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David T Kirkpatrick - One of the best experts on this subject based on the ideXlab platform.
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A Whole Genome Screen for Minisatellite Stability Genes in Stationary Phase Yeast Cells
2016Co-Authors: Bonnie Alver, Peter A Jauert, Laura Brosnan, David T KirkpatrickAbstract:Alver et al. Yeast stationary phase DNA stability pg 2 Repetitive elements comprise a significant portion of most eukaryotic genomes. Minisatellites, a type of repetitive element composed of repeat units 15- 100bp in length, are stable in actively dividing cells but change in composition during meiosis and in stationary phase cells. Alterations within Minisatellite tracts have been correlated with the onset of a variety of diseases, including diabetes mellitus, myoclonus epilepsy and several types of cancer. However, little is known about the factors preventing Minisatellite alterations. Previously, our lab developed a color segregation assay in which a Minisatellite was inserted into the ADE2 gene in the yeast Saccharomyces cerevisiae to monitor alteration events. We demonstrated that Minisatellite alterations that occur in stationary phase cells give rise to a specific colony morphology phenotype known as blebbing. Here, we performed a modified version of the Synthetic Genetic Array (SGA) analysis to screen for mutants that produce a blebbing phenotype. Screens were conducted using two distinctly different Minisatellite tracts: the ade2-min3 construct consisting of three identical 20bp repeats, and the ade2-h7.5 construct, consisting of 7.5 28bp variabl
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INVESTIGATION A Whole Genome Screen for Minisatellite Stability Genes in Stationary-Phase Yeast Cells
2016Co-Authors: Bonnie Alver, Peter A Jauert, Laura Brosnan, Chad L. Myers, David T KirkpatrickAbstract:ABSTRACT Repetitive elements comprise a significant portion of most eukaryotic genomes. Minisatellites, a type of repetitive element composed of repeat units 152100 bp in length, are stable in actively dividing cells but change in composition during meiosis and in stationary-phase cells. Alterations within Minisatellite tracts have been correlated with the onset of a variety of diseases, including diabetes mellitus, myoclonus epilepsy, and several types of cancer. However, little is known about the factors preventing Minisatellite alterations. Previously, our laboratory developed a color segregation assay in which a Minisatellite was inserted into the ADE2 gene in the yeast Saccharomyces cerevisiae to monitor alteration events. We demonstrated that Minisatellite alterations that occur in stationary-phase cells give rise to a specific colony morphology phenotype known as blebbing. Here, we performed a modified version of the synthetic genetic array analysis to screen for mutants that produce a blebbing phenotype. Screens were conducted using two distinctly different Minisatellite tracts: the ade2-min3 construct consisting of three identical 20-bp repeats, and the ade2-h7.5 construct, consisting of seven-and-a-half 28-bp variable repeats. Mutations in 102 and 157 genes affect the stability of the ade2-min3 and ade2-h7.5 alleles, respectively. Only seven hits over-lapped both screens, indicating that different factors regulate repeat stability depending upon Minisatellite size and composition. Importantly, we demonstrate that mismatch repair influences the stability of the ade2-h7.5 allele, indicating that this type of DNA repair stabilizes complex Minisatellites in stationary phase cells. Our work provides insight into the factors regulating Minisatellite stability
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the role of csm3 mrc1 and tof1 in Minisatellite stability and large loop dna repair during meiosis in yeast
Fungal Genetics and Biology, 2013Co-Authors: Andrea R Leclere, John K Yang, David T KirkpatrickAbstract:Abstract Double-stranded break (DSB) repair during meiotic recombination in yeast Saccharomyces cerevisiae leads to the formation of heteroduplex DNA, a hybrid DNA molecule composed of single strands from two homologous chromosomes. Differences in sequence between the strands within heteroduplex DNA generate mismatches or large unpaired loops that are substrates for repair. At least two pathways function to repair large loops that form within heteroduplex DNA: the RAD1 -dependent large loop repair (LLR) pathway and another as yet uncharacterized RAD1 -independent LLR pathway. Repair of large loops during meiotic recombination is especially important for the genomic stability of the repetitive DNA sequences known as Minisatellites. Minisatellite DNA tracts are generally stable during mitotic cell divisions but frequently alter in length during meiosis. Using a yeast Minisatellite system in which the human Minisatellite associated with the HRAS1 proto-oncogene has been inserted into the recombination hotspot region upstream of HIS4 in S. cerevisiae , our lab previously showed that the RAD1 -dependent LLR pathway controls Minisatellite length expansions, but not contractions. Here we show that Minisatellite length expansions are controlled by the products of the CSM3 and TOF1 genes, while contractions are controlled by MRC1 . By examining meiotic segregation patterns in yeast strains heterozygous for the 26 bp his4-lopd insert, we found that deleting CSM3 caused a loss of LLR activity similar to that seen in a RAD1 mutant. Double mutant analysis revealed that failure to repair loops is exacerbated upon deleting both RAD1 and CSM3 – specifically the type of repair that fills in loops, which would generate Minisatellite length expansions. A model for Minisatellite length alteration based on these results is presented.
