Microsatellites and associated repetitive elements in the sheep genome

To determine the frequency and clustering of a variety of simple di-and trinucleotide repeats, an Artiodactyl short interspersed element (SINE), an ovine satellite repeat, and a human Alu 1 repeat were used to screen a random selection of cosmids containing inserts of ovine genomic DNA. In total, 197 individual cosmids were digested with EcoRI and the fragments separated on 0.7% agarose gels. Southern blots of these gels were then sequentially probed with (AC)₇, (CT)₉, and (CAC)₆ oligonucleotides, and the repeats described above. The frequency at which (AC)₁, (CT)_n, and (CAC)_n repeats were found in the cosmids indicated that they occurred at average intervals of 65 kb, 367 kb, and 213 kb respectively within the ovine genome. The Artiodactyl SINE was the most common, occurring at an average interval of 20 kb. No human Alu 1 sequences were detected. There was a significant positive association between the (AC)_n and the Artiodactyl SINE. This association is quite strong as there was significant clustering of the two repeats both within cosmids and also within the EcoRI fragments of the digested genomic fragments. With the exception of the sheep satellite sequence, which occurs in tandem arrays, none of the other repeats showed significant clustering within the 41-kb (average size) cosmid inserts. The first 25 ovine microsatellites we characterized had an average polymorphic information content (PIC) of 0.65. The different microsatellite types, containing either perfect, imperfect, or compound repeats, had similar average PICs of 0.64, 0.65, and 0.66 respectively. There was a weak regression relationship (R²(adj)%=21.9) between the length of the longest uninterrupted dinucleotide repeat in the largest allele and the PIC of the microsatellite.     

Pms2 Suppresses Large Expansions of the (GAA·TTC)n Sequence in Neuronal Tissues

Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)_n sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)_n sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)_n sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)_n sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)_n sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway.     

Differential stability of the (GAA)n tract in the Friedreich ataxia (STM7) gene

Friedreich ataxia (FA) is an autosomal recessive, neurodegenerative disorder characterized by polypurine trinucleotide expansion. The (GAA)_n motif is located in intron 18 of the STM7 gene (previously considered as intron 1 of the X25 gene) on chromosome 9q13. We studied the distribution profile of the polymorphic (GAA)_n repetitive tract in 178 healthy individuals. The number of repeats of the trinucleotide block ranged from 7 to 29. In three individuals there were more than 29 repetitions of the GAA motif. While two of the individuals would be diagnosed as carriers of the FA mutation (GAA size > 90), the status of the third person, with a (GAA)₅₈ tract, appears less clear at present. Thus an FA carrier rate of 1/60 to 1/90 can be assumed for the German population. In addition an intermediate-sized allele, (GAA)₃₈ was identified in a mother with two affected children. The (GAA)₃₈ allele appears to be expanded during transmission to at least (GAA)₆₆ and (GAA)_{> 400} in her two FA-affected offspring. Therefore the shortest known STM7 allele conferring FA is (GAA)₆₆. These novel facts have to be considered for differential diagnosis and definition of the FA carrier state. Received: 7 February 1997     

105 Activity of DNA ligase on substrates containing non-canonical structures

The expansion of trinucleotide repeat tracts (e.g. (CAG)_n tracts) has been shown to contribute to genomic instability and has been implicated in the pathogenesis of several neurodegenerative diseases, including Huntington’s Disease and Fragile X syndrome (Kovtun et al., 2008). While the molecular mechanism of this expansion is unknown, the ability of trinucleotide repeat sequences to form non-canonical secondary structures, such as hairpins, has been implicated as a multifaceted source of error (Gacy et al., 1995). Non-canonical DNA secondary structures have been shown to impact the action of enzymes in the base excision repair (BER) pathway, by which oxidatively damaged bases are removed. More specifically, there is evidence that trinucleotide repeat-containing DNA mistakenly enters long-patch BER, which can potentially lead to the incorporation of extra nucleobases by DNA polymerase (Jarem et al., 2011). The final enzyme in the BER pathway is DNA Ligase, which catalyses the formation of a phosphodiester bond to seal a nick site (Taylor et al., 2011). When extra nucleotides have been added during an erroneous long-patch BER process, the action of DNA ligase may expand the repeat tract by incorporating these additional bases into duplex DNA. In this study, DNA constructs containing (CAG)_n hairpins at various distances from a nick site are used to investigate the ability of DNA Ligase to ligate substrates containing non-canonical secondary structure back into duplex DNA.     

