Genomic characterization of recent human LINE-1 insertions: evidence supporting random insertion.

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.

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

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.]

Many human L1 elements are capable of retrotransposition.

Using a selective screening strategy to enrich for active L1 elements, we isolated 13 full-length elements from a human genomic library. We tested these and two previously-isolated L1s (L1.3 and L1.4) for reverse transcriptase (RT) activity and the ability to retrotranspose in HeLa cells. Of the 13 newly-isolated L1s, eight had RT activity and three were able to retrotranspose. L1.3 and L1.4 possessed RT activity and retrotransposed at remarkably high frequencies. These studies bring the number of characterized active human L1 elements to seven. Based on these and other data, we estimate that 30-60 active L1 elements reside in the average diploid genome.

LINE-1: a human transposable element.

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.

Binding of the ubiquitous nuclear transcription factor YY1 to a cis regulatory sequence in the human LINE-1 transposable element.

The first step of the currently favored model for the mechanism of transposition of the human LINE-1 element involves the synthesis of full length LINE-1 mRNA. Previous work demonstrated that the 5'-terminal 100 base pairs of the human LINE-1 element (L1Hs) has an important role in regulating it's expression. Here we report further deletion analysis revealing the presence of a cis regulatory element overlapping the region between base pairs +12 and +18. Oligonucleotides containing this sequence form a specific complex with a nuclear protein extracted from NTera2D1 and Jurkat cells, and with recombinant YY1 produced in E. coli. The complex is competed by YY1 binding sites found in other genes, and is ablated by anti-YY1 serum. These results suggest that YY1 is involved in the regulation of L1Hs transcription and therefore transposition.

Studies on p40, the leucine zipper motif-containing protein encoded by the first open reading frame of an active human LINE-1 transposable element.

Full-length human LINE-1 retrotransposons encode p40 proteins with varying electrophoretic mobilities under denaturing conditions. The p40 expressed from the first open reading frame in the LINE-1 copy designated L1.2A co-electrophoreses with the endogenous p40 in human teratocarcinoma cells. This finding is consistent with previous data indicating that L1.2A is an active element. The amino acid sequence in the central region of the L1.2A p40 accounts, at least in part, for its characteristic mobility. This region includes sequences which can, in principle, form a leucine zipper.

Transmissible familial Creutzfeldt-Jakob disease associated with five, seven, and eight extra octapeptide coding repeats in the PRNP gene.

The PRNP gene, encoding the amyloid precursor protein that is centrally involved in Creutzfeldt-Jakob disease (CJD), has an unstable region of five variant tandem octapeptide coding repeats between codons 51 and 91. We screened a total of 535 individuals for the presence of extra repeats in this region, including patients with sporadic and familial forms of spongiform encephalopathy, members of their families, other neurological and non-neurological patients, and normal controls. We identified three CJD families (in each of which the proband's disease was neuropathologically confirmed and experimentally transmitted to primates) that were heterozygous for alleles with 10, 12, or 13 repeats, some of which had "wobble" nucleotide substitutions. We also found one individual with 9 repeats and no nucleotide substitutions who had no evidence of neurological disease. These observations, together with data on published British patients with 11 and 14 repeats, strongly suggest that the occurrence of 10 or more octapeptide repeats in the encoded amyloid precursor protein predisposes to CJD.

Identification, characterization, and cell specificity of a human LINE-1 promoter.

A constructed human LINE-1 (L1Hs) element containing intact 5' and 3' untranslatable regions and an in-frame fusion between the L1Hs open reading frame 1 and the bacterial lacZ gene (p1LZ) was found to promote the expression of beta-galactosidase in a variety of transiently transfected cell types in tissue culture. Full-length RNA was detected in the transfected cells. Most of the RNA transcripts initiated at or near the beginning of the L1Hs segment. Sequences within the L1Hs segment of p1LZ were sufficient for expression of the reporter gene; however, modulation of the transcriptional regulatory region by upstream sequences was not ruled out. Deletion analysis revealed that the sequences most critical for transcription were located within the first 100 bp of L1Hs. Other sequences within the first 668 bp of L1Hs also contributed to overall expression. Expression of p1LZ was high in human teratocarcinoma cells and low in all other cell types. This pattern of cell-type-specific expression matches the known pattern of endogenous L1Hs transcription in cultured cells.

Translation of LINE-1 DNA elements in vitro and in human cells.

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.

High-performance gel-permeation chromatography of polypeptides in a volatile solvent: rapid resolution and molecular weight estimations of proteins and peptides on a column of TSK-G3000-PW.

A gel-permeation column of TSK-G3000-PW that was equilibrated and developed with 36 or 45% acetonitrile in 0.1% trifluoroacetic acid fractionated mixtures of peptides with high resolving power. In addition, the elution volumes of 11 standard peptides and proteins were linearly related to the logarithms of their molecular weights in the acetonitrile-trifluoroacetic acid solvent at both low and high flow rates. Since the solvent is volatile and relatively transparent to short-wavelength ultraviolet light this high-performance gel-permeation system offers a rapid and highly sensitive method for the analysis, characterization, and purification of peptides and proteins from complex mixtures.