Multilocus Minisatellite Probes Research Articles

Molecular Fingerprinting of Leishmania infantum Strains following an Outbreak of Visceral Leishmaniasis in Central Israel

Human and canine visceral leishmaniasis caused by Leishmania infantum emerged in central Israel after an absence of over 30 years. The origin of this outbreak was investigated by examining genetic polymorphisms in 37 strains isolated from dogs and patients with visceral leishmaniasis in the continuously active northern Israeli and West Bank foci and in a new Israeli focus using DNA fingerprinting with the human multilocus minisatellite probe 33.15. Analysis of the patterns obtained by DNA fingerprinting separated the strains geographically into northern (clade B) and central (clades A and C) genotypic groups. These results suggest that the emergence of visceral leishmaniasis in central Israel is due not to parasite spread from northern Israel to the new focus but rather to increased parasite transmission in central Israel and the West Bank coupled with changes in the ecoepidemiology of this region.

Mini-and microsatellite mutations in children from Chernobyl accident cleanup workers

Knowledge about possible genotoxic effects of low-dose radiation on the human germline is limited and relies primarily on extrapolations from high-dose exposures. To test whether ionizing radiation can cause paternal genetic mutations that are transmitted to offspring, we enrolled families of 88 Chernobyl cleanup workers exposed to ionizing radiation. We analyzed DNA isolated from lymphocytes for mutations via DNA blotting with the multi-locus minisatellite probes 33.6 and 33.15 and via PCR in a panel of six tetranucleotide repeats. Children conceived before and children conceived after their father's exposure showed no statistically significant differences in mutation frequencies. We saw an increase in germline microsatellite mutations after radiation exposure that was not statistically significant. We found no dependence of mutation rate on increasing exposure. A novel finding was that the tetranucleotide marker D7S1482 demonstrated germline hypermutability. In conclusion, our results do not support an increased level of germline minisatellite mutations but suggest a modest increase in germline mutations in tetranucleotide repeats. Small sample size, however, limited statistical power.

Urbanisation and the genetic structure of Passer italiae (Vieillot 1817) populations in the South of Italy

Sparrows have colonized areas modified by humans since the onset of agriculture and seed storage. In fact, present day distributions of the sparrow populations are directly related to urban areas, farms and villages. Consequently the sedentary behaviour of sparrows, in pristine areas, with low densities of specimens, can act as a barrier to gene flow among populations. We analyzed the genetic variability and gene flow rates in four Italian sparrow populations sampled in areas affected by different levels of human activity. Population variability was analyzed by DNA fingerprinting with M13 multilocus minisatellite probe, which yielded multibanded fingerprints (the average number of bands was 25, ranging in size from 23 kb to 0.3 kb). The population located within the pristine area of the Cilento-Vallo di Diano National Park (San Marco) showed the higher value of within-population similarity (Sw = 0.73). This suggests a probable higher level of inbreeding in comparison with the other localities. The genetic isolation of the San Marco population is also imputable to the between-populations similarity values (Sb), that seem to be related with a preferential direction of gene circulation along urbanised corridors. The results show that population variability and gene flow rate in the Italian sparrow are directly related to the presence of urbanised areas. The results also show that the M13 multilocus fingerprinting probe represents a advantageous marker for the detection of genetic variability within a small geographical scale in passerine birds.

Multilocus DNA profiling using the probe 33.6 in the Tamil Nadu population of South India.

Using the multilocus minisatellite probe 33.6 in combination with the HinfI restriction enzyme, the extent of genetic variation detected by DNA fingerprinting was estimated in 102 unrelated individuals of the Dravidian Hindu population of Tamil Nadu, South India. In this first study of its kind on an anthropologically defined Indian population, DNA fragments of size >2.5 kb could be reliably scored. Results indicate that the Tamilian Hindus show an average number of bands per individual somewhat smaller (15.69 for fragments of size >2.5 kb) than that in other Caucasian populations. For comparable molecular weight of fragment sizes, the Tamilian Hindus show a lower level of band sharing probabilities between unrelated individuals compared with other Caucasians. Nevertheless, the probe 33.6 offers a high level of individualization of DNA fingerprints for unrelated individuals in Tamil Nadu. Computations on expected band sharing frequencies between various biological relatives and expected DNA fingerprint identity indicate that this multilocus minisatellite probe can be efficiently used in resolving forensic identification and parentage testing cases in this South Indian population in spite of its high level of inbreeding. Am. J. Hum. Biol. 10:87-93, 1998. © 1998 Wiley-Liss, Inc.

