Minisatellite Loci Research Articles

Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy.

To determine whether the measurement of repeat number mutations at a minisatellite locus could detect human germline mutations induced by chemotherapy, we performed a longitudinal study of the mutation frequencies in sperm from 10 patients treated for Hodgkin's disease. Polymerase chain reaction on small pools of DNA equivalent to 100 sperm and Southern blotting were used to screen at least 7900 sperm in each sample to quantify the mutation frequency at the minisatellite MS205 locus. Pretreatment and posttreatment semen samples were obtained at least 2 months after completion of therapy from 4 patients treated with a regimen (Novantrone, Oncovin, vinblastine and prednisone [NOVP]) that lacks alkylating agents and from three patients treated with regimens (Cytoxan, vinblastine, procarbazine and prednisone/Adriamycin, bleomycin, dacarbazine, lomustine, and prednisone [CVPP/ABDIC] or mechlorethamine, Oncovin, procarbazine and prednisone [MOPP]) containing alkylating agents. There were no effects of NOVP or CVPP/ABDIC on the mutation frequencies. In the 1 patient treated with MOPP, the treatment with the highest dose of gonadotoxic alkylating agents, there was a statistically significant increase in mutation frequency from 0.79% pretreatment to 1.14% posttreatment, indicating induction of mutations in stem spermatogonia. During-treatment semen samples obtained from 2 patients treated with ABVD, which does not contain gonadotoxic alkylating agents, and 1 with NOVP also did not show any increases above the baseline mutation frequencies, indicating no increase in the minisatellite mutation frequency in spermatocytes. Thus, measurement of repeat number changes at minisatellite MS205 appears to be able to detect induced germline mutations in human sperm. However, most chemotherapy regimens do not significantly increase this class of mutations.

Genetic implications of translocation and stocking of fish species, with particular reference to Western Australia

Species or strains of fish may be translocated for farming, where the only access to the wild is via inadvertent escapes, or for stocking, where deliberate releases are undertaken. In either case, it is important that the translocated animals are representative of the donor population(s) in terms of genetic composition and level of variability. Many studies have shown that this ideal is difficult to achieve, the major reason being the use of inadequate numbers or composition of broodstock as founders of a strain. Also, where more than one conspecific population is involved, there may be outbreeding depression problems. In the case of farming, measures to improve the introduced strain genetically are likely to be undertaken, e.g. breeding programmes, manipulation of sex and ploidy, transgenic techniques. Such approaches are necessary economically, but can alter genetic make-up. Thus, stringent attempts must be made to minimize escapes or reduce their impact should they occur. With stocking, genetic change during captive rearing should be avoided. No strain manipulation should be undertaken, and other agents of change should be minimized. Stocking may result in hybridization with related species or with endemic populations of the same species. In either case, there can be detrimental genetic effects on the native forms. To be able to identify subsequently any genetic changes in reared strains, whether intended for farming or stocking, wild population composition should be determined, using appropriate molecular techniques. Such molecular methods will demonstrate the degree of interpopulation differentiation and, thus, reproductive isolation. The same markers should then be used in each subsequent generation (in the hatchery and after escape or reintroduction to the wild) to monitor any changes in genetic composition or variability. Markers should include microsatellite DNA loci, but the inclusion of more than one type of marker is recommended. However, as the aforementioned markers are not considered to be influenced by natural selection, they give no information on the adaptive nature of such differences. For this reason, it is suggested that markers influenced by selection should be investigated. Monitoring a strain subsequent to deliberate or inadvertent release can be undertaken using genetic markers, either deliberately enhanced by breeding or occurring naturally. Highly variable minisatellite DNA loci have been used as family markers in farmed escape studies with Atlantic salmon. These investigations have demonstrated significantly superior survival of native strains compared with farmed salmon in natural stream conditions. These latter results, demonstrating fitness differences, were strongly indicative of local adaptation. Thus, methods exist to monitor the genetic effects of translocation and stocking. However, a holistic approach should be taken to such exercises, where genetics forms part of a wider suite of considerations.

Genetic variation at the ApoB 3' HVR minisatellite locus in the Mbenzele Pygmies from the Central African Republic.

