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Among the 10(5) LINE-1 sequences (L1Hs) in the human genome are one or more 6-kb segments that are active retrotransposons. Expression of these retrotransposons appears to be favored in cells of germ line origin, as well as in some other tumor cells of epithelial origin. In such cells, the product of the first L1Hs open reading frame (ORF), a protein called p40, is detectable; p40 has no apparent similarity to gag proteins, but contains a leucine zipper region which may be responsible for the occurrence of p40 multimers. Transcription of L1Hs initiates at residue 1 although the transcriptional regulatory regions are downstream in the first 670 bp of the 5' untranslated region; deletion of a YY1-binding site in the first 20 bp reduces transcription by fivefold. Translation of the second ORF, which encodes reverse transcriptase, is independent of the translation of the frame encoding p40.

The LINE-1 (L1) family of interspersed DNA sequences found throughout the human genome (L1 Homo sapiens, L1Hs) includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. We report that an antiserum against the polypeptide encoded by the L1Hs 5' open reading frame (ORF1) detects, in human cells, an endogenous ORF1 protein as well as the ORF1 product of an appropriate transfecting recombinant vector. The endogenous polypeptide is most abundant in teratocarcinoma and choriocarcinoma cells, among those cell lines tested; it appears to be a single species of approximately 38 kDa. In contrast, RNAs synthesized in vitro from cDNAs representing full-length, polyadenylylated cytoplasmic L1Hs RNA yield, upon in vitro translation, ORF1 products of slightly different sizes. This is consistent with the fact that the various cDNAs are different and represent transcription of different genomic L1Hs elements. In vitro studies additionally suggest that translation of ORF1 is initiated at the first AUG codon. Finally, in no case was an ORF1-ORF2 fusion protein detected.

The amplification of DNA by LINE-1 (L1) retrotransposons has created a large fraction of the human genome. To better understand their role in human evolution we endeavored to delineate the L1 elements that have amplified since the emergence of the hominid lineage. We used an approach based on shared sequence variants to trace backwards from the currently amplifying Ta subfamily. The newly identified groups of insertions account for much of the molecular evolution of human L1s. We report the identification of a L1 subfamily that amplified both before and after the divergence of humans from our closest extant relatives. Progressively more modern groups of L1s include greater numbers of insertions. Our data are consistent with the hypothesis that the rate of L1 amplification has been increasing during recent human evolution.

LINE-1 (L1) elements play an important creative role in genomic evolution by distributing both L1 and non-L1 DNA in a process called retrotransposition. A large percentage of the human genome consists of DNA that has been dispersed by the L1 transposition machinery. L1 elements are not randomly distributed in genomic DNA but are concentrated in regions with lower GC content. In an effort to understand the consequences of L1 insertions, we have begun an investigation of their genomic characteristics and the changes that occur to them over time. We compare human L1 insertions that were created either during recent human evolution or during the primate radiation. We report that L1 insertions are an important source for the creation of new microsatellites. We provide evidence that L1 first strand cDNA synthesis can occur from an internal priming event. We note that in contrast to older L1 insertions, recent L1s are distributed randomly in genomic DNA, and the shift in the L1 genomic distribution occurs relatively rapidly. Taken together, our data indicate that strong forces act on newly inserted L1 retrotransposons to alter their structure and distribution.