Female-Specific Restriction Fragments Revealed by Dna-Fingerprinting and Implications for Extra-Pair Fertilisations in the Short-Tailed Shearwater (Puffinus-Tenuirostris, Procellariiformes, Procellariidae)

We report a female-specific restriction fragment in the DNA fingerprints of short-tailed shearwaters (Puffinus tenuirostris) that hybridises to a derivative of the human multilocus minisatellite probe 33.6. This genetic method of assigning sex has relevance to studies of population biology, reproductive success and mating systems in this species. The presence or absence of the female-specific restriction fragment has allowed us to reinterpret results from a previous DNA fingerprinting study of mating systems in the short-tailed shearwater.

Parentage analysis of multi-male social groups of tasmanian native hens (Tribonyx mortierii): genetic evidence for monogamy and polyandry

Accurate estimates of male reproductive success are essential to understanding the evolution of polyandrous mating systems. Here, we use multilocus DNA fingerpinting to assess parentage in an island population of Tasmanian native hens (Tribonyx mortierii), which often live in multi-male and/or multi-female social groups. This isolated population presented special challenges to this technique because it was artificially founded from a small number of individuals in the recent past. DNA profiles from four multilocus minisatellite probes were analyzed for adults and offspring from six social groups using two methods: (1) significance of band-sharing coefficients and (2) distribution among a group's offspring of fragments unique to certain adults. Traditional band-sharing analyses did not provide sufficient resolution to establish parentage in this population due to the high level of band-sharing between adults within groups. In contrast, the distribution of unique fragments suggests that in most cases, all offspring within a group have the same male and female genetic parents, so that monogamy may be the predominant “genetic” mating system of this species. This forces a rexamination of the evolutionary basis of polyandry in these birds. It also demonstrates some of the difficulties in using these highly polymorphic genetic markers for parentage analyses when putative parents are closely related.

Genetic variability among root voles (Microtus oeconomus) from different geographic regions: populations can be distinguished by DNA fingerprinting

Genetic variability among root voles (Microtus oeconomus) from different geographic regions: populations can be distinguished by DNA fingerprinting

Genetic variability among root voles (Microtus oeconomus) from different geographic regions: populations can be distinguished by DNA fingerprinting

Genetic variability among root voles (Microtus oeconomus [Pallas, 1776]) originating from two distantly separate regions of Norway (Valdres and Finnmark) was studied by DNA fingerprinting using the probes 33.15, 33.6 and M13. All three probes revealed polymorphic, although relatively simple, patterns. DNA fingerprint banding patterns were clearly diagnostic of the animals' region of origin. Notably, Valdres animals display a high molecular-weight cluster of bands not found in Finnmark, reflective of the isolation, and possibly an indication of separate colonization events, of the two groups. On the local level in Finnmark, bands associating with a specific trap site were observed in trappings on consecutive years. Comparisons of Finnmark animals taken at three trap sites at approximately 10 km intervals show a gradient of genetic similarity. Captive bred siblings were also compared, yielding average values significantly higher than those seen from same-site comparisons. We suggest that the sensitivity provided by DNA fingerprinting with multi-locus minisatellite probes is appropriate for population genetic studies in M. oeconomus. Also, because band-sharing correlates to spacing in M. oeconomus, we propose that DNA fingerprinting may be used to study dispersal, recruitment and other population processes in this and possibly other rodent species.

Demonstration by DNA fingerprint analysis of genomic instability in mouse BALB 3T3 cells during cell transformation

