Identification and characterization of new long conserved noncoding sequences in vertebrates

Abstract

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENUinduced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results Electronic supplementary materialThe online version of this article

Ultraconserved Elements in the Human Genome: Association and Transmission Analyses of Highly Constrained Single-Nucleotide Polymorphisms

“…Their extreme conservation reflects functional constraints, rather than mutational coldspots (Drake et al 2006;Chen et al 2007;Katzman et al 2007;Sakuraba et al 2008). Yet, paradoxically, these elements have produced no detectable phenotypes when deleted in mice (Ahituv et al 2007).…”

“…Multiple lines of evidence have shown that ultraconserved elements are under negative selection (Drake et al 2006;Chen et al 2007;Katzman et al 2007;Sakuraba et al 2008), although the strength of the negative selection may be relatively weak and the consequence not immediate, as mice homozygous for a deletion of the UCE showed no overt phenotype (Ahituv et al 2007). We approached the same question from a human genetics perspective: If the effects of the derived alleles of hcSNPs are strongly and immediately deleterious and affecting the viability of offspring, then one should expect to observe an undertransmission of the derived allele from parents to offspring.…”

“…These ultraconserved or nearly ultraconserved elements are expected to harbor important biological functions as they are dosage sensitive (Derti et al 2006;Chiang et al 2008), are enriched in or near specific classes of genes (Bejerano et al 2004), and are under purifying selection (Drake et al 2006;Chen et al 2007;Katzman et al 2007;Sakuraba et al 2008). Indeed, a number of reports have demonstrated the ability of intergenic and intronic highly conserved elements to direct tissue-specific expression (Woolfe et al 2005;McEwen et al 2006;Pennacchio et al 2006;Ahituv et al 2007;Paparidis et al 2007;Visel et al 2008).…”

ENU-Based Gene-Driven Mutagenesis in the Mouse: A Next-Generation Gene-Targeting System

“…We conducted a feasibility study of screening of ENU-induced mutations in G1 genomic DNAs and the ENU-induced mutation rate was primarily estimated to be 1 mutation per megabase pairs (Mb) in G1 genomic DNA [13,30,31]. In other words, 3,000 ENUinduced mutations may be randomly induced in a given G1 mouse, since the size of the mouse genome is approximately 3 × 10 9 bp.…”

Next-generation gene targeting in the mouse for functional genomics

“…ENU equally induced the mutations in the LCNS as well (57,58). We also analyzed ENU-induced mutation spectrum in details (57,59).…”