DNA polymerase-catalyzed elongation of repetitive hexanucleotide sequences: application to creation of repetitive DNA libraries

We demonstrate the elongation of various hexanucleotide sequences with thermophilic DNA polymerase, under isothermal or thermal cyclic reaction conditions. We prepared 10 types of double repeat hexanucleotide duplexes with various GC compositions containing between 0 and 6 GC nucleotides per repeat and incubated these duplexes with thermophilic Taq DNA polymerase and dNTPs at various temperatures. All of the model repetitive short duplexes were elongated under the isothermal incubation conditions, although there were some differences in the elongation efficiencies derived from the GC composition in the repetitive sequences. It was also found that all of the model repetitive duplexes were extended more effectively by a 3-step thermal cyclic reaction involving denaturation, annealing, and extension. On the basis of this technique, we prepared a glutamate-encoding short repetitive duplex and created long repetitive DNAs under isothermal and thermal cyclic reaction conditions. DNA sequencing analysis of the cloned repetitive DNA revealed that well-ordered long repetitive DNAs of various chain lengths were created by this DNA polymerase-catalyzed ligation method, and these were easily cloned into vectors by the TA-cloning method. This method could be useful for obtaining DNAs encoding arbitrary long repetitive amino acid sequences more effectively than the conventional T4 ligase-catalyzed ligation method.     

Prototypic sequences for human repetitive DNA

Summary We report a collection of 53 prototypic sequences representing known families of repetitive elements from the human genome. The prototypic sequences are either consensus sequences or selected examples of repetitive sequences. The collection includes: prototypes for high and medium reiteration frequency interspersed repeats, long terminal repeats of endogenous retroviruses, alphoid repeats, telomere-associated repeats, and some miscellaneous repeats. The collection is annotated and available electronically.[/ap ]Offprint requests to: J. Jurka     

Analysis of rat repetitive DNA sequences.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.     

A new approach to cloning of tandem repetitive DNA sequences

A new approach to screening of the repeated human DNA sequences tandemly arranged in the genome is described. Efficiency of the developed approach for search of tandemly arranged DNA sequences is corroborated by the obtained experimental data.     

Neurological proteins are not enriched for repetitive sequences

Proteins associated with disease and development of the nervous system are thought to contain repetitive, simple sequences. However, genome-wide surveys for simple sequences within proteins have revealed that repetitive peptide sequences are the most frequent shared peptide segments among eukaryotic proteins, including those of Saccharomyces cerevisiae, which has few to no specialized developmental and neurological proteins. It is therefore of interest to determine if these specialized proteins have an excess of simple sequences when compared to other sets of compositionally similar proteins. We have determined the relative abundance of simple sequences within neurological proteins and find no excess of repetitive simple sequence within this class. In fact, polyglutamine repeats that are associated with many neurodegenerative diseases are no more abundant within neurological specialized proteins than within nonneurological collections of proteins. We also examined the codon composition of serine homopolymers to determine what forces may play a role in the evolution of extended homopolymers. Codon type homogeneity tends to be favored, suggesting replicative slippage instead of selection as the main force responsible for producing these homopolymers.     

Interspersion of repetitive sequences in rat liver DNA

In rat liver DNA, which contains only 20% repetitive sequences, a close interspersion of repetitive and unique sequences is found in about 35 % of the total DNA. The mean length of repetitive and unique alternating sequences is respectively 230 and 400 base pairs.     

Centromeric repetitive DNA sequences in the genus Brassica

Representatives of two major repetitive DNA sequence families from the diploid Brassica species B. campestris and B. oleracea were isolated, sequenced and localized to chromosomes by in situ hybridization. Both sequences were located near the centromeres of many chromosome pairs in both diploid species, but major sites of the two probes were all on different chromosome pairs. Such chromosome specificity is unusual for plant paracentromeric repetitive DNA. Reduction of stringency of hybridization gave centromeric hybridization sites on more chromosomes, indicating that there are divergent sequences present on other chromosomes. In tetraploid species derived from the diploids, the number of hybridization sites was different from the sum of the diploid ancestors, and some chromosomes had both sequences, indicating relatively rapid homogenization and copy number evolution since the origin of the tetraploid species.     

Similarity and divergence among rodent repetitive DNA sequences

Summary We have analyzed the sequence of 63 B1 and 71 B2 repetitive elements from published data base sequences. The sequences conform to previously published consensus sequences, but are not identical to them. The B2 sequences show seven regions of high variability between family members, which we show points to the B2 family containing subfamilies; no similar evidence is found for subfamilies of the B1 family. The comparisons show no evidence for the emergence of species-specific variants of B1 or B2 sequences since the separation of murine and hamster lines of descent, nor of their concerted evolution within species in the last 10 million years.     

Highly repetitive DNA sequences in cyanobacterial genomes.

We characterized three distinct families of repeated sequences in the genome of the cyanobacterium Calothrix sp. strain PCC 7601. These repeated sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides. These elements were named short tandemly repeated repetitive (STRR) sequences. We used the three different Calothrix STRR sequences as probes to perform Southern hybridization experiments with DNAs extracted from various cyanobacterial strains, Bacillus subtilis, and Escherichia coli. The three different STRR sequences were found as repetitive genomic DNA components specific to the heterocystous strains tested. The role of the STRR sequences, as well as their possible use in taxonomic studies, is discussed.     