This study analyzes the polymorphic minisatellite ApoB 3' HVR in the Mbenzele Pygmies from the Central African Republic. A total of 14 alleles was observed, with frequencies ranging from 0.020 (19, 21, 27, and 45 repeat unit alleles) to 0.210 (37 repeat unit allele). Departure from Hardy-Weinberg equilibrium was not statistically significant. The estimated heterozygosity was 0.874 +/- 0.016. Taking data from the literature into consideration, the results support the hypothesis that the Africans are different from non-Africans due to greater ApoB 3' HVR genetic diversity and a unimodal profile of ApoB 3' HVR allele frequency distribution. Interpopulational relationships were also analyzed using an F(ST) based genetic distance. The results highlight the similarity between the Mbenzele Pygmies and Bantu-speaking groups (Ewondo and Zulu), and the divergence between the Mbenzele and San, the two groups which are often considered to be the most direct descendants of proto-Africans. Am. J. Hum. Biol. 12:588-592, 2000. Copyright 2000 Wiley-Liss, Inc.

Differential Methylation of Microsatellite and Minisatellite Loci in Rice

Differential Methylation of Microsatellite and Minisatellite Loci in Rice

Minisatellite mutational processes reduce F(st) estimates.

We have used a new method for binning minisatellite alleles (semi-automated allele aggregation) and report the extent of population diversity detectable by eleven minisatellite loci in 2,689 individuals from 19 human populations distributed widely throughout the world. Whereas population relationships are consistent with those found in other studies, our estimate of genetic differentiation (F(st)) between populations is less than 8%, which is lower than comparative estimates of between 10%-15% obtained by using other sources of polymorphism data. We infer that mutational processes are involved in reducing F(st) estimates from minisatellite data because, first, the lowest F(st) estimates are found at loci showing autocorrelated frequencies among alleles of similar size and, second, F(st) declines with heterozygosity but by more than predicted assuming simple models of mutation. These conclusions are consistent with the view that minisatellites are subject to selective or mutational constraints in addition to those expected under simple step-wise mutation models.

Meiotic instability of human minisatellite CEB1 in yeast requires DNA double-strand breaks.

Minisatellites are tandemly repeated DNA sequences of 10-100-bp units. Some minisatellite loci are highly unstable in the human germ line, and structural analysis of mutant alleles has suggested that repeat instability results from a recombination-based process. To provide insights into the molecular mechanism of human minisatellite instability, we developed Saccharomyces cerevisiae strains carrying alleles of the most unstable human minisatellite locus, CEB1 (ref. 2). We observed that CEB1 is destabilized in meiosis, resulting in a variety of intra- and inter-allelic gains or losses of repeat units, similar to rearrangements described in humans. Using mutations affecting the initiation of recombination (spo11) or mismatch repair (msh2 pms1 ), we demonstrate that meiotic destabilization depends on the initiation of homologous recombination at nearby DNA double-strand break (DSBs) sites and involves a 'rearranged heteroduplex' intermediate. Most of the human and yeast data can be explained and unified in the context of DSB repair models.

Physical mapping of three minisatellite sequences in the Atlantic salmon (Salmo salar) genome

We have determined the localization of three minisatellite loci SsBglIIL.6, SsBglIIU.20 and SsPstL.26 in the Atlantic salmon (Salmo salar) genome by fluorescent in situ hybridization (FISH). FISH analysis of the SsBglIIL.6 and SsBglIIU.20 minisatellites revealed that they are both located in single chromosome pairs allowing their direct identification; in contrast, the SsPstIL.26 probe hybridizes to four different chromosomal pairs. The analysis of chromosomal location of minisatellite sequences could be very useful for studying structural changes that have taken place during chromosome evolution in the karyotype of Salmo salar.

Description of Three New Alleles at the D1S80 (MCT118) Locus

Abstract The minisatellite locus D1S80 is a variable number of tandem repeat (VNTR) locus which is commonly typed for forensic and paternity testing in a large number of laboratories all over the world. Here we describe three new alleles at the D1S80 locus; one small allele composed of 13 units of the repeated 16 mer, a further large allele with approximately 45 repeat units which we found two times in our population sample and another large allele with approximately 50 repeats.

Linkage and physical mapping of rat microsatellites derived from minisatellite loci.

Linkage and physical mapping of rat microsatellites derived from minisatellite loci.

