Alu-mediated inactivation of the human CMP- N-acetylneuraminic acid hydroxylase gene

Abstract

Inactivation of the CMP-N-acetylneuraminic acid hydroxylase gene has provided an example of human-specific genomic mutation that results in a widespread biochemical difference between human and nonhuman primates. We have found that, although a region containing a 92-bp exon and an AluSq element in the hydroxylase gene is intact in all nonhuman primates examined, the same region in the human genome is replaced by an AluY element that was disseminated at least one million years ago. We propose a mechanistic model for this Alu-mediated replacement event, which deleted the 92-bp exon and thus inactivated the human hydroxylase gene. It is suggested that Alu elements have played potentially important roles in genotypic and phenotypic evolution in the hominid lineage.

Figure 1

Comparison of genomic nucleotide sequences around the 92-bp exon of various primate CMP-Neu5Ac hydroxylase genes. Hs, Pt, Gg, and Mm refer to the human, chimpanzee, gorilla, and rhesus monkey, respectively. The shaded boxes represent the 92-bp exon deleted in the hominid lineage. The Alu element is represented by the open box. The direct repeats of the sialic acid hydroxylase AluSq (sahAluSq) are underlined. The arrowheads indicate replacement boundaries. The 5′-TAAAGATTAATTTTTATTTTT-3′ sequence, which would have a strong preference to the target-priming by the Alu poly(A) tail, is located in the 5′ region immediately adjacent to the upstream replacement boundary. Dots refer to identical nucleotides in the other primates; dashes indicate gaps used for sequence alignment. In the gap corresponding to the human deletion, the complete sequences of the other primate genes are shown.

Figure 2

Schematic comparison of chimpanzee and human CMP-Neu5Ac hydroxylase genomic DNA. In the human genome, the exon to AluSq (E-A) region in the chimpanzee genome is replaced by the sahAluY.

Figure 3

Phylogenetic tree of Alu subfamilies. Human Alu elements having intact head and tail were randomly selected from both the GenBank database and the on-line database of Alu pairs (http://dir.niehs.nih.gov./ALU/). The tree was made by the neighbor-joining method (20). Distances were calculated with Kimura's two-parameter method (21). The poly(A) tails of sequences were not used in tree-making. The sequence of an AluJb element, which belongs to the old AluJb subfamily, was used as an outgroup. The average age of AluJb, AluSq, AluY, and AluYb8 subfamilies has been estimated at 81, 44, 19, and 3 million years, respectively (ref. 22). The Alu elements shown in Fig. 1 are represented by Hs sahAluY, Pt sahAluSq, Gg sahAluSq, and Mm sahAluSq. msAluY indicates the sequence most similar to the one of sahAluY. Hs, Homo sapiens; Pt, Pan troglodytes; Gg, Gorilla gorilla; Mm, Macaca mulatta.

Figure 4

Model of the Alu-mediated replacement event that occurred in the CMP-Neu5Ac hydroxylase gene in the hominid lineage. (A) Double-strand break indicated by vertical arrows. The solid boxes represent the sahAluSq elements; the shaded box represents the 92-bp exon. The Alu target region containing A/T stretch is located in the 5′ immediately adjacent region of the double-strand break point. (B) Homologous recombination between the injured and intact alleles, after 5′-to-3′ exonucleolytic digestion, which generates a 3′-single-stranded tail. (C) Target-priming to the target site by free sahAluY RNA transcript. Free sahAluY RNA transcript is indicated by both a cross-hatched box and the letter “A,” representing the poly(A) tail. (D) Reverse transcription. An arrow indicates reverse transcription. The open box represents the sahAluY cDNA. (E) Elimination of RNA. (F) Annealing between the sahAluY cDNA and genomic sahAluSq. (G) DNA synthesis. An arrow indicates DNA synthesis. (H) Production of the allele that lacks the E-A region by DNA replication. This allele is derived from the upper strand shown in G.