DNA sequence organization in the genome of Petroselinum sativum (Umbelliferae)

The genome of parsley was studied by DNA/DNA reassociation to reveal its spectrum of DNA reiteration frequencies and sequence organization. The reassociation of 300 nucleotide DNA fragments indicates the presence of four classes of DNA differing in repetition frequency. These classes are: highly repetitive sequences, fast intermediate repetitive sequences, slow intermediate repetitive sequences, and unique sequences. The repeated classes are reiterated on average 136,000, 3000, and 42 times respectively. A minor part of the genome is made up of palindromes. — The organization of DNA sequences in the P. sativum genome was determined by the reassociation kinetics of DNA fragments of varying length. Further information was derived from S1 nuclease resistance and from hyperchromicity measurements on DNA fragments reassociated to defined C₀t values. — The portion of the genome organized in a short period interspersion pattern amounts to 47%, with the unique sequences on an average 1000 nucleotides long, and most of the repetitive sequences about 300 nucleotides in length, whereas the weight average length may be up to 600 nucleotides. — About 5% unique DNA and 11% slow intermediate repetitive DNA consist of sequences from 10³ up to 10⁴ nucleotides long; these are interspersed with repetitive sequences of unknown length. Long repetitive sequences constitute 33% of the genome, 13% are satellite-like organized, and 20% in long stretches of intermediate repetitive DNA in which highly divergent sequences alternate with sequences that show only minimal divergence. — The results presented indicate remarkable similarities with the genomes of most animal species on which information is available. The most intriguing pecularity of the plant genome derives from its high content of repetitive DNA and the presumed organization of the latter.     

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.     

Constraints upon the organisation and evolution of chromosomes in Allium

Summary In terms of chromosome morphology, karyotype organisation, taxonomy and genetic relationship as judged from chromosome pairing in the Fl hybrid, A. cepa and A.fistulosum are two closely related species. But large variation in nuclear DNA amounts has occurred during the evolution of the two species. A comparison of the molecular composition of DNA in the two species has confirmed that the excess DNA acquired during evolution was predominantly repetitive sequences (sequences which do not encode genetic information). However, its distribution within the chromosome complements was equal in all chromosomes irrespective of the differences in chromosome size. The even distribution of the excess DNA within complements suggests strong constraints underlying evolutionary changes in genome organisation. The nature of the constraints is discussed, and it is shown that such constraints can influence the direction of karyotype evolution during speciation.     

Dissecting (CAC)5/(GTG)5 multilocus fingerprints from man into individual locus-specific, hypervariable components

Individual components of multilocus fingerprints from man produced by (CAC)₅/(GTG)₅ oligonucleotides have been scrutinized to characterize their peculiar properties. Successful cloning and changes occurring during the propagation of recombinant simple repetitive DNA in prokaryotic hosts are described. The isolated locus-specific probes were characterized with respect to their formal (and population genetic) properties and their usefulness for individualization and linkage studies. The localization was determined on chromosomes 8, 9, 11, and 22. Repeat flanking sequences were characterized and analyzed for their coding potential because of significant open reading frames and apparent evolutionary conservation among vertebrates. The organization of the repeats and their flanking regions in the human genome is discussed with respect to the sequence (fine) architecture that developed during evolution. Classical “minisatellite” sequences were not detected near hypervariable (cac)_n/(gtg)_n repeats. The singlecopy probes described herein are a convenient complement to the oligonucleotides employed for multilocus fingerprinting. Many practical applications are apparent.     

