The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

We previously isolated two human L1 elements (L1.2 and LRE2) as the progenitors of disease-producing insertions. Here, we show these elements can actively retrotranspose in cultured mammalian cells. When stably expressed from an episome in HeLa cells, both elements retrotransposed into a variety of chromosomal locations at a high frequency. The retrotransposed products resembled endogenous L1 insertions, since they were variably 5' truncated, ended in poly(A) tracts, and were flanked by target-site duplications or short deletions. Point mutations in conserved domains of the L1.2-encoded proteins reduced retrotransposition by 100- to 1000-fold. Remarkably, L1.2 also retrotransposed in a mouse cell line, suggesting a potential role for L1-based vectors in random insertional mutagenesis.

Human L1 elements are highly abundant poly(A) (non-LTR) retrotransposons whose second open reading frame (ORF2) encodes a reverse transcriptase (RT). We have identified an endonuclease (EN) domain at the L1 ORF2 N-terminus that is highly conserved among poly(A) retrotransposons and resembles the apurinic/apyrimidinic (AP) endonucleases. Purified L1 EN protein (L1 ENp) makes 5'-PO4, 3'-OH nicks in supercoiled plasmids, shows no preference for AP sites, and preferentially cleaves sequences resembling L1 in vivo target sequences. Mutations in conserved amino acid residues of L1 EN abolish its nicking activity and eliminate L1 retrotransposition. We propose that L1 EN cleaves the target site for L1 insertion and primes reverse transcription.

Although LINE-1 (long interspersed nucleotide element-1, L1) retrotransposons comprise 17% of the human genome, an exhaustive search of the December 2001 ''freeze'' of the haploid human genome working draft sequence (95% complete) yielded only 90 L1s with intact ORFs. We demonstrate that 38 of 86 (44%) L1s are polymorphic as to their presence in human populations. We cloned 82 (91%) of the 90 L1s and found that 40 of the 82 (49%) are active in a cultured cell retrotransposition assay. From these data, we predict that there are 80 -100 retrotransposition-competent L1s in an average human being. Remarkably, 84% of assayed retrotransposition capability was present in six highly active L1s (hot L1s). By comparison, four of five full-length L1s involved in recent human insertions had retrotransposition activity comparable to the six hot L1s in the human genome working draft sequence. Thus, our data indicate that most L1 retrotransposition in the human population stems from hot L1s, with the remaining elements playing a lesser role in genome plasticity.

Revealing the mechanisms for neuronal somatic diversification remains a central challenge for understanding individual differences in brain organization and function. Here we show that an engineered human LINE-1 (for long interspersed nuclear element-1; also known as L1) element can retrotranspose in neuronal precursors derived from rat hippocampus neural stem cells. The resulting retrotransposition events can alter the expression of neuronal genes, which, in turn, can influence neuronal cell fate in vitro. We further show that retrotransposition of a human L1 in transgenic mice results in neuronal somatic mosaicism. The molecular mechanism of action is probably mediated through Sox2, because a decrease in Sox2 expression during the early stages of neuronal differentiation is correlated with increases in both L1 transcription and retrotransposition. Our data therefore indicate that neuronal genomes might not be static, but some might be mosaic because of de novo L1 retrotransposition events.