We employed DNA fingerprint analysis to monitor DNA rearrangements in BALB 3T3 cells transformed spontaneously or by treatment with 3-methylcholanthrene (MCA) and UV-C. The effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in combination with MCA was also examined. Twenty-three spontaneously transformed cells, 28 induced transformed cells (18 by 1 microgram/ml MCA, six by 5 micrograms/ml MCA, and four by UV-C), and 31 non-transformed subclones were isolated from parental BALB 3T3 A31-1-1 cells. The DNAs were digested with HinfI and subjected to DNA fingerprint analysis with three multi-locus minisatellite probes, Per-6, Core, and Ins. Per-6 was the most effective probe for detecting DNA rearrangements. Rearranged bands detected by the Per-6 probe were observed in 9/31 (29%) of non-transformed subclones, 14/23 (61%) of spontaneously transformed cells, 16/18 (89%) of cells transformed by 1 microgram/ml of MCA, 6/6 (100%) of cells transformed by 5 micrograms/ml MCA, and 4/4 (100%) of UV-C-transformed cells. Higher numbers of DNA rearrangements (> or = 3) occurred most frequently in the induced transformed cells. TPA enhanced the frequency of DNA rearrangements in cells transformed by MCA. These data indicate that (1) genomic DNA in BALB 3T3 cells is unstable and susceptible to rearrangement, (2) its instability is elevated during cell transformation, and (3) MCA and UV-C induce DNA rearrangements, and TPA enhances the effect of the former, probably via the recombination process. DNA fingerprint analysis is valuable for monitoring genomic instability during cell transformation.

Detection of recombinational mutations in cultured human cells by Southern blot analysis with minisatellite DNA probes

Using the human acute monocytic leukemia cell line, THP-1, a hypermutability of minisatellite loci was demonstrated in cell culture by Southern blot analysis with minisatellite DNA probes. DNA was isolated from 98 subclones and hybridized to a panel of minisatellite probes consisting of three multilocus minisatellite probes (ML probes) and seven locus-specific minisatellite probes (LS probes). The Southern blot patterns of the hybridized subclones were compared with those of the parental THP-1. Four mutated bands with two ML probes and two mutated bands with two LS probes were detected. The mutation frequency was estimated roughly at 0.1% based on the total number of bands analyzed, and it was much higher than that expected for other DNA regions. Four of these mutations were thought to be alterations of repetitions caused by insertion or deletion of tandem repeats, and one mutant lost a complete minisatellite allele. The nature of the sixth mutant was unclear. Because of the hypermutability of minisatellite DNA, Southern blot analysis using minisatellite DNA probes can be used as a mutation assay system directly based on the DNA.

Short-term study of chimaerism after bone marrow transplantation for severe aplastic anaemia.

Chimaerism was studied early (2 weeks to 3 months) during haematopoietic reconstitution after bone marrow transplantation in 18 severe aplastic anaemia patients (acquired SAA: 14 patients; Fanconi anaemia: four patients). Fourteen patients received marrow from an identical sibling donor, one from the phenoidentical father and three from a matched unrelated donor. Peripheral blood cell DNA was first analysed by Southern blotting with a multilocus minisatellite probe (33.6.3) or a Y chromosome specific probe (pHY2.1). For all 14 patients grafted with a genotypically identical sibling donor, the post-graft DNA profile strictly matched the respective donor profile (minisatellite probe) or disclosed the Y chromosome specific band in the case of female patients grafted with a male donor. In contrast, for the one patient grafted in a mismatched situation and for two out of three patients grafted with a matched unrelated donor, the results indicated autologous bone marrow recovery. This difference between patients grafted with an identical sibling donor and those grafted in other situations is statistically significant (P less than 0.01). The 15 patients with circulating cells of donor origin were then studied by polymerase chain reaction amplification of the DNA samples. The three male patients with a female donor were studied by amplification of a Y chromosome specific sequence (DYZ1), allowing the detection of one male cell in 10(4) female cells. In all three cases, residual male nucleated cells were detected. The analysis was performed by amplification of the 33.6.3 minisatellite sequence for the 12 remaining patients. No residual recipient cells were detected within the sensitivity limit of the method which is 1% in that case. This suggests that detection of residual host cells depends on the sensitivity of the technique used.

Restriction Fragment Length Polymorphism Analysis of Forensic Science Casework in the People's Republic of China

Restriction fragment length polymorphism (RFLP) analysis for the purpose of individualization is now being used in casework in the People's Republic of China. This report describes the use of the multilocus minisatellite probe 33.15 to solve three cases, including two homicides and a rape. In the third case, fetal tissue was analyzed to prove that the alleged rapist was, in fact, the father. In each case, analysis of deoxyribonucleic acid (DNA) resulted in a positive match. The probability of chance association of the DNA fingerprint was calculated as 5.6 x 10(-12), which is similar to the figures reported in the literature.