DNA sequence organisation in avian genomes

By means of renaturation kinetics of DNA of the three avian species Cairina domestica, Gallus domesticus and Columba livia domestica the following major DNA repetition classes were observed: a very fast reannealing fraction comprising about 15% of the DNA, a fast or intermediate reannealing fraction that makes up 10%, and a slow reannealing fraction of about 70%, which apparently renatures with single copy properties. — Comparing the reassociation behaviour of short (0.3 kb) and long (>2 kb) DNA fragments of duck and chicken it becomes apparent that only 12% (duck) and 28% (chicken) of the single copy DNA are interspersed with repetitive elements on 2 to 3 kb long fragments. The lengths of the repetitive sequences were estimated by optical hyperchromicity measurements, by agarose A-50 chromatography of S₁ nuclease resistant duplexes and by electron microscopic measurements of the S₁ nuclease resistant duplexes. It was found that in the case of the chicken DNA the single copy sequences alternating with middle repetitive ones are at least 2.3 kb long; the interspersed moderate repeats have a length average of at least 1.5 kb. The sequence length of the moderate repeats in duck DNA is smaller. The results show that the duck and the chicken genomes do not follow the short period interspersion pattern of genome organisation, characteristic of the eucaryotic organisms studied so far.     

Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH

More than 15 human genetic diseases have been associated with the expansion of trinucleotide DNA repeats, which may involve the formation of non-duplex DNA structures. The slipped-strand nucleation of duplex DNA within GC-rich trinucleotide repeats may result in the changes of repeat length; however, such a mechanism seems less likely for the AT-rich (GAA)_n·(TTC)_n repeats. Using two-dimensional agarose gels, chemical probing and atomic force microscopy, we characterized the formation of non-B-DNA structures in the Friedreich ataxia-associated (GAA)_n·(TTC)_n repeats from the FRDA gene that were cloned with flanking genomic sequences into plasmids. For the normal genomic repeat length (n = 9) our data are consistent with the formation of a very stable protonated intramolecular triplex (H-DNA). Its stability at pH 7.4 is likely due to the high proportion of the T·A·T triads which form within the repeats as well as in the immediately adjacent AT-rich sequences with a homopurine· homopyrimidine bias. At the long normal repeat length (n = 23), a family of H-DNAs of slightly different sizes has been detected. At the premutation repeat length (n = 42) and higher negative supercoiling, the formation of a single H-DNA structure becomes less favorable and the data are consistent with the formation of a bi-triplex structure.     

Replication fork regression in repetitive DNAs

Among several different types of repetitive sequences found in the human genome, this study has examined the telomeric repeat, necessary for the protection of chromosome termini, and the disease-associated triplet repeat (CTG)·(CAG)_n. Evidence suggests that replication of both types of repeats is problematic and that a contributing factor is the repetitive nature of the DNA itself. Here we have used electron microscopy to investigate DNA structures formed at replication forks on large model DNAs containing these repeat sequences, in an attempt to elucidate the contributory effect that these repetitive DNAs may have on their replication. Visualization of the DNA revealed that there is a high propensity for a paused replication fork to spontaneously regress when moving through repetitive DNAs, and that this results in a four-way chickenfoot intermediate that could present a significant block to replication in vivo, possibly leading to unwanted recombination events, amplifications or deletions.     

An alternative view of mammalian DNA sequence organization: II. Short repetitive sequences are organized into scrambled tandem clusters in Syrian hamster DNA

