Survey of plant short tandem DNA repeats

Length variations in simple sequence tandem repeats are being given increased attention in plant genetics. Some short tandem repeats (STRs) from a few plant species, mainly those at the dinucleotide level, have been demonstrated to show polymorphisms and Mendelian inheritance. In the study reported here a search for all of the possible STRs ranging from mononucleotide up to tetranucleotide repeats was carried out on EMBL and GenBank DNA sequence databases of 3026 kb nuclear DNA and 1268 kb organelle DNA in 54 and 28 plant species (plus algae), respectively. An extreme rareness of STRs (4 STRs in 1268 kb DNA) was detected in organelle compared with nuclear DNA sequences. In nuclear DNA sequences, (AT)_n sequences were the most abundant followed by (A)_n · (T)_n, (AG)_n · (CT)_n, (AAT)_n · (ATT)_n, (AAC)_n · (GTT), (AGC)_n · (GCT)_n, (AAG)_n · (CTT)_n, (AATT)_n · (TTAA)_n, (AAAT)_n · (ATTT)_n and (AC)_n · (GT)_n sequences. A total of 130 STRs were found, including 49 (AT)_n sequences in 31 species, giving an average of 1 STR every 23.3 kb and 1 (AT)_n STR every 62 kb. An abundance comparable to that for the dinucleotide repeat was observed for the tri- and tetranucleotide repeats together. On average, there was 1 STR every 64.6 kb DNA in monocotyledons versus 1 every 21.2 kb DNA in dicotyledons. The fraction of STRs that contained G-C basepairs increased as the G+C contents went up from dicotyledons, monocotyledons to algae. While STRs of mono-, di- and tetranucleotide repeats were all located in non coding regions, 57% of the trinucleotide STRs containing G-C basepairs resided in coding regions.     

Modulation of polymorphic loci detection with synthetic tandem repeat variants

The modifications of hybridization patterns were studied when Southern blots, carrying stallions DNA samples, were probed with eight synthetic tandem repeats (STRs), related by sequence variations in the basic unit. Because STRs preferentially crosshybridize with genomic VNTRs, they usually give patterns looking more like DNA fingerprints, but we found that even small modifications in the STR monomer could cause major changes in the hybridization profiles and could induce a shift of fingerprint pattern towards the detection of only one or two loci. This enables the use of STRs as direct genetic markers for linkage analysis, without cloning of the corresponding DNA fragment. Moreover, the set of STR variants can suggest consensus sequences allowing some prediction of the banding pattern.     

Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules.

We have found abundant telomere-specific terminal transferase activity in crude macronuclear extracts from vegetatively growing cells of the hypotrichous ciliate Oxytricha nova. This activity adds two to seven tandem repeats of the sequence GGGGTTTT (the Oxytricha telomeric repeat) to the 3' end of oligonucleotide primers ending in repeats of G4T4 and always adds the repeats in the proper phase. The activity requires the presence of micromolar amounts of dGTP and dTTP as well as single-stranded oligomer primers ending 3' with repeats of the Oxytricha telomeric sequence. A nuclease activity is present in the extracts which is closely balanced with telomere terminal transferase activity. We propose a simple model for replication of the ends of linear DNA molecules based on the telomere terminal transferase.     

Copy number of tandem direct repeats within the inverted repeats of Marek's disease virus DNA

We previously reported that DNA of the oncogenic strain BC-1 of Marek's disease virus serotype 1 (MDV1) contains three units of tandem direct repeats with 132 base pair (bp) repeats within the inverted repeats of the long regions of the MDV1 genome, whereas the attenuated, nononcogenic viral DNA contains multiple units of tandem direct repeats (Maotani et al., 1986). In the present study, the difference in the copy numbers of 132 bp repeats of oncogenic and nononcogenic MDV1 DNAs in other strains of MDV1 was investigated by Southern blot hybridization. The main copy numbers in different oncogenic MDV1 strains differed: those of BC-1, JM and highly oncogenic Md5 were 3, 5 to 12 and 2, respectively. The viral DNA population with two units of repeats was small, but detectable, in cells infected with either the oncogenic BC-1 or JM strain. The MDV1 DNA in various MD cell lines contained either two units or both two and three units of repeats. The significance of the copy number of repeats in oncogenicity of MDV1 is discussed.     

A novel method for determining linkage between DNA sequences: hybridization to paired probe arrays

Cooperative hybridization has been used to establish physical linkage between two loci on a DNA strand. Linkage was detected by hybridization to a new type of high-density oligonucleotide array. Each synthesis location on the array contains a mixture of two different probe sequences. Each of the two probes can hybridize independently to a different target sequence, but if the two target sequences are physically linked there is a cooperative increase in hybridization yield. The ability to create and control non-linear effects raises a host of possibilities for applications of oligonucleotide array hybridization. The method has been used to assign linkage in 50:50 mixtures of DNA containing single nucleotide polymorphisms (SNPs) separated by 17, 693, 1350 and 2038 bp and to reconstruct haplotypes. Other potential uses include increasing the specificity of hybridization in mutation detection and gene expression monitoring applications, determining SNP haplotypes, characterizing repetitive sequences, such as short tandem repeats, and aiding contig assembly in sequen-cing by hybridization.     

Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21

We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human-rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes.     

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA. The use of such stacking increases the effective length of microchip oligonucleotides, enhances sequencing accuracy and allows the sequencing of longer DNA. The effects of mismatches, base composition, length and other factors on the stacking are evaluated. Contiguous stacking hybridization of DNA with immobilized 8mers and one or two 5mers labeled with two different fluorescent dyes increases the effective length of sequencing oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation of all four bases or 5-nitroindole as a universal base into different positions of the 5mers permitted a decrease in the number of additional rounds of hybridization. Contiguous stacking hybridization appears to be a promising approach to significantly increasing the efficiency of sequencing by hybridization.     

Characterization and genetic mapping of simple repeat sequences in the tomato genome

Tomato genomic libraries were screened for the presence of simple sequence repeats (SSRs) with seventeen synthetic oligonucleotide probes, consisting of 2- to 5-basepair motifs repeated in tandem. GAn and GTn sequences were found to occur most frequently in the tomato genome (every 1.2 Mb), followed by ATTn and GCCn (every 1.4 Mb and 1.5 Mb, respectively). In contrast, only ATn and GAn microsatellites (n > 7) were found to be frequent in the GenBank database, suggesting that other motifs may be preferentially located away from genes. Polymorphism of microsatellites was measured by PCR amplification of individual loci or by Southern hybridization, using a set of ten tomato cultivars. Surprisingly, only two of the nine microsatellite clones surveyed (five GTn, three GAn and one ATTn), showed length variation among these accessions. Polymorphism was also very limited betweenLycopersicon esculentum andL. pennelli, two distant species. Southern analysis using the seventeen oligonucleotide probes identified GATAn and GAAAn as useful motifs for the detection of multiple polymorphic fragments among tomato cultivars. To determine the structure of microsatellite loci, a GAn probe was used for hybridization at low stringency on a small insert genomic library, and randomly selected clones were analyzed. GAn based motifs of increasing complexity were found, indicating that simple dinucleotide sequences may have evolved into larger tandem repeats such as minisatellites as a result of basepair substitution, replication slippage, and possibly unequal crossing-over. Finally, we genetically mapped loci corresponding to two amplified microsatellites, as well as nine large hypervariable fragments detected by Southern hybridization with a GATA8 probe. All loci are located around putative tomato centromeres. This may contribute to understanding of the structure of centromeric regions in tomato.     

Sequence analysis of genomic regions containing trinucleotide repeats isolated by a novel cloning method

A method was developed for effective isolation of trinucleotide repeats from genomic DNA. This method is based on the DNA polymerase reaction, which is restricted with only two or three nucleotide substrates and primed by biotinylated oligonucleotide probes. Sequences are then isolated by a streptavidin biotin-trapping method. More than 80% of the clones from each library contained more than eight trinucleotide repeats. Sequence analysis showed that the characteristic dinucleotide flanking sequences usually confronting various trinucleotide repeats are not found in the vicinity of CAG repeats, suggesting that CAG repeats may have been generated through a mechanism different from that of other trinucleotide repeats.     

Human identity testing with PCR-based systems

Large numbers of repetitive stretches of DNA are present within the human genome that are associated with human individuality due to their polymorphic character. Approximately one-third of these repeat sequences is arranged as microsatellites or short tandem repeats (STRs) whose valuable application as state-of-the-art technique in human identity testing will be briefly summarized in this review. Prerequisties for successful DNA typing using STRs amplified by polymerase chain reaction (PCR) are outlined and particular attention is paid to the molecular structure of STRs from autosomes as well as from the Y chromosome. A comprehensive overview about current and emerging methods of STR analysis is given as well.     

Polymers of random short oligonucleotides detect polymorphic loci in the human genome.

Polymers of random 14 mer oligonucleotides are shown to detect discrete loci in the human genome. Eighteen different synthetic tandem repeats of random 14 base-pair units (STRs) have been generated and all of them turn out to detect polymorphic loci on southern blots of human DNA samples, presumably corresponding to a variable number of tandem repeats (VNTR). This finding suggests that minisatellites are a major component of the human genome and are strongly associated with the generation of genetic variability. In addition, it should open new strategies to make new polymorphic probes available.     

Fractionation, phosphorylation and ligation on oligonucleotide microchips to enhance sequencing by hybridization.