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zinc regulates the stability of repetitive Minisatellite dna tracts during stationary phase
Genetics, 2007Co-Authors: Maire K Kelly, Peter A Jauert, Linnea E Jensen, Christine L Chan, Chinh S Truong, David T KirkpatrickAbstract:Repetitive Minisatellite DNA tracts are stable in mitotic cells but unstable in meiosis, altering in repeat number and repeat composition. As relatively little is known about the factors that influence Minisatellite stability, we isolated mutations that destabilize a Minisatellite repeat tract in the ADE2 gene of Saccharomyces cerevisiae. One mutant class exhibited a novel color segregation phenotype, “blebbing,” characterized by Minisatellite instability during stationary phase. Minisatellite tract alterations in blebbing strains consist exclusively of the loss of one 20-bp repeat. Timing experiments suggest that these tract alterations occur only after cells have entered stationary phase. Two complementation groups identified in this screen have mutations in either the high-affinity zinc transporter ZRT1 or its zinc-dependent transcriptional regulator ZAP1. The Δzrt1 mutant specifically affects the stability of Minisatellite tracts; microsatellites or simple insertions in the ADE2 reading frame are not destabilized by loss of ZRT1. The Δzrt1 blebbing phenotype is partially dependent on a functional RAD50. Zinc is known for its role as an essential cofactor in many DNA-binding proteins. We describe possible models by which zinc can influence Minisatellite stability. Our findings directly implicate zinc homeostasis in the maintenance of genomic stability during stationary phase.
Alec John Jeffreys - One of the best experts on this subject based on the ideXlab platform.
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Further evidence for elevated human Minisatellite mutation rate in Belarus eight years after the Chernobyl accident.
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 1997Co-Authors: Valeri N Nesterov, Nicolay G Krouchinsky, Valdislav A Ostapenko, Gilles Vergnaud, F Giraudeau, J Buard, Alec John JeffreysAbstract:Analysis of germline mutation rate at human Minisatellites among children born in areas of the Mogilev district of Belarus heavily polluted after the Chernobyl accident has been extended, both by recruiting more families from the affected region and by using five additional Minisatellite probes, including multi-locus probe 33.6 and four hypervariable single-locus probes. These additional data confirmed a twofold higher mutation rate in exposed families compared with non-irradiated families from the United Kingdom. An elevated rate was seen at all three independent sets of Minisatellites (detected separately by multi-locus probes 33.15, 33.6 and six single-locus probes), indicating a generalised increase in Minisatellite germline mutation rate in the Belarus families. Within the Belarus cohort, mutation rate was significantly greater in families with higher parental radiation dose estimated for chronic external and internal exposure to caesium-137, consistent with radiation induction of germline mutation. The spectra of mutation seen in the unexposed and exposed families were indistinguishable, suggesting that increased mutation observed over multiple loci arises indirectly by some mechanism that enhances spontaneous Minisatellite mutation.
