Reading between the LINEs: human genomic variation induced by LINE-1 retrotransposition - PubMed (original) (raw)

Reading between the LINEs: human genomic variation induced by LINE-1 retrotransposition

F M Sheen et al. Genome Res. 2000 Oct.

Abstract

The insertion of mobile elements into the genome represents a new class of genetic markers for the study of human evolution. Long interspersed elements (LINEs) have amplified to a copy number of about 100,000 over the last 100 million years of mammalian evolution and comprise approximately 15% of the human genome. The majority of LINE-1 (L1) elements within the human genome are 5' truncated copies of a few active L1 elements that are capable of retrotransposition. Some of the young L1 elements have inserted into the human genome so recently that populations are polymorphic for the presence of an L1 element at a particular chromosomal location. L1 insertion polymorphisms offer several advantages over other types of polymorphisms for human evolution studies. First, they are typed by rapid, simple, polymerase chain reaction (PCR)-based assays. Second, they are stable polymorphisms that rarely undergo deletion. Third, the presence of an L1 element represents identity by descent, because the probability is negligible that two different young L1 repeats would integrate independently between the exact same two nucleotides. Fourth, the ancestral state of L1 insertion polymorphisms is known to be the absence of the L1 element, which can be used to root plots/trees of population relationships. Here we report the development of a PCR-based display for the direct identification of dimorphic L1 elements from the human genome. We have also developed PCR-based assays for the characterization of six polymorphic L1 elements within the human genome. PCR analysis of human/rodent hybrid cell line DNA samples showed that the polymorphic L1 elements were located on several different chromosomes. Phylogenetic analysis of nonhuman primate DNA samples showed that all of the recently integrated "young" L1 elements were restricted to the human genome and absent from the genomes of nonhuman primates. Analysis of a diverse array of human populations showed that the allele frequencies and level of heterozygosity for each of the L1 elements was variable. Polymorphic L1 elements represent a new source of identical-by-descent variation for the study of human evolution. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF242435-AF242451.]

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Figures

Figure 1

Figure 1

Schematic diagram of the L1Hs display and results. (A) L1 display protocol. A truncated or full-length L1Hs-Ta (rectangle) is depicted surrounded by flanking DNA (solid lines). The relative locations of the ACA and _Acc_I sites are indicated. The dashed lines represent the products of two rounds of PCR amplifications. The arrows below indicate the relative positions and orientations of the arbitrary decamers and the 19- or 20-bp-long flanking primers that were synthesized to match the non-L1 flanking DNA sequences (3FPa, 3FPb, and 5FP). (B) L1 display results. A typical L1 display experiment performed with a single decamer on genomic DNA from six individuals is shown. The figure is an autoradiograph of the gel after Southern blotting and hybridization with oligonucleotide Hb. Ca-1 and 2, European/Caucasian 1 and 2; Ch, Chinese; Dr, Druse; Py, Pygmy; Me, Melanesian. The mobilities of the DNA size markers are indicated.

Figure 1

Figure 1

Schematic diagram of the L1Hs display and results. (A) L1 display protocol. A truncated or full-length L1Hs-Ta (rectangle) is depicted surrounded by flanking DNA (solid lines). The relative locations of the ACA and _Acc_I sites are indicated. The dashed lines represent the products of two rounds of PCR amplifications. The arrows below indicate the relative positions and orientations of the arbitrary decamers and the 19- or 20-bp-long flanking primers that were synthesized to match the non-L1 flanking DNA sequences (3FPa, 3FPb, and 5FP). (B) L1 display results. A typical L1 display experiment performed with a single decamer on genomic DNA from six individuals is shown. The figure is an autoradiograph of the gel after Southern blotting and hybridization with oligonucleotide Hb. Ca-1 and 2, European/Caucasian 1 and 2; Ch, Chinese; Dr, Druse; Py, Pygmy; Me, Melanesian. The mobilities of the DNA size markers are indicated.

