Characterization of two unique Long Interspersed Nuclear Elements (LINEs), colonist1 and colonist2
--Lutz, S and Gengenbach, B

Maize acetyl-CoA carboxylase (ACCase) is encoded by a small gene family, of which four genes have been characterized: A1, A2, B1, and B2. Type A and B genes are 96% identical, with conserved introns and 3' non coding regions. Differences within the A1-A2 and B1-B2 pairs occur mainly in flanking sequences. The Type B genes are also distinguishable from the Type A genes, and from each other, by the presence of an insertion into an intron 1400 bp from the translational start site of A1. Type A genes do not contain this insertion and the insertion in the B1 and B2 genes varies in size and sequence arrangement.

The insertion in Type B1 is at least 6kb and is flanked by a 3-bp direct repeat. Nucleotide sequence of this insertion shows the presence of two unique domains encoding polypeptides with homology to the reverse transcriptase (RT) domains of LINE-like non-viral retrotransposons, which include three LINEs from plants: Cin4 from maize (Schwarz-Sommer et al., EMBO J. 6:3873-3880, 1987) , del2 from lily (Leeton et al., Mol. Gen. Genet. 237:97-104, 1993), and BNR from sugar beet (Schmidt et al., Chromosome Research 3:335-345, 1995). Genomic library screening with each of the two RT domains from maize ACCase Type B1 resulted in two different sets of positive clones (none of the positives from either set contain both RT domains together, as in B1), suggesting these RT domains are part of unique LINEs. These unique elements are designated colonist1 and colonist2.

LINEs were first discovered in mammals and have now been found in every eukaryotic species examined. LINEs are believed to move via an RNA intermediate and are characterized by the following features: lengths of 6-7kb, frequent deletions of the 5' end, two open reading frames (one coding for a reverse transcriptase), two cysteine-binding motifs, short direct repeats usually <20bp, and an adenine rich terminus. LINE copy number is variable with mammalian LINEs being highly abundant (104 to 105 copies per genome) while Cin4 is moderately abundant (50-100 copies per genome) (Hutchinson, In: Mobile DNA, DH Berg, MM Howe, eds., Amer. Soc. Microbiology, Washington DC, pp 593-617, 1989; Z. Schwarz-Sommer et al., 1987).

Characterization of colonist1 and colonist2 suggests that colonist1 inserted first into this ACCase intron with colonist2 subsequently inserting into colonist1. Sequence from the 3' end of colonist1 has 73% identity over 480 nucleotides, in reverse orientation, to the largest (1.8kb) intron from shrunken2 of maize (Hannah et al., Plant Physol. 98:1214-1216, 1992). Colonist1 is characterized by a RT domain having much greater amino acid identity (40% in 102 amino acid overlap) to Q, a LINE from mosquito (Besansky, Insect Mol. Biol. 3(1):49-56, 1994), than it does to Cin4 from maize. Neither of the two copies of colonist1 so far studied contain an adenine rich terminus. Colonist2 has an RT domain with 44% identity to Cin4 over 198 amino acids of overlap and contains the consensus CX1-3CX7-8HX4C cysteine motif characteristic of the 3' end of this open reading frame. Colonist2 appears to have an adenine rich terminus of variable length. Genomic Southerns showed colonist1 and colonist2 to be present in the genome at a copy number of 100-500.

LINEs are generally present as a single family within a given species with the exception of Drosophila melanogaster which has several families of LINE-like sequence (Di Nocera et al., Genetica 94:173-180, 1994). With the addition of colonist1 and colonist2 to the list of characterized LINEs, maize becomes the first plant genome shown to contain more than one family of LINE sequences. 


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