Hyperchromicity, S₁ nuclease digestion, and reassociation studies of Syrian hamster repetitive DNA have led to novel conclusions about repetitive sequence organization. Re-evaluation of the hyperchromicity techniques commonly used to determine the average length of genomic repetitive DNA regions indicates that both the extent of reassociation, and the possibility of non-random elution of hyperpolymers from hydroxyapatite can radically affect the observed hyperchromicity. An alternative interpretation of hyperchromicity experiments, presented here, suggests that the average length of repetitive regions in Syrian hamster DNA must be greater than 4000 nucleotides.S₁ nuclease digestion of reassociated 3200 nucleotide Syrian hamster repetitive DNA, on the other hand, yields both long (>2000 nucleotides) and short (300 nucleotides) resistant DNA duplexes. Calculations indicate that the observed mass of short nuclease-resistant duplexes (>60%) is too large to have arisen only from independent short repetitive DNA sequences alternating with non-repetitive regions. Reassociation experiments using long and short S₁ nuclease-resistant duplexes as driver DNA indicate that all repetitive sequences are present in both fractions at approximately the same concentration. Isolated long S₁ nuclease-resistant duplexes, after denaturation, renaturation, and a second S₁ nuclease digestion, again produce both long and short DNA duplexes. Reassociation experiments indicate that all repetitive DNA sequences are still present in the “recycled” long S₁ nuclease-resistant duplexes. These experiments imply that many of the short S₁ nuclease-resistant repetitive DNA duplex regions present in reassociated Syrian hamster DNA were initially present in the genome as part of longer repetitive sequence blocks. This conclusion suggests that the majority of “short” repetitive regions in Syrian hamster DNA are organized into scrambled tandem clusters rather than being individually interspersed with non-repetitive regions.     

Heterochromatin and microsatellites detection in karyotypes of four sea turtle species: Interspecific chromosomal differences

The wide variation in size and content of eukaryotic genomes is mainly attributed to the accumulation of repetitive DNA sequences, like microsatellites, which are tandemly repeated DNA sequences. Sea turtles share a diploid number (2n) of 56, however recent molecular cytogenetic data have shown that karyotype conservatism is not a rule in the group. In this study, the heterochromatin distribution and the chromosomal location of microsatellites (CA)_n, (GA)_n, (CAG)_n, (GATA)_n, (GAA)_n, (CGC)_n and (GACA)_n in Chelonia mydas, Caretta caretta, Eretmochelys imbricata and Lepidochelys olivacea were comparatively investigated. The obtained data showed that just the (CA)_n, (GA)_n, (CAG)_n and (GATA)_n microsatellites were located on sea turtle chromosomes, preferentially in heterochromatic regions of the microchromosomes (mc). Variations in the location of heterochromatin and microsatellites sites, especially in some pericentromeric regions of macrochromosomes, corroborate to proposal of centromere repositioning occurrence in Cheloniidae species. Furthermore, the results obtained with the location of microsatellites corroborate with the temperature sex determination mechanism proposal and the absence of heteromorphic sex chromosomes in sea turtles. The findings are useful for understanding part of the karyotypic diversification observed in sea turtles, especially those that explain the diversification of Carettini from Chelonini species.     

DNA sequence organization in the genome of Cycas revoluta

The pattern of DNA sequence organization in the genome of Cycas revoluta was analyzed by DNA/DNA reassociation. Reassociation of 400 base pair (bp) fragments to various C₀t values indicates the presence of at least four kinetic classes: the foldback plus very highly repetitive sequences (15%), the fast repeats (24%), the slow repeats (44%), and the single copy (17%). The latter component reassociates with a rate constant 1×10^–4 M^–1S^–1 corresponding to a complexity of 1.6× 10⁶ kb per haploid genome. A haploid C. revoluta nucleus contains approximately 10.3 pg DNA. The single-copy sequences account for about 28% of the DNA, but only 17% reassociate with single-copy kinetics because of interspersion with repetitive sequences. — The interspersion of repetitive and single-copy sequences was examined by reassociation of DNA fragments of varying length to C₀t values of 70 and 500. A major (65%) and homogeneous class of single-copy sequences averaging 1,100 bp in length is interspersed in a short period pattern with repeated sequences. A minor (35%) heterogeneous single-copy component is interspersed in a long-period pattern. The majority of repetitive sequences have a length distribution of 100–350 bp with subclasses averaging 150 and 300 bp in length. Repeat sequences with a wide range in sizes exceeding 2 kilobase pair (kb) are also present in this genome. — The size and distribution of inverted repeat (ir) sequences in the DNA of C. revoluta were studied by electron microscopy. It is estimated that there are approximately 4 × 10⁶ ir pairs (one per 2.33 kb) that form almost equal numbers of looped and unlooped palindromes. This high value is 2.5 times that found in wheat DNA. These palindromes are in general randomly distributed in the genome with an average interpalindrome distance of 1.6 kb. The majority (about 85%) of ir sequences of both types of palindromes belong to a main-size class, with an average length of 210 bp in the unlooped and and 163 bp in the looped type. These values are comparable to those reported for some other plant and animal genomes. Distribution of length of single stranded loops showed a main-size class (75%) with an average length of 220 bp.     