Figure 2

Figure 2

Identification of LID 1–6 by L1 display and verification of LID dimorphism. (a) L1 display. The products of the second round of PCR amplifications were Southern blotted and probed with oligonucleotide Hb (Fig. 1_a_). Digital photographs (Kodak DC40) of the sections of the autoradiograms that depict LID 1–6 are shown. Each lane represents the results obtained from one individual. (b) PCR amplification with primers ACA and 3FPa. Digital photographs of ethidium bromide-stained gels (Kodak DC40) are shown. (c) Southern blot of _Acc_I-digested genomic DNA hybridized with 3′-flanking probes. The probes were generated by amplifying the non-L1Hs 3′-flanking DNA of the cloned LIDs with primers 3FPa and 3FPb and tailing the products by the addition of [α-32P]dCTP with terminal transferase. Digital photographs of the autoradiograms of the hybridized blots are depicted. Fragments representing both the empty alleles (slower mobility) and the occupied alleles (faster mobility) can be seen in the blots hybridized with the 3′-flanking probes from LID 1, 2, 4, 5. The two bands in the Ca-2 and Dr samples of the LID-1 blot (positions indicated by short lines) are located extremely close to one another. The absence of fragments for the Ca-1 samples in the LID-2 and LID-4 blots was due to an insufficient loading of DNA. (d,e) PCR amplification of LID 1–6 with 5′- and 3′-flanking primers. Genomic DNA was amplified with primers 3FPa and 5FP. For each LID, 200 ng genomic DNA was amplified with the LID-specific primers 5FP and 3FPa. The arrowheads indicate the location of the amplified products of the empty alleles. The larger bands are the amplified products of the filled alleles. Digital photographs of the ethidium bromide-stained gels (d) or the autoradiograms of the gel after blotting and hybridization with probe Hb (e) are shown.

Figure 3

Figure 3

Quantification of L1Hs-Ta 3′ UTRs in the human genome. Southern blot quantification. Genomic DNA from (1) individual Ca-2, (2) mouse LMTK-cells, and (3) LMTK-cells to which plasmid pL1.2A, which contains a subset Ta L1Hs), was added were digested with _Sau_3AI and _Acc_I to release the L1Hs 3′ UTRs. Samples 2 and 3 were mixed in varying ratios to represent 0, 700, 1050, 1400, and 2100 relative copies of L1Hs per haploid genome. Samples (1 μg) of each were Southern blotted and hybridized to oligomer C, a L1Hs-Ta-specific probe. The relative activity of the hybridized bands was measured on a PhosphorImager (Molecular Dynamics). Results indicate a relative copy number of 2250 for the Ca-2 band and a linear relationship of copy number to signal in the standard lanes.

Figure 4

Figure 4

Schematic diagram of the LID-insertion PCR assay. The diagram displays the LI-insertion dimorphism assay. The L1 element is in dark green, with the flanking unique sequence regions in yellow. The 5′- and 3′-flanking unique sequence primers are in red stripe and black, respectively. The internal Ta subfamily specific primer is shown in light green. The PCR amplicons generated from the L1-occupied and empty alleles are shown as green and red lines. In the assay, two PCR reactions are utilized to genotype each L1 insertion. In the first PCR reaction, 5′- and 3′-flanking unique sequence oligonucleotide primers are used to assay individual loci for empty alleles that do not contain Ta L1 elements. In the second PCR reaction, the 3′-flanking unique sequence oligonucleotide is used for the PCR, along with Ta L1 element subfamily specific primer ACA. With this approach, the size of the PCR-based amplicons generated from L1-occupied alleles is minimized and individual loci are tested for L1-occupied sites. The expected results of the PCR reactions are shown for the three potential genotypes at the bottom of the figure.

Figure 5

Figure 5

Principal components analysis of LID elements in humans. A principal coordinate (PC) genetic map of 14 human populations as defined by variation in six LINE elements is presented in three views. The top two panels (a,b) show two-dimensional views of the data by plotting PC1 against PC2 and PC3, respectively. The lower panel (c) shows a three-dimensional view of the genetic distances. The first, second, and third PC axes account for 59.1%, 20.6%, and 11.4% of the variation in the samples. Thus, panel a captures 79.7% of the sample variation, panel b 70.5%, and panel c 91.1%. Population classifications–African: Bantu (BAN), African American (AFRAM), !Kung (!Kung); Asian: Armenian (ARM); European/Caucasian: Syrian (SYR), Turkish Cypriot (TUR), French (FRE), Breton (BRE), German (GER), Swiss (SWI), European-American (CAU), Hispanic American (HIS); Native American: Greenland Native (GREEN), Alaska Native (ALAS). A hypothetical ancestral population (ROOT) with a frequency of 0.0 for all LINE insertions was added into the analysis and serves as a point of initial dispersion for all other points on the map.

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