Oligonucleotide microchips are manufactured by immobilizing presynthesized oligonucleotides within 0.1 x 0.1 x 0.02 mm or 1 x 1 x 0.02 mm polyacrylamide gel pads arranged on the surface of a microscope slide. The gel pads are separated from each other by hydrophobic glass spacers and serve as a kind of 'microtest tube' of 200 pl or 20 nl volume, respectively. Fractionation of single-stranded DNAs is carried out by their hybridization with chip pads containing immobilized 10mers. DNA extracted separately from each pad is transferred onto a sequencing chip and analyzed thereon. The chip, containing a set of 10mers, was enzymatically phosphorylated, then hybridized with DNA and ligated in a site-directed manner with a contiguously stacked 5mer. Several cycles of successive hybridization-ligation of the chip-bound 10mers with different contiguously stacked 5mers and hybridized with DNA were carried out to sequence DNA containing tetranucleotide repeats. Combined use of these techniques show significant promise for sequence comparison of homologous regions in different genomes and for sequence analysis of comparatively long DNA fragments or DNA containing internal repeats.     

Polar alteration of short tandem repeats (STRs) in mammalian cells

Instability of short tandem repeats (STRs) in DNA during replication is observed in all organisms examined, and is causatively involved in various human diseases. We explore the mechanisms involved in instability by examining length changes occurring during the replication of [(CA)(20)TA](n) and [(CAG)(20)TAG](n), in human cells. We show that the majority of alterations consist of an insertion or deletion of one repeat unit, and base substitutions or length changes involving many repeat units are rare. We also show that length changes of two-tract STRs are biased toward the 3'-end of the repeat tract, in reference to lagging strand synthesis. There are some differences between our observations and previous observations in microbes, e.g. the orientation effect was not observed in this study. The results of this study are discussed in terms of the molecular mechanisms leading to alterations in repeat tracts.     

Complex structure of knobs and centromeric regions in maize chromosomes

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of individual maize chromosomes via the isolation and characterization of chromosome-specific cosmid clones. Restriction fragment fingerprinting, sequencing, and in situ hybridization were applied to discover a new family of knob associated tandem repeats, the TR1, which are capable of forming fold-back DNA segments, as well as a new family of centromeric tandem repeats, CentC. Analysis of knob and centromeric DNA segments revealed a complex organization in which blocks of tandemly arranged repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration/association of certain retrotransposable elements into knobs or centromere regions as well as for integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. DNA hybridization to a blot panel of eight individual maize chromosome addition lines revealed that CentC, TR1, and 180-bp tandem repeats are found in each of these maize chromosomes, but the copy number of each can vary significantly from about 100 to 25,000. In situ hybridization revealed variation among the maize chromosomes in the size of centromeric tandem repeats as well as in the size and composition of knob regions. It was found that knobs may be composed of either 180-bp or TR1, or both repeats, and in addition to large knobs these repeated elements may form micro clusters which are detectable only with the help of in situ hybridization. The association of the fold-back elements with knobs, knob polymorphism and complex structure suggest that maize knob may be consider as megatransposable elements. The discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes.     

A brief review of short tandem repeat mutation

Short tandem repeats （STRs） are short tandemly repeated DNA sequences that involve a repetitive unit of 1-6 bp. Because of their polymorphisms and high mutation rates, STRs are widely used in biological research. Strand-slippage replication is the predominant mutation mechanism of STRs, and the stepwise mutation model is regarded as the main mutation model. STR mutation rates can be influenced by many factors. Moreover, some trinucleotide repeats are associated with human neurodegenerative diseases. In order to deepen our knowledge of these diseases and broaden STR application, it is essential to understand the STR mutation process in detail. In this review, we focus on the current known information about STR mutation.     

Isolation and identification of a novel tandemly repeated DNA sequence in the centromeric region of human chromosome 8

EcoRI subclones, designated as 50E1 and 50E4, were independently obtained from a cosmid clone previously mapped to the centromeric region of human chromosome 8. Southern blot hybridization analyses suggested that both subclones contain repetitive DNA sequences different from the chromosome 8 specific alphoid DNA. DNA sequence analysis of the 704 bp insert of 50E1 and the 1, 962 bp insert of 50E4 revealed that both inserts contained tandemly repeated units of 220 bp. Fluorescence in situ hybridization studies confirmed these two subclones to be specifically located on the centromeric region of chromosome 8. A 220 bp consensus sequence, derived from nine monomeric repeats, showed no significant homology to alphoid consensus sequences or to other currently known human centromeric DNA sequence. Furthermore, no significant homology was found with any other DNA sequence deposited in the EMBL or GenBank databases, indicating that this chromosome 8 specific repetitive DNA sequence is novel. From slot blot experiments it was estimated that 0.013% of the human genome comprises 1,750 of these monomeric repeats, residing on the centromeric region of chromosome 8 in tandem array(s).