Experimentally observed germline mutations at human micro- and minisatellite loci.

We have analysed close to 30,000 human germline transmission events at five microsatellite loci (D3S1359, HumTH01, HumvWA, HumTPO and HumFES) and four minisatellite loci (D1S80, ApoB, Col2A1 and D17S30). At these loci the mutation rates are similar at the microsatellite and the minisatellite loci, varying from 0.2 x 10(-3) to < 3.3 x 10(-3) and from 0.5 x 10(-3) to 1.5 x 10(-3), respectively. Interestingly, paternal mutations appeared to be dominant at the microsatellite loci, whilst maternal mutations are dominant at minisatellite loci. Based on our data, no unequivocal support for a strict strand-slippage mutation mechanism (gain or loss of a single repeat) was found, although the vast majority of the mutational events were small gains or losses of one to three repeats, and only few unequivocal large gains or losses were observed.

Brief communication. Transmission distortion at a minisatellite locus in the harlequin beetle riding pseudoscorpion

Brief communication. Transmission distortion at a minisatellite locus in the harlequin beetle riding pseudoscorpion

New method of PCR amplification of two human minisatellite loci and reliable method of DNA isolation

A method of DNA isolation and purification from highly contaminated sources usual for forensic medicine and criminalistic practice was developed. Oligonucleotide primers were synthesized and the conditions of PCR amplification were optimized for two human minisatellite (VNRT) loci — apolipoprotein B and D1S30 widely used in PCR-based DNA typing. The proposed procedure of DNA isolation includes standard treatment with proteinase K followed by phenol-chloroform extraction and further purification from cations and low molecular weight compounds on a Dowex/Sephadex G-50 minicolumn. The length of the primers and optimal PCR conditions determine stability and specificity of amplification of the VNTR-loci. The sensitivity of the proposed method is 2–4 ng DNA template in the reaction mixture.

Inbreeding of bottlenecked butterfly populations. Estimation using the likelihood of changes in marker allele frequencies.

Polymorphic enzyme and minisatellite loci were used to estimate the degree of inbreeding in experimentally bottlenecked populations of the butterfly, Bicyclus anynana (Satyridae), three generations after founding events of 2, 6, 20, or 300 individuals, each bottleneck size being replicated at least four times. Heterozygosity fell more than expected, though not significantly so, but this traditional measure of the degree of inbreeding did not make full use of the information from genetic markers. It proved more informative to estimate directly the probability distribution of a measure of inbreeding, sigma2, the variance in the number of descendants left per gene. In all bottlenecked lines, sigma2 was significantly larger than in control lines (300 founders). We demonstrate that this excess inbreeding was brought about both by an increase in the variance of reproductive success of individuals, but also by another process. We argue that in bottlenecked lines linkage disequilibrium generated by the small number of haplotypes passing through the bottleneck resulted in hitchhiking of particular marker alleles with those haplotypes favored by selection. In control lines, linkage disequilibrium was minimal. Our result, indicating more inbreeding than expected from demographic parameters, contrasts with the findings of previous (Drosophila) experiments in which the decline in observed heterozygosity was slower than expected and attributed to associative overdominance. The different outcomes may both be explained as a consequence of linkage disequilibrium under different regimes of inbreeding. The likelihood-based method to estimate inbreeding should be of wide applicability. It was, for example, able to resolve small differences in sigma2 among replicate lines within bottleneck-size treatments, which could be related to the observed variation in reproductive viability.

Distinct mutation patterns of breast cancer-associated alleles of the HRAS1 minisatellite locus

DNA sequence analysis of 130 alleles of the HRAS1 minisatellite has demonstrated that breast cancer-associated variants arise as a consequence of both replication errors and gene conversions. Unlike mutations at other variable number of tandem repeats (VNTRs), high-risk variants of the HRAS1 minisatellite do not demonstrate positional polarity. Instead, most mutations occur at three hotspots, with replication errors confined to one hotspot, gene conversions to a second and a mixed pattern of mutation at the third. DNA sequence analysis of 66 low-risk a1 alleles revealed no evidence for hypermutation. Therefore, while the HRAS1 minisatellite may serve as a reporter for a broad-based group of mutational mechanisms, these results are consistent with a direct pathogenetic contribution by high-risk alleles as the biological basis underlying cancer association of this VNTR.