Long interspersed element-1 (LINE-1) sequences are a large family of transposable elements found in the genomes of all mammals (Burton et al. 1986; Xiong and Eickbush 1990). They belong to the poly(A)-containing (also called the non-long-terminal-repeat) class of retrotransposons. The consensus human LINE-1 (L1Hs) is 6.0 kb long, contains two nonoverlapping reading frames, terminates in an A-rich tail, and is surrounded by a short (4–20 bp) duplication of non-LINE-1 (L1) sequence, the target site duplication (Fanning and Singer 1987). The human genome contains an estimated 105 truncated and 4 × 103 full-length L1Hs elements (Adams et al. 1980; Grimaldi et al. 1984; Hwu et al. 1986), which together constitute ∼15% of the genome (Smit 1996). The majority of L1Hs elements are not capable of transposition because they are truncated or rearranged or contain other significant mutations. Nevertheless, abundant evidence indicates that L1Hs transposition continues to occur. Several examples of recent de novo transposition events have been identified largely as the result of mutations caused by the insertion of new L1Hs elements into functional genes (Kazazian et al. 1988; Woods-Samuels et al. 1989; Miki et al. 1992; Narita et al. 1993; Bleyl et al. 1994; Holmes et al. 1994). All but one of the newly transposed L1Hs sequences in the human genome belongs to a subfamily of L1 elements called Ta (transcribed, subset a). This subfamily was first recognized as a group of expressed elements with a high degree of sequence identity to one another (Skowronski et al. 1988). The Ta subfamily of L1 elements (L1Hs-Ta) are characterized by the presence of the sequence ACA in the 3′ untranslated region at position 5930–5932; numbers refer to the actively transposing element LRE-1 [Dombroski et al. 1991]). Elements with the genomic L1Hs consensus sequence have a GAG sequence at this position (Skowronski et al. 1988). Recent experiments have suggested that the human genome may contain 30–60 active L1Hs retrotransposons (Sassaman et al. 1997). The de novo insertion of a transposable element into the genome creates a new polymorphic genetic marker with a number of unique properties, as first described for Alu-insertion polymorphisms (Batzer and Deininger 1991; Perna et al., 1992; Deininger and Batzer 1993, 1995, 1999; Batzer et al. 1994, 1996; Stoneking et al. 1997). As with Alu-insertion polymorphisms, each L1Hs insertion represents a unique historic event. This is a result of the large number of potential target sites (theoretically equal to 3 × 109, the number of base pairs in the human genome) for the integration of new mobile elements. Thus, there is an extremely low likelihood that two independent L1Hs insertions would land between the exact same base pairs, and in the unlikely event that this should occur, the two L1 elements would probably differ in length. Accordingly, individual loci bearing the same L1Hs insertion are identical by descent. In addition, the ancestral state of an L1Hs insertion is the absence of the element, because the direction of mutation is the insertion of a new mobile element into the genome. Orthologous loci in nonhuman primates may also be analyzed for the presence of the mobile element insertion to verify the ancestral state. Once inserted, most L1Hs elements are stable over long periods of time (Smit et al. 1995). In rare instances when a transposable element is deleted from the genome, the process is often imperfect, and a “footprint” of the original mobile element is left behind (Edwards and Gibbs 1992). Finally, L1 transposition has been occurring in mammals for millions of years and continues to this day, suggesting that a series of dimorphic L1Hs insertions that have arisen throughout human evolution may be found in the present-day human population; loci that are dimorphic in a population via the presence or absence of an L1 element are called LINE-1 insertion dimorphisms (LIDs). Most other types of genetic markers do not share these properties (Batzer et al. 1994, 1996; Stoneking et al. 1997). Thus, dimorphic transposable elements, such as Alu or L1, have a number of unique, useful properties for the study of human population genetics. Previously, dimorphic Alu elements have been used to provide insights into human genetic diversity and evolution (Perna et al. 1992; Batzer et al. 1994, 1996; Hammer 1994; Novick et al. 1995, 1998; Tishkoff et al. 1996; Stoneking et al. 1997). Dimorphic L1 elements could present another potentially useful class of genetic polymorphisms if a large number of such dimorphisms could be readily identified. Here, we describe the identification of dimorphic LINE elements from the human genome using a method called L1 display to ascertain the LIDs. Using this approach we have identified six LIDs from six individuals of diverse geographic backgrounds. In addition, we developed PCR-based assays to genotype these six individual LIDs in 850 individuals from 14 worldwide populations. Our results show that evolutionarily young LIDs can be readily identified by the L1 display assay and that these elements are a novel source of genomic variation for the study of human population genetics and forensics.

The recent insertion of mobile elements, of the Alu and L1 families, in the human genome provides a distinct class of polymorphism in the human genome. Because the insertion of these elements in the genome is so rare and once they are inserted they are stable; they represent a unique group of markers that are identical by descent. This type of marker is among the most informative in ascertaining relationships between individuals and populations. The assays for these markers are extremely robust, easy to perform, and readily adaptable to mass analysis and automation. In addition, as the insertion alleles are all newly arisen, the ancestral allele is always the allele missing the insertion. This information allows estimations of the roots of trees, which are not possible with all types of markers. The greatest potential for these markers is with upcoming developments that will allow the identification of new insertions in many different genomes simultaneously. These procedures will allow investigators to isolate markers that are particularly informative for specific populations and allow development of panels of markers tailored for particular populations.

The first moveable DNA elements to be studied at the molecular level were the transposable elements found in prokaryotes. Although several different types are now known, they all share two properties: first, the DNA within the element encodes a gene or genes that are required for transposition, and, second, specific DNA sequences are repeated in inverted orientation at the two ends of the element and are required for transposition.

The Ta (transcribed, subset a) subfamily of L1 LINEs (long interspersed elements) is characterized by a 3-bp ACA sequence in the 3' untranslated region and contains approximately 520 members in the human genome. Here, we have extracted 468 Ta L1Hs (L1 human specific) elements from the draft human genomic sequence and screened individual elements using polymerase-chain-reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity. One hundred twenty-four of the elements amenable to complete sequence analysis were full length ( approximately 6 kb) and have apparently escaped any 5' truncation. Forty-four of these full-length elements have two intact open reading frames and may be capable of retrotransposition. Sequence analysis of the Ta L1 elements showed a low level of nucleotide divergence with an estimated age of 1.99 million years, suggesting that expansion of the L1 Ta subfamily occurred after the divergence of humans and African apes. A total of 262 Ta L1 elements were screened with PCR-based assays to determine their phylogenetic origin and the level of human genomic variation associated with each element. All of the Ta L1 elements analyzed by PCR were absent from the orthologous positions in nonhuman primate genomes, except for a single element (L1HS72) that was also present in the common (Pan troglodytes) and pygmy (P. paniscus) chimpanzee genomes. Sequence analysis revealed that this single exception is the product of a gene conversion event involving an older preexisting L1 element. One hundred fifteen (45%) of the Ta L1 elements were polymorphic with respect to insertion presence or absence and will serve as identical-by-descent markers for the study of human evolution.