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Minisatellite mutation rate variation associated with a flanking dna sequence polymorphism
Nature Genetics, 1994Co-Authors: Darren G Monckton, Rita Neumann, Tara Guram, N Fretwell, Annette Macleod, Keiji Tamaki, Alec John JeffreysAbstract:Human Minisatellite mutation in the male germline frequently involves complex inter-allelic gene conversion events restricted to one end of the tandem repeat array. Some alleles at Minisatellite MS32 show reduced variability in human populations and are associated with a G to C transversion upstream of the array. Analysis of single sperm demonstrated a frequently profound reduction in mutation rate at alleles carrying the C variant. This mutation suppression acts in cis, but does not affect the ability of an allele to act as sequence donor during gene conversion. This mutation rate polymorphism provides strong evidence for elements near the Minisatellite that regulate tandem repeat instability.
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digital dna typing at a second hypervariable locus by Minisatellite variant repeat mapping
Human Molecular Genetics, 1993Co-Authors: David L Neil, Alec John JeffreysAbstract:: Minisatellite variant repeat unit mapping by PCR (MVR-PCR) assays the interspersion pattern of variant repeat units along Minisatellite alleles. Mapping such internal variation in the highly polymorphic Minisatellite MS31A (locus D7S21), reveals extreme levels of allelic variability, far in excess of that detectable by allele length analysis. Flanking base substitutional polymorphisms have enabled the 5' structure of large numbers of MS31A alleles to be derived from genomic DNA by allele-specific MVR-PCR. More than 100 alleles have now been mapped and all are different. Several alleles show related internal structures and some of these provide evidence of polarity in allelic variation reminiscent of that seen at two other hypervariable Minisatellites, D1S8 (MS32) and D16S309 (MS205). We also describe the diploid digital coding of MS31A, including the simultaneous coding of MS31A and a second locus, MS32, by duplex MVR-PCR, which greatly enhances the potential forensic applications of this technique.
Terry Burke - One of the best experts on this subject based on the ideXlab platform.
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Minisatellite dna markers in the chicken genome ii isolation and characterization of Minisatellite loci
Animal Genetics, 2009Co-Authors: Michael William Bruford, Olivier Hanotte, Terry BurkeAbstract:The isolation of chicken Minisatellites is reported. Two charomid libraries were constructed from fractions of size-selected MboI-digested genomic DNA. A total of 5985 colonies were screened with the multilocus probes 33.6 and 33.15; 137 positives were obtained (2.35%). A total of 55 clones were tested against four unrelated chickens; 30 revealed variable single locus patterns. More detailed characterization was made of 15 probes, which were tested against four families comprising a total of 53 offspring. Of parent/offspring comparisons 51% were informative for segregation. No mutations were detected. Three Minisatellites were linked on the same autosome, implying that these loci may occur in clusters, and one was Z chromosome-linked. Heterozygosity and allelic variability were measured in 67 individuals from several different strains and breeds. Mean heterozygosity ranged from 50 to 84%. Minisatellite loci appear to be highly variable in the chicken and should provide highly informative markers in intraspecific crosses in genome mapping studies.
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Minisatellite dna markers in the chicken genome i distribution and abundance of Minisatellites in multilocus dna fingerprints
Animal Genetics, 2009Co-Authors: Michael William Bruford, Terry BurkeAbstract:This paper reports the detailed characterization of multilocus Minisatellite DNA fingerprints in the chicken. Results are presented of DNA fingerprint segregation analyses carried out in three chicken pedigrees, calculating the number of detected loci, testing for Mendelian inheritance, and cosegregation among fingerprint bands. Two pedigrees (families 1 and 2) were analysed using the Jeffreys probes 33.6 and 33.15 only, and one pedigree (family 3) was analysed using 33.6, 33.15. 3′α-globin HVR and M13 protein III gene repeat. Mean band transmission frequencies in families 1 and 2 were near to the Mendelian expectation of 0.5 and no mutations were observed. Family 3 showed transmission frequencies slightly exceeding 0.5. Linkage among bands was higher than observed in some other avian species, with each allele represented by a mean of 1.48 HaeIII fragments. Cosegregation of heterozygous parental fragments representing distinguishable loci followed the expected binomial distribution. The number of Minisatellites detectable by the four probes was estimated to be 217. The pattern of cosegregation among those Minisatellite loci was tested against that expected for different levels of recombination through the use of a simulation model. We conclude that most Minisatellites are unlinked and probably widely dispersed in the chicken genome.