Genetic Structure Among Breeding Herring Gulls ( Larus argentatus) from the Great Lakes and Eastern Canada

Herring gulls ( Larus argentatus) have been used as sentinel species for exposure to toxic chemicals since the 1960s. Reference populations in these studies have never been characterized to determine whether genetic differences might explain some of the effects seen in gulls at contaminated locations. Previously it was shown that there were higher rates of germline minisatellite DNA mutation rates in herring gulls colonizing sites located near steel industries in urban areas in the Great Lakes. It was suggested that population substructuring among gull colonies could account for differences in the mutation rates observed as a result of surveying different minisatellite loci. Here this explanation is dismissed by showing that genetic exchange among herring gull populations within the Great Lakes appears to be sufficient to ensure genetic homogeneity of these populations; in contrast, differences exist at the minisatellite loci scored between the gulls in the Great Lakes and the Maritimes. It is suggested that reference samples for future Great Lakes herring gull studies be selected from within the Great Lakes, and that potential genetic divergence from Maritime gulls should be investigated in more detail.

Reference database of hypervariable genetic markers of Argentina: application for molecular anthropology and forensic casework.

The population of Argentina is mostly composed of people of European ancestry. Aboriginal communities are at present very reduced in number and restricted to small geographically isolated patches. Three aboriginal communities, the Mapuche, Tehuelche and Wichi, were selected for short tandem repeat (STR) investigation. The metropolitan population of the city of Buenos Aires was analyzed, with both micro- and minisatellites. The minisatellite loci D1S7, D2S44, D4S139, D5S110, D8S358, D10S28, and D17S26 were typed on HaeIII-digested DNA obtained from unrelated individuals. D1S80 was typed by polymerase chain reaction (PCR). The autosomal STRs THO1, FABP, D6S366, CSF1PO, TPOX, F13A1, FES/FPS, vWA, MBPA/B, D16S539, D7S820, D13S317, and RENA4 and the sex chromosome STRs HPRTB, DYS385, DYS3891, DYS38911, DYS19, DYS390, DYS391, DYS392, DYS393 and YCAII were also investigated. As a by-product of our investigations, a reference database was created that is routinely used in forensic casework and paternity testing. STR allele frequency distributions are characterized by significant differences within and also between different populations. In contrast, the minisatellite bin distribution of the metropolitan population is not significantly different from other Caucasian populations.

Reference database of hypervariable genetic markers of Argentina: Application for molecular anthropology and forensic casework

The population of Argentina is mostly composed of people of European ancestry. Aboriginal communities are at present very reduced in number and restricted to small geographically isolated patches. Three aboriginal communities, the Mapuche, Tehuelche and Wichi, were selected for short tandem repeat (STR) investigation. The metropolitan population of the city of Buenos Aires was analyzed, with both micro- and minisatellites. The minisatellite loci D1S7, D2S44, D4S139, D5S110, D8S358, D10S28, and D17S26 were typed on HaeIII-digested DNA obtained from unrelated individuals. D1S80 was typed by polymerase chain reaction (PCR). The autosomal STRs THO1, FABP, D6S366, CSF1PO, TPOX, F13A1, FES/FPS, vWA, MBPA/B, D16S539, D7S820, D13S317, and RENA4 and the sex chromosome STRs HPRTB, DYS385, DYS389I, DYS389II, DYS19, DYS390, DYS391, DYS392, DYS393 and YCAII were also investigated. As a by-product of our investigations, a reference database was created that is routinely used in forensic casework and paternity testing. STR allele frequency distributions are characterized by significant differences within and also between different populations. In contrast, the minisatellite bin distribution of the metropolitan population is not significantly different from other Caucasian populations.

