A family of repeated sequences dispersed through the genome of Lilium henryi

A family of dispersed repeats longer than 7 kilobase pairs (kbp) has been identified in the very large genome of Lilium henryi, and two subregions cloned. Initially a rapidly reannealing probe (C₀t<1 M s) was prepared by hydroxyapatite chromatography. Half the copies of all sequences repeated 15000 times per genome are expected to reanneal by this C₀t value. The probe hydridized to abundant fragments of 2, 5, and 7 kbp released from genomic DNA by Bam HI digestion. Twelve 2-kb fragments and ten 5-kb sequences were cloned into pBR322. Restriction mapping of the two sets of clones showed individual members to be quite similar. Length variation was no more than 200 base pairs (bp) between repeats, and consensus sites were present on 80%–90% of occasions. In situ hybridization using representative 2-kbp and 5-kbp clones showed each sequence to be dispersed throughout all chromosomal regions. Studies on the genomic organization suggested that the 2-kbp and 5-kbp sequences are usually adjacent, and that occasional absence of the internal Bam HI site results in the release of the 7-kbP fragment. There are at least 13000 copies of the full repeat per L. henryi genome, thus accounting for approximately 0.3% of the total of 32 million kbp.     

Distribution of repeated DNA families in the human genome

By means of restriction enzymes analysis and molecular hybridization, the distribution of repeated DNA families has been studied in the different DNA components into which the human genome can be fractionated by density gradient techniques. Three classes of DNA molecules have been analyzed: i) an homogeneous DNA component (satellite-like sequences; Q = 1.696 g/cm3, 3% of total DNA, AT repeated), ii) AT rich (Q = 1.698 g/cm3, 30% of total DNA, AT main-band) and GC rich (Q = 1.708 g/cm3, 6% of total DNA, GC main-band) DNA components. By this approach we have observed that Sau3A digestion of GC main-band gives rise to two bands of 75bp and 150bp, absent or under-represented in both AT rich DNA components. A preliminary characterization of these DNA fragments suggests that they contain one or more families of repeated sequences which fail to hybridize to EcoRI, HindIII and AluI families of repeats. In addition, we have observed that EcoRI sequences (alpha-RI DNA) are under-represented in GC main-band and show the same clustered organization in both AT rich DNA components.     

Replication pattern of human repeated DNA sequences

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the EcoRI 340 bp family (αRI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [³H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [³H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.     

A family of repeated sequences in Drosophila genome with telomeric localization: properties and polymorphism

The previously cloned Drosophila genome fragment Dm665 (2.4 kb) hybridizing with telomers on polytene chromosomes is a representative of the family of repeats, a part of which being organized in tandem clusters. The repeats are not transcribed in cell culture, are species-specific and represented in 200-250 copies per haploid genome. In natural and laboratory Drosophila lines polymorphism has been revealed with regard to homology with Dm665 in the telomeres.     

Simple repeated sequences in human satellite DNA.

In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.     

Species-specific homogeneity of the primate Alu family of repeated DNA sequences

We have determined the base sequence of several cloned Alu family members from the DNAs of a new world monkey (owl monkey) and a prosimian (galago). The three owl monkey Alu family members reported here belong to a single 300 base pair consensus sequence which closely resembles the human Alu family consensus. The galago Alu family members can best be represented as belonging to either of two related but distinct consensus sequences. One of the two galago Alu family subgroups (Type I) more accurately resembles the human consensus sequence than does the other subgroup (Type II). In this work we compare base sequences of human and galago Type I Alu family members. There are several examples of species-specific differences between the human and Type I galago sequences indicating that the Alu family members are effectively homogenized within a species.     

Characterization of a family of tandemly repeated DNA sequences in Triticeae

The recombinant plasmid dpTa1 has an insert of relic wheat DNA that represents a family of tandemly organized DNA sequences with a monomeric length of approximately 340 bp. This insert was used to investigate the structural organization of this element in the genomes of 58 species within the tribe Triticeae and in 7 species representing other tribes of the Poaceae. The main characteristic of the genomic organization of dpTa1 is a classical ladder-type pattern which is typical for tandemly organized sequences. The dpTa1 sequence is present in all of the genomes of the Triticeae species examined and in 1 species from a closely related tribe (Bromus inermis, Bromeae). DNA from Hordelymus europaeus (Triticeae) did not hybridize under the standard conditions used in this study. Prolonged exposure was necessary to obtain a weak signal. Our data suggest that the dpTa1 family is quite old in evolutionary terms, probably more ancient than the tribe Triticeae. The dpTa1 sequence is more abundant in the D-genome of wheat than in other genomes in Triticeae. DNA from several species also have bands in addition to the tandem repeats. The dpTa1 sequence contains short direct and inverted subrepeats and is homologous to a tandemly repeated DNA sequence from Hordeum chilense.     