Long Interspersed Element-1 (LINE-1 or L1) retrotransposons have dramatically impacted the human genome. L1s must retrotranspose in the germ-line or during early development to ensure their evolutionary success; yet the extent to which this process impacts somatic cells is poorly understood. We previously demonstrated that engineered human L1s can retrotranspose in adult rat hippocampus progenitor cells (NPCs) in vitro and in the mouse brain in vivo 1 . Here, we demonstrate that NPCs isolated from human fetal brain and NPCs derived from human embryonic stem cells (hESCs) support the retrotransposition of engineered human L1s in vitro. Furthermore, we developed a quantitative multiplex polymerase chain reaction that detected an increase in the copy number of endogenous L1s in the hippocampus and in several regions of adult human brains when compared to the copy number of endogenous L1s in heart or liver genomic DNAs from the same donor. These data suggest that de novo L1 retrotransposition events may occur in the human brain and, in principle, have the potential to contribute to individual somatic mosaicism.The human nervous system is complex, containing approximately 10 15 synapses with a vast diversity of neuronal cell types and connections that are influenced by complex and incompletely understood environmental and genetic factors 2 . Neural progenitor cells (NPCs) give rise to the three main lineages of the nervous system: neurons, astrocytes, and oligodendrocytes. To determine if human NPCs can support L1 retrotransposition, we transfected human fetal brain stem cells (hCNS-SCns) (Fig. 1A) 3 with an expression construct containing a retrotransposition-competent human L1 driven from its native promoter (RC-L1; L1 RP ). The RC-L1 also contains a retrotransposition indicator cassette in its 3′ UTR, consisting of a reversed copy of the enhanced green fluorescent protein (EGFP) expression cassette, which is interrupted by an intron in the same transcriptional orientation as the RC-L1 4-7 . The

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the ϳ400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.Retrotransposons are DNA sequences that can move (i.e., retrotranspose) to different genomic locations via an RNA intermediate. They are present in the genomes of virtually all eukaryotes and can be subdivided into two general structural classes. Long terminal repeat (LTR) retrotransposons resemble simple retroviruses but lack a functional envelope (Env) gene (2). Non-LTR retrotransposons lack LTRs and generally terminate in a polyadenylic acid [poly(A)] tail (20,23).L1s are the most abundant non-LTR retrotransposons in the human genome and comprise approximately 17% of nuclear DNA (42). The overwhelming majority of L1s are retrotransposition defective (RD-L1s) and cannot retrotranspose because they are 5Ј truncated, internally rearranged, or mutated (23); however, an estimated 30 to 60 human L1s remain retrotransposition competent (RC-L1s) (40). RC-L1s are 6.0 kb in length and contain a 5Ј untranslated region (UTR) harboring an internal promoter (43), two nonoverlapping open reading frames (open reading frame 1 [ORF1] and ORF2) (7, 41), and a 3Ј UTR ending in an unorthodox poly(A) tail (20,46). In addition, these elements are flanked by variable-length target site duplications, which are hallmarks of the retrotransposition process (20).Non-LTR retrotransposons encode endonuclease activities, which can generate either site-specific (4, 11, 47) or relatively non-site-specific nicks in chromosomal DNA (5, 10). The liberated 3Ј hydroxyl residue then acts as a primer for reverse transcription of the retrotransposon RNA by the retrotransposon-encoded reverse transcriptase (RT) by a mechanism termed target site-primed reverse transcription (TPRT) (28, 29). Thus, the processes of integration and reverse transcription are coupled for non-LTR retrotransposons.Biochemical studies revealed that ORF1 encodes a 40-kDa RNA binding protein that colocalizes with L1 RNA in cytoplasmic ribonucleoprotein particles (RNPs) (17, 18). ORF2 encodes a multifunctional protein containing endonuclease and RT activities (10, 34) and also has a carboxyl-terminal cysteine-rich domain (C) of unknown function (9). Using an assay to monitor L1 ret...

Summary Long Interspersed Element-1 (LINE-1 or L1) sequences comprise the bulk of retrotransposition activity in the human genome; however, the abundance of highly active or ‘hot’ L1s in the human population remains largely unexplored. Here, we used a fosmid-based, paired-end DNA sequencing strategy to identify 68 full-length L1s which are differentially present among individuals but are absent from the human genome reference sequence. The majority of these L1s were highly active in a cultured cell retrotransposition assay. Genotyping 26 elements revealed that two L1s are only found in Africa and that two more are absent from the H952 subset of the Human Genome Diversity Panel. Therefore, these results suggest that ‘hot’ L1s are more abundant in the human population than previously appreciated, and that ongoing L1 retrotransposition continues to be a major source of inter-individual genetic variation.