De novo mutations and allelic diversity at minisatellite locus D7S22 investigated by allele-specific four-state MVR-PCR analysis

We have studied the allelic diversity and de novo mutations at the hypervariable minisatellite locus D7S22. A four-state minisatellite variant repeat unit mapping by PCR (MVR-PCR) method was developed for this purpose, and a substitution polymorphism close to the repeat array was used to design allele-specific flanking primers to study individual haplotypes in genomic DNA. A total of 150 alleles from different allele size groups and flanking haplotypes were mapped. On average, MVR-codes extending 65 repeats (2.4 kb) into the repeat array were obtained. The interspersion patterns of variant repeats were highly polymorphic. However, subgroups of alleles close in size and with identical flanking haplotype revealed common MVR-code characteristics indicating a close evolutionary relationship. Unlike the situation in many other hypervariable minisatellites, no polarized variability was revealed at this minisatellite locus. Fifty four small families with D7S22 de novo mutations were analysed by MVR-PCR. The sites where the length change occurred were revealed in 22 cases, while in 32 cases the mutation obviously occurred further into the repeat array. In agreement with a non-polar distribution of the allelic variation, there was no evidence for a hypermutable hot spot for mutation within the repeat array. Comparison of MVR-codes in the mutant and progenitor in gain mutations indicated that at least one, possibly four cases, reflected inter-allelic events. Together with evidence from DNA sequencing of alleles of <2 kb, this indicates that as many as half of the gain mutations might be inter-allelic events in D7S22. Based on these results, different factors which might affect the mutation rate are discussed.

Evaluation of seven PCR-based assays for the analysis of microchimerism

Objective: The presence of small numbers of cells of donor origin in the circulation of recipients of organ transplants (microchimerism) may correlate with immunologic tolerance. As part of our ongoing studies on microchimerism, we evaluated the utility of seven PCR-based assays for the detection of the less abundant DNA in paired mixtures (100 ng total DNA). Design and methods: DNA samples were screened to identify pairs informative for one or more PCR assays. DNA mixtures from the informative pairs were then analyzed using at least one assay. The assays were based on the X-Y homologous region; a Y chromosome microsatellite locus; three autosomal microsatellite loci; the D1S80 minisatellite locus; and sequence specific oligonucleotide probe (SSOP) analysis of the HLA DRB1 locus. Results: About 0.1% of male DNA against a background of female DNA was detectable using primers for the X-Y homologous region, but the sensitivity was increased to 0.0001% using nested primers for the Y chromosome microsatellite marker. Analysis of the minor DNA component was difficult with the three autosomal microsatellite assays because of the presence of shadow bands. Similar problems with the D1S80 assay were resolved using more stringent PCR conditions, and the sensitivity was 0.1%. Using the DRB1 locus, we were able to detect 1% DNA in the mixed samples. Conclusions: These studies show that: (a) nested PCR for the Y chromosome is the most sensitive assay for the detection of microchimerism; (b) D1S80 is a useful marker for microchimerism; (c) additional optimization of analytical conditions is required if autosomal microsatellite markers and the SSOP assay are to be used for microchimerism analysis.

Prevalence of minisatellite and microsatellite instability in radiation-induced post-Chernobyl pediatric thyroid carcinomas.

Exposure to ionizing radiation induces different forms of genomic instability in cultured cells and experimental animals. A higher rate of germline mutations at human hypervariable minisatellite loci was reported in children born from parents exposed to radiation after Chernobyl, implicating genome destabilization as a possible mechanism responsible for late radiation effects in humans. To test if radiation-induced carcinogenesis in the thyroid gland may be associated with somatic minisatellite instability or microsatellite instability, we utilized a PCR-based approach to study normal and tumor DNA from 17 pediatric post-Chernobyl papillary thyroid carcinomas for mutations at three different minisatellite loci (D1S80, D17S30, ApoB), and 27 microsatellite loci of di-, tri-, or tetranucleotide repeats. Minisatellite instability was found in three (18%) tumors, with one of them exhibiting mutations in all three minisatellite loci, whereas two others showed mutations in one of two informative markers. By contrast, none of 20 sporadic thyroid cancers from patients with no history of radiation exposure was positive for minisatellite instability. Microsatellite analysis of post-Chernobyl tumors revealed a mutation in one (6%) tumor only at the locus of D10S1412, whereas all other 26 microsatellite markers showed identical patterns in each normal/tumor pair. Our results suggest that somatic cell microsatellite instability does not contribute to radiation-induced thyroid carcinogenesis. However, somatic minisatellite mutation events are present in a subset of radiation-induced, but not sporadic, thyroid cancers, suggesting that this type of genomic instability may play a role in radiation-induced tumorigenesis in the thyroid gland.