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identification of hypervariable single locus Minisatellite dna probes in the blue tit parus caeruleus
Molecular Ecology, 1994Co-Authors: G R Verheyen, Terry Burke, Bart Kempenaers, M Van Den Broeck, C Van Broeckhoven, Andre A DhondtAbstract:We report the isolation of a set of hypervariable Minisatellite DNA sequences from a blue tit Parus caeruleus genomic DNA library. In our strategy, we cloned a Minisatellite-rich DNA fraction into a charomid vector. The resulting cosmid library was screened with the two Minisatellite DNA probes 33.6 and 33.15 for recombinants containing a Minisatellite DNA insert. A total of 233 positive clones were isolated. Of 37 clones that have been analysed, nine gave polymorphic signals and can be used as single locus probes (SLPs). Four of the SLPs were investigated in more detail. The number of alleles, the heterozygosity and the mutation rate were estimated. Linkage analysis revealed that two of these loci were linked. The SLPs are of value to studies of the mating system and reproductive success in the blue tit, and may also be useful in population genetic studies.
M Nesje - One of the best experts on this subject based on the ideXlab platform.
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reduced genetic variation in norwegian peregrine falcons falco peregrinus indicated by Minisatellite dna fingerprinting
Ibis, 2002Co-Authors: Jan T. Lifjeld, Gro Bjornstad, Oodd F Steen, M NesjeAbstract:The Scandinavian Peregrine Falcon Falco peregrinus population went through a severe population bottleneck during the second half of the twentieth century, and was almost extinct during the 1970s. This event may have reduced the amount of genetic variation in the population. With this background, a comparative study, using multilocus, Minisatellite DNA fingerprinting, was carried out on broods of the Peregrine Falcon, the Merlin Falco columbarius and the Eurasian Hobby Falco subbuteo from south-east Norway. Band-sharing analysis of DNA fingerprints was used to test whether broods of Peregrine Falcons showed a greater between-nest similarity in their fingerprint profiles than did broods of the two congeneric species breeding in the same region, which have not undergone any recent population bottlenecks. The results show that broods of Peregrine Falcons were significantly more similar to each other genetically than were broods of either Merlins or Eurasian Hobbies. Furthermore, there was a positive correlation between the similarity in Minisatellite DNA and the similarity in a set of 11 microsatellite loci analysed for a subset of the Peregrine Falcon samples. The correlation supports the assumption that Minisatellite fingerprints provide a reliable indicator of overall genetic similarity, i.e. relatedness, between breeding pairs in the population. Hence we can conclude that broods of Peregrine Falcons were genetically more related to each other than were broods of the other two species. The high similarity in Minisatellite DNA between broods indicates a loss of genetic variation in the Peregrine Falcon population caused by the bottleneck, but this explanation can only be verified through a comparative genetic study of individuals sampled before and after the bottleneck event.
Alec J Jeffreys - One of the best experts on this subject based on the ideXlab platform.
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factors influencing recombination frequency and distribution in a human meiotic crossover hotspot
Human Molecular Genetics, 2005Co-Authors: Alec J Jeffreys, Rita NeumannAbstract:Little is known about the factors that influence the frequency and distribution of meiotic recombination events within human crossover hotspots. We now describe the detailed analysis of sperm recombination in the NID1 hotspot. Like the neighbouring MS32 hotspot, the NID1 hotspot is associated with a Minisatellite, suggesting that hotspots predispose DNA to tandem repetition. Unlike MS32, crossover resolution breakpoints in NID1 avoid the Minisatellite, producing a cold spot within the hotspot. This avoidance may be related to the palindromic nature of the Minisatellite interfering with the generation and/or processing of recombination intermediates. The NID1 hotspot also contains a single nucleotide polymorphism (SNP) close to the centre, which appears to directly influence the frequency of crossover initiation. Quantitative gene conversion assays show that this SNP affects the frequency of gene conversion and crossover to a very similar extent, providing evidence that conversions and crossovers are triggered by the same recombination initiating events. The recombination-suppressing allele is over-transmitted to recombinant progeny, and provides the most dramatic example to date of recombination-mediated meiotic drive, of a magnitude sufficient to virtually guarantee that the recombination suppressor will eventually replace the more active allele in human populations.