Simple sequences are ubiquitous repetitive components of eukaryotic genomes

Simple sequences are stretches of DNA which consist of only one, or a few tandemly repeated nucleotides, for example poly (dA) X poly (dT) or poly (dG-dT) X poly (dC-dA). These two types of simple sequence have been shown to be repetitive and interspersed in many eukaryotic genomes. Several other types have been found by sequencing eukaryotic DNA. In this report we have undertaken a systematical survey for simple sequences. We hybridized synthetical simple sequence DNA to genome blots of phylogenetically different organisms. We found that many, probably even all possible types of simple sequence are repetitive components of eukaryotic genomes. We propose therefore that they arise by common mechanisms namely slippage replication and unequal crossover and that they might have no general function with regards to gene expression. This latter inference is supported by the fact that we have detected simple sequences only in the metabolically inactive micronucleus of the protozoan Stylonychia, but not in the metabolically active macronucleus which is derived from the micronucleus by chromosome diminution.     

DNA ligase I competes with FEN1 to expand repetitive DNA sequences in vitro

Repeat sequences in various genomes undergo expansion by poorly understood mechanisms. By using an oligonucleotide system containing such repeats, we recapitulated the last steps in Okazaki fragment processing, which have been implicated in sequence expansion. A template containing either triplet or tandem repeats was annealed to a downstream primer containing complementary repeats at its 5'-end. Overlapping upstream primers, designed to strand-displace varying numbers of repeats in the downstream primer, were annealed. Human DNA ligase I joined overlapping segments of repeats generating an expansion product from the primer strands. Joining efficiency decreased with repeat length. Flap endonuclease 1 (FEN1) cleaved the displaced downstream strand and together with DNA ligase I produced non-expanded products. However, both expanded and non-expanded products formed irrespective of relative nuclease and ligase concentrations tested or enzyme addition order, suggesting the pre-existence and persistence of intermediates leading to both outcomes. FEN1 activity decreased with the length of repeat segment displaced presumably because the flap forms structures that inhibit cleavage. Increased MgCl(2) disfavored ligation of substrate intermediates that result in expansion products. Examination of expansion in vitro enables dissection of substrate and replication enzyme dynamics on repeat sequences.     

A series of repetitive DNA sequences are associated with human core and H1 histone genes

Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following³²P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)₁₆ simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.     

Matrix-associated DNA from maize is enriched in repetitive sequences

In order to elucidate some features of the topological organization of DNA within the plant nucleus, DNA fragments involved in the attachment of the DNA loops to the nuclear matrix in maize were studied. The matrix-associated DNA from dry embryo and meristematic cells after extensive digestion with DNase I and high salt treatment was about 2% of the total DNA, sized within the range of 50 and 250 bp. This DNA was found to be enriched in repetitive DNA sequences, both for nuclei from dry embryo and meristematic cells. The loop size of the DNA in cells of Zea mays appeared to be between 5 and 25 kbp.Abbreviations EDTA Diamino-ethanetetraacetic acid - EtBr Ethidium bromide - LIS Lithium diiodosalicylate - PMSF Phenylmethylsulfonyl fluoride - SDS Sodium dodecyl sulfate     

DNA distribution and intrachromosomal organization of moderately repetitive sequences

The DNA content of individual subregions along the 4th chromosome of Drosophila hydei has been measured. 51% of the subregions have a DNA content averaging 0.7-0.8 pg; 31% a mean DNA content of 0.25-0.35 pg and 18% a mean DNA content of 1.7 pg. Moreover the structural chromosomal distribution of moderately repetitive DNA is not random since the specific activity of the chromosomal segments in terms of those sequences is not the same. 9% of the subregions are very poor in repetitive sequences and 18% rich in repetitive DNA while being very poor in DNA.     

Nucleotide sequences of highly repetitive DNA from scleractinian corals

The staghorn coral genome contains 5% of a satellite DNA, consisting of 80 to 300 x 10(3) copies of a 118-bp repeat unit per haploid Acropora genome.     

Small RNA molecules related to the Alu family of repetitive DNA sequences.

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.     

Binding of netropsin to DNA in complexes with polypeptides containing repetitive lysine sequences

The interaction of the antibiotic netropsin with calf thymus DNA, T4 DNA and poly(dA-dT) . poly(dA-dT) in complexes with sequential polypeptides containing repetitive lysine sequences and histone H1 was investigated using circular dichroism spectroscopy and equilibrium dialysis. Both soluble DNA-polypeptide complexes and insoluble complexes showed binding of netropsin. The possibility of displacement of polypeptides from DNA binding sites by competition with netropsin molecules was eliminated by experiments using 14C-labelled polypeptides. From the analysis of CD titration behavior as well as from the results of equilibrium dialysis studies it follows that netropsin does not compete with polypeptides for DNA binding sites, which suggests that these two ligands occupy different sites. Various explanations for minor differences in the CD behavior of the bound netropsin in the saturation region are also discussed.