Long interspersed repeated sequences of the mouse genome

Long interspersed repeated sequences of the mouse genome can be prepared by digesting reassociated DNA with single-strand nuclease. Length resolution reveals many discrete bands that can be assigned to 15 kbp and 6 kbp groups. The reassociated 6 kbp group (which we identify with the MIF-1 family) possesses significant sequence heterogeneity, evidenced by the production of several smaller fragments upon single-strand nuclease digestion of heteroduplexes. The sites of sequence heterogeneity are relatively few and can be mapped using additional restriction endonuclease cuts. We have mapped additional restriction sites into this group, particularly within a cloned HindIII 400 bp fragment, and have also clearly mapped one end of this relatively homogeneous long interspersed repeated sequence.     

Linkage in human heterochromatin between highly divergent Sau3A repeats and a new family of repeated DNA sequences (HaeIII family)

The hybridization of human DNA with three non-cross-hybridizing monomers (68 bp in length) of the heterochromatic Sau3A family of DNA repeats, indicates the coexistence within a Sau3A-positive genomic block of divergent Sau3A units as well as of unrelated sequences. To gain some insight into the structure of these human heterochromatic DNA regions, three previously cloned Sau3A-positive genomic fragments (with a total length of approximately 1900 base-pairs (bp] were sequenced. The analysis of the sequences showed the presence of clustered Sau3A units with different degrees of divergence and of two DNA regions of approximately 100 bp and 291 bp in length, unrelated to the family of repeats. A consensus sequence derived from the 24 identified Sau3A monomers presents, among highly variable regions, two less variant regions of 8 bp and 10 bp in length, respectively. The Sau3A-unrelated DNA fragment 291 bp in length, used as a probe on genomic DNA digested with a series of restriction enzymes, defines a "new" family of DNA repeats possessing periodicities for HaeIII (HaeIII family). Sau3A and HaeIII repeats display a high degree of linkage in a collection of Sau3A-positive genomic recombinant phages.     

Interspersion of different repeated sequences in the wheat genome revealed by interspecies DNA/DNA hybridisation.

The repeated sequences in oats DNA have been used to study chromosomal repeated sequence organisation in wheat. Approximately 75% of the wheat genome consists of repeated sequences but only approximately 20% will form heteroduplexes with repeated sequences from oats DNA at 60 degrees C in 0.18 M Na+. The proportion of wheat DNA that forms heteroduplexes with oats DNA is shown to be independent of the wheat DNA fragment length. However, the proportion of wheat DNA that is retained with the heteroduplexes when fractionated on hydroxyapatite is very dependent upon the wheat fragment length up to 3500 nucleotides. This is because more non-renatured wheat DNA is attached to the heteroduplexes with longer fragments. The results indicate that the repeated sequences in the wheat genome homologous to repeated sequences in oats are not clustered in the chromosomes but distributed amongst other repeated and possible non-repeated sequences.     

Conservation of repeated DNA sequences in aneuploid human tumor cells

A series of human neuroectodermal tumors, all containing more than the normal diploid DNA, and each with its own distinct chromosome mode, were studied using restriction enzyme cleavage and specific DNA sequence hybridization. Methods described were quite sensitive and quantitative and as few as 40 molecules with a given restriction site were reproducibly detected in total nuclear DNA. Analysis of several fluorescent gel bands associated with different chromosomal domains revealed no changes between any of the tumor and normal cells. Specific probe hybridization, using purified complex repeating sequences, indicated fidelity of base sequence, as well as preservation of the relative amounts of each of a number of minor related multimers in both the tumor and normal cells. Centromeric regions containing arrays of such sequences may be maintained in these tumor cells and furthermore it is possible that some of these cells are polyploid with respect to DNA sequences, rather than aneuploid as their chromosome profiles suggest.This paper is dedicated to the late H.S.N. Greene, our inspired teacher     

Members of the KpnI family of long interspersed repeated sequences join and interrupt alpha-satellite in the monkey genome.