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stage specificity dose response and doubling dose for mouse Minisatellite germ line mutation induced by acute radiation
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Yuri E Dubrova, Mark Plumb, Julia Brown, Jan Fennelly, Philippe Bois, D T Goodhead, Alec J JeffreysAbstract:Germ-line mutation induction at mouse Minisatellite loci by acute irradiation with x-rays was studied at premeiotic and postmeiotic stages of spermatogenesis. An elevated paternal mutation rate was found after irradiation of premeiotic spermatogonia and stem cells, whereas the frequency of Minisatellite mutation after postmeiotic irradiation of spermatids was similar to that in control litters. In contrast, paternal irradiation did not affect the maternal mutation rate. A linear dose–response curve for paternal mutation induced at premeiotic stages was found, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. High frequencies of spontaneous and induced mutations at Minisatellite loci allow mutation induction to be evaluated at low doses of exposure in very small population samples, which currently makes Minisatellite DNA the most powerful tool for monitoring radiation-induced germ-line mutation.
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Somatic mutation processes at a human Minisatellite
1997Co-Authors: Alec J Jeffreys, Rita NeumannAbstract:Germline instability at human Minisatellites frequently involves complex inter-allelic transfers of repeat units usually restricted to one end of the repeat array and apparently regulated by flanking DNA. In contrast, nothing is known about the structural basis of somatic instability at Minisatellites. An electrophoretic size-enrichment strategy was therefore developed at Minisatellite MS32 (D1S8) to enable rare abnormal-length mutants to be detected, validated and quanti-tated in blood DNA by single molecule PCR. Structural analysis of rare mutant alleles in blood revealed simple deletions/duplications of repeat unit blocks located at random along the tandem repeat array, a mode of mutation completely different from that seen in sperm. Furthermore, allele-specific suppression of sperm instability at MS32 did not affect somatic instability. These data suggest that conversion-based Minisatellite mutation in sperm is completely germline-specific and most likely meiotic in origin. Somatic instability appears to occur by a separate pathway involving replication slippage or, more likely, intra-allelic unequal crossing over
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four state mvr pcr increased discrimination of digital dna typing by simultaneous analysis of two polymorphic sites within Minisatellite variant repeats at d1s8
Web Science, 1993Co-Authors: Keiji Tamaki, Darren G Monckton, Annette Macleod, Maxine J Allen, Alec J JeffreysAbstract:: Minisatellite variant repeat mapping by PCR (MVR-PCR) provides a digital approach to DNA typing that can reveal huge levels of variation at Minisatellite loci. MVR-PCR has so far been applied to three human Minisatellites, including the hypervariable locus D1S8. Previous analysis at D1S8 was based on the discrimination of repeat unit types that differ by a single base substitution. We now show that a second polymorphic site within D1S8 repeats may be assayed simultaneously with the first to define four classes of repeat units ('four-state MVR-PCR'). This approach can also be applied to the other end of D1S8 alleles in 'reverse four-state MVR-PCR'. Both of these procedures substantially increase the informativeness of MVR analysis at D1S8 and should prove useful in studies of Minisatellite biology and potentially in forensic DNA typing.
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Minisatellite variant repeat mapping application to dna typing and mutation analysis
Experientia. Supplementum, 1993Co-Authors: Alec J Jeffreys, John A L Armour, Andrew Collick, Darren G Monckton, Annette Macleod, David L Neil, Keiji Tamaki, Maxine J Allen, Mark A JoblingAbstract:Most DNA typing systems assay allele length variation at tandemly repeated loci such as Minisatellites and microsatellites. Allele length measurements are approximate, which impedes the use of such loci in forensic analysis and in studies of allelic variability at hypervariable loci. We now review progress in the development of alternative DNA typing systems based on allelic variation in the interspersion patterns of variant repeat units along Minisatellite alleles. Minisatellite variant repeat mapping by PCR (MVR-PCR) not only provides a powerful new digital approach to DNA typing, but also for the first time allows investigation of the true level of allelic variability at Minisatellite loci and of the mutational mechanisms that generate ultravariability.