Friedreich's ataxia (GAA)n•(TTC)n repeats strongly stimulate mitotic crossovers in Saccharomyces cerevisae

Expansions of trinucleotide GAA•TTC tracts are associated with the human disease Friedreich''s ataxia, and long GAA•TTC tracts elevate genome instability in yeast. We show that tracts of (GAA)₂₃₀•(TTC)₂₃₀ stimulate mitotic crossovers in yeast about 10,000-fold relative to a “normal” DNA sequence; (GAA)_n•(TTC)_n tracts, however, do not significantly elevate meiotic recombination. Most of the mitotic crossovers are associated with a region of non-reciprocal transfer of information (gene conversion). The major class of recombination events stimulated by (GAA)_n•(TTC)_n tracts is a tract-associated double-strand break (DSB) that occurs in unreplicated chromosomes, likely in G1 of the cell cycle. These findings indicate that (GAA)_n•(TTC)_n tracts can be a potent source of loss of heterozygosity in yeast.     

垂穗披碱草与达乌里披碱草基因组细胞遗传学比较

采用顺序FISH-GISH技术,12个重复序列探针,包括9个三核苷酸简单重复序列、2个卫星DNA重复序列pSc119.2和pAs1以及5S rDNA,通过重复序列的物理定位对达乌里披碱草和垂穗披碱草基因组中部分重复序列的分布特征进行了比较分析,为进一步研究垂穗披碱草和达乌里披碱草的物种形成及演化提供新的分子细胞遗传学证据。结果表明:(1)所有的序列在这2个物种的染色体上都能产生可检测的杂交信号,且在2个物种中(AAC)_(10)、(ACT)_(10)、(CAT)_(10)都表现为共分布,(AAG)_(10)与(AGG)_(10)表现为近似共分布;2个物种的H基因组除5S rDNA序列外,其他序列都产生强烈且丰富的杂交位点,St与Y基因组不同重复序列探针的荧光位点数目有所差别,表现为5S rDNA、pSc119.2、(AAC)_(10)、(CAT)_(10)、(ACT)_(10)、(CAC)_(10)探针的信号位点较少或无信号,其余的探针信号位点稍多。(2)达乌里披碱草的第2对染色体上具有(AAC)_(10)、(CAT)_(10)、(ACT)_(10)的杂交位点、第6对染色体上具有(CAC)_(10)的杂交位点,而在垂穗披碱草的St基因组中未观察到上述序列杂交位点;达乌里披碱草St基因组仅有第4对染色体的端部具有pSc119.2杂交位点,而在垂穗披碱草St基因组中的pSc119.2杂交位点位于第5对染色体长臂的间隔区;相对于达乌里披碱草,垂穗披碱草St和Y基因组染色体含有更多的重复序列杂交位点。(3)达乌里披碱草的H/Y基因组间易位在不同材料间是稳定存在的,达乌里披碱草基因组相对稳定,不同材料间H基因组重复序列杂交信号多态性高于St和Y基因组;垂穗披碱草基因组的变异较大,不同材料间St和Y基因组重复序列杂交信号多态性高于H基因组。研究认为,垂穗披碱草和达乌里披碱草的H基因组均起源于布顿大麦,St基因组可能起源于不同的拟鹅观草属物种;与达乌里披碱草相比垂穗披碱草St与Y基因组可能具有更高的染色体结构变异性,而垂穗披碱草St与Y基因组变异较大的原因可能是与同区域分布的含StY基因组的物种发生了种间渗透杂交。     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997