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Telomeres gradually shorten as human somatic cells divide and a correlation has been observed between the average telomere length and cell senescence. It has been proposed that the genes responsible for cell senescence are located near the telomere and are activated when telomere length reaches a critical point. This is consistent with evidence from Saccharomyces cerevisiae, in which genes are regulated differently depending on their distance from the telomere. We investigated the possibility that differential gene expression is conferred by telomere length in human cells. A plasmid containing the neomycin phosphotransferase (neo) gene was transfected into the SV40-transformed human fibroblast cell line LM217. In one transfectant the plasmid was integrated at the telomere of chromosome 13. Subclones of this cell line that had various lengths of telomeric repeat sequences on the end of this chromosome were isolated. No effect on neo gene expression was found when the length of the telomere varied between 25 and 0.5 kb, as demonstrated by colony forming ability, growth rates and RNA blot analysis. These results therefore suggest that putative chromatin structural differences conferred by telomere length do not affect the expression of genes located near telomeres.

The mammalian interspersed repetitive (MIR) element was amplified in mammals 130 million years ago. The MIR element is at least 260 bp in length and is found in approximately 105 copies in the mammalian genome. We analyzed copies of the MIR element in the DNA of various mammals to determine its relationship to the structure and function of genes, in an attempt to identify specific uses of the MIR element within the mammalian genome. We found that alternative splicing within the acetylcholine receptor gene in humans takes place within the MIR element and results in the incorporation of part of the MIR element into the coding sequence of this gene. Furthermore, the polyadenylation signal (AATAAA) at the 3' end of four different mammalian genes is derived from the MIR element. These uses of the MIR element suggest that other regulatory sequences found within the mammalian genome originated from ancient transposable elements, many of which may no longer be recognizable.

Extrachromosomal circular DNA (eccDNA) generated from chromosomal DNA is found in all mammalian cells and increases with cell stress or aging. Studies of eccDNA structure and mode of formation provide insight into mechanisms of instability of the mammalian genome. Previous studies have suggested that eccDNA is generated through a process involving recombination between repetitive sequences. However, we observed that approximately one half of the small eccDNA fragments cloned from HeLa S3 cells were composed entirely of nonrepetitive or low-copy DNA sequences. We analyzed four of these fragments by polymerase chain reaction and nucleotide sequencing and found that they were complete eccDNAs. We then screened a human genomic library with the eccDNAs to isolate the complementary chromosomal sequences. Comparing the recombination junctions within the eccDNAs with the chromosomal sequences from which they were derived revealed that nonhomologous recombination was involved in their formation. One of the eccDNAs was composed of two separate sequences from different parts of the genome. These results suggest that rejoining of ends of fragmented DNA is responsible for the generation of a substantial portion of the eccDNAs found in HeLa S3 cells.

The mechanisms of recombination responsible for random integration of transfected DNA into the genome of normal human cells have been investigated by analysis of plasmid-cell DNA junctions. Cell clones containing integrated plasmid sequences were selected by morphological transformation of primary human fibroblasts after transfection with a plasmid containing simian virus 40 sequences. Nucleotide sequence analysis of the plasmid-cell DNA junctions was performed on cloned DNA fragments containing the integration sites from two of these cell clones. Polymerase chain reaction was then performed with human cell DNA from primary fibroblasts to isolate the cell DNA from the same sites before plasmid integration. Comparison of the sequences at the plasmid-cell DNA junctions with those of both the original plasmid and the cell DNA demonstrated short sequence similarities and additional nucleotides, typical of nonhomologous recombination. Evidence of short deletions in the cell DNA at the plasmid integration sites suggests that integration occurred by a mechanism similar to that used for repair of spontaneous or gamma ray-induced strand breaks. Plasmid integration occurred within nonrepetitive cell DNA with no major rearrangements, although rearrangements of the cell DNA at the integration site occurred in one of the clones after integration.