purified and end-sequenced to confirm its identity.

The amplified 873 bp ACCase fragment was used as an RFLP probe to genetically map the two structural genes that code for maize ACCase. Two families of recombinant inbred plants derived from the crosses Tx303 x CO159 and T232 x CM37 (Burr et al., Genetics 118:519, 1988) were used. Southern blot analyses of EcoRI-digested DNA isolated from each recombinant plant revealed a strongly hybridizing band that was polymorphic in both families. The segregation pattern of this polymorphic band enabled the mapping of one ACCase structural gene (accA) to near the centromere of chromosome 2, in the interval defined by the RFLP markers umc131 and uox (ssu1B) (Figure 1). [Ed. note: accA and accB are considered temporary symbols pending clarification of the relationships of functions and mapsites]
 
In addition to the accA band, these blots revealed a second, less intensely hybridizing band that was polymorphic only in the T232 x CM37-derived family. The segregation pattern of this band enabled the mapping of the second ACCase structural gene (accB) to chromosome 10L, in the interval defined by the RFLP markers ncsu2 and umc155. The position of the accB locus was confirmed by analyzing the segregation of a polymorphism revealed by EcoRV digestion of DNA isolated from the same individual recombinant plants.

The accA locus corresponds in position to the ACCase gene mapped by Egli et al. (MNL 68:92, 1994) to chromosome 2. A graminicide-resistance locus, termed Acc1-S2, has been previously mapped to 10L, 6.3 cM from umc155 (van Dee et al., Agron. Abstracts, p.198, 1992). This resistance locus probably corresponds in position to the accB locus that we have mapped here. These data support the conclusion that such herbicide-tolerant mutations occur in the structural genes for ACCase. Furthermore, the data imply that the accB locus codes for the plastid-located, graminicide-sensitive ACCase I isoform, and the accA locus codes for the nonplastid-located, graminicide-insensitive ACCase II isoform.

Figure 1. Genetic location of the two maize 240 kD ACCase genes on the RFLP map of the maize genome. The accA gene is located near the centromere on chromosome 2, in the interval defined by the RFLP loci, umc131 and uox. The accB gene is located on the long arm of chromosome 10, in the interval defined by the RLFP loci, umc155 and ncsu2. The thick bars on each map represent the genetic location of the centromeres of chromosomes 2 and 10.


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