Rearranged sequences of a human Kpn I element.

Abstract

The complete nucleotide sequence of a human Kpn I element inserted into a-satellite DNA is presented.This sequence reveals several features of interest. First, a large block of DNA normally associated with Kpn I elements has been deleted. Second, the order of the remaining sequences is permuted in a manner that cannot be accounted for by simple inversion. Third, a significant open reading frame of 675 bases is detected. (5,8,11,12). This laboratory has isolated and characterized human DNA segments carrying unusual monomeric domains of asatellite sequence (13). Of particular interest are the nonsatellite sequences that have been found occasionally interrupting the long tandem arrays of satellites (10,13,14). Because satellite sequences undergo continual rectification by unequal crossover (15) and gene conversion, it seems possible that the interrupting sequences represent mobile elements that have inserted into satellite relatively recently in evolutionary time. In this study I focused on a part of the pa7 clone, which has been shown to carry a nonsatellite sequence inserted into a-satellite DNA (13). The invading sequence is shown here to be an unusual Kpn I element.The Kpn I elements are abundant (about 30,000 copies per haploid genome) and particularly interesting structurally. No terminal repeats have yet been found, making them unlike most transposable elements (16,17), and their variable constructions include deletions and inversions (11). In this paper the complete nucleotide sequence of an element inserted into human a-satellite DNA is presented. The sequence reveals the presence of a large sequence permutation, thereby describing yet another type of polymorphism found in the Kpn I family of primate dispersed repetitive elements. Moreover, a large open reading frame is found, suggesting a coding function. MATERIALS AND METHODSEnzymes and polynucleotide linkers were purchased from New England BioLabs, and isotopes were from New England Nuclear. DNA Sequence Determination. DNA sequence was determined by using the partial chemical cleavage method of Maxam and Gilbert (18). DNA was labeled at the 3' end with the Klenow fragment of DNA polymerase 1 and at the 5' end by polynucleotide kinase.Subcloning. The 4.0-kilobase (kb) Sal I-EcoRI DNA segment carrying a Kpn I element with flanking a-satellite DNA was purified from the previously described pa7 clone (13) and ligated into pBR322 DNA cut with Sal I and EcoRI.In order to facilitate the sequence determination, two experiments designed to introduce restriction sites at useful locations were performed. In the first experiment the subclone was opened at the Bgl II site and then treated for various periods with the exonuclease BAL-31. Progress of the digestion was followed by gel electrophoresis. Cla I linkers were blunt-end-ligated to the ends and cleaved by Cla I, and the molecules were recircularized by ligation. After transformation of Escherichia coli HB101 and selection on ampicillin plates, random colonies were used for "miniprep" (19) and restrict...

Human centromeric DNAs

“…The mammalian Kpn LINE family (also known as LINE-1 or L1) has members of 6-7 kb in length located every 30-60 kb in the human genome. Presumably, such LINE sequences are also substantially underrepresented in human centromeres, accounting for their limited observance in human centromeric DNA arrays (e.g., Potter 1984;Wevrick et al 1992;Prades et al 1996).…”

SINE and LINE within human centromeres

“…However, certain Alu and L1 elements have been detected within the centromeric alphoid DNA sequences of man and primates (Grimaldi and Singer 1983;Potter 1984;Higgins et al, 1985). A more systematic search for such repeats within the human chromosome 21 centromeric sequences has shown that they were quite frequent in this region, too (Sainz et al 1992; Fig.…”

The sequence of a large L1Md element reveals a tandemly repeated 5' end and several features found in retrotransposons.

“…Primate Li sequences have been shown to be evolutionarily related to the LlMd family by DNA hybridization and sequence analysis (36,37,57 (35,37,46). Martin et al (37) compared a 312-bp region of monkey and mouse Li sequences, finding a ratio of silent versus replacement differences indicating that this portion of Li has evolved under selection for protein function.…”