Three different members of a family (KpnI-family) of interspersed repeated DNA sequences were found linked to alpha-satellite sequences in cloned segments of the African green monkey genome. In two of these segments the KpnI-family member is over 6 kbp in length and one of them is flanked by alpha-satellite on both sides indicating that it was inserted into a satellite array. Hybridization of subcloned portions of the family members to restriction endonuclease digests of monkey and human DNA and to a genomic library of African green monkey DNA indicate that 1) family members are interspersed in both the monkey and human genomes, 2) some family members may include sequences in addition to those in the three characterized here, 3) some family members may contain only parts of the sequences characterized here and 4) while the overall organization of the family is similar in the human and monkey genome the majority of the family members in each of the two genomes are distinctly identified by the variant position of certain restriction endonuclease sites. This last observation suggests that within each genome there is a tendency to maintain particular versions of the sequence. Observations 2) and 3) suggest that the KpnI family is complex and includes a variety of subfamilies.     

Female-specific DNA sequences in the chicken genome

Eight in silico W-specific sequences from the WASHUC1 chicken genome assembly gave female-specific PCR products using chicken DNA. Some of these fragments gave female-specific products with turkey and peacock DNA. Sequence analysis of these 8 fragments (3077 bp total) failed to detect any polymorphisms among 10 divergent chickens. In contrast, comparison of the DNA sequences of chicken with those of turkey and peacock revealed a nucleotide difference every 25 and 28 bp, respectively. Radiation hybrid mapping verified that these amplicons exist only on chromosome W. The homology of 6 W-specific fragments with chromo-helicase-DNA-binding gene and expressed sequenced tags from chicken and other species indicate that these fragments may have or have had a biological function. These fragments may be used for early sexing in commercial chicken and turkey flocks.     

A genome-wide analysis of FRT-like sequences in the human genome

Efficient and precise genome manipulations can be achieved by the Flp/FRT system of site-specific DNA recombination. Applications of this system are limited, however, to cases when target sites for Flp recombinase, FRT sites, are pre-introduced into a genome locale of interest. To expand use of the Flp/FRT system in genome engineering, variants of Flp recombinase can be evolved to recognize pre-existing genomic sequences that resemble FRT and thus can serve as recombination sites. To understand the distribution and sequence properties of genomic FRT-like sites, we performed a genome-wide analysis of FRT-like sites in the human genome using the experimentally-derived parameters. Out of 642,151 identified FRT-like sequences, 581,157 sequences were unique and 12,452 sequences had at least one exact duplicate. Duplicated FRT-like sequences are located mostly within LINE1, but also within LTRs of endogenous retroviruses, Alu repeats and other repetitive DNA sequences. The unique FRT-like sequences were classified based on the number of matches to FRT within the first four proximal bases pairs of the Flp binding elements of FRT and the nature of mismatched base pairs in the same region. The data obtained will be useful for the emerging field of genome engineering.     

Long poly(A) tracts in the human genome are associated with the Alu family of repeated elements

Long poly(dA).poly(dT) tracts (poly(A) tracts), regions of DNA containing at least 20 contiguous dA residues on one strand and dT residues on the complementary strand, are found in about 2 X 10(4) copies interspersed throughout the human genome. Using poly(dA).poly(dA) as a hybridization probe, we identified recombinant lambda phage that contained inserts of human DNA with poly(A) tracts. Three such tracts have been characterized by restriction mapping and sequence analysis. One major poly(A) tract is present within each insert and is composed of from 28 to 35 A residues. In each case, the poly(A) tract directly abuts the 3' end of the human Alu element, indicating that the major class of poly(A) tracts in the human genome is associated with this family of repeats. The poly(A) tracts are also adjacent to A-rich sequences and, in one case, to a polypurine tract, having the structure GA3-GA3-GA4-GA6-GA5-GA4. We suggest that repetitive cycles of unequal crossing over may give rise to both the long poly(A) and polypurine tracts observed in this study.