"Major minisatellite loci" detected by minisatellite clones 33.6 and 33.15 correspond to the cognate loci D1S111 and D7S437

G. Chimini et al. (1989, Genomics 5: 316-324) have recently reported that the two multilocus DNA fingerprinting probes 33.6 and 33.15 each detect a single major site in the human genome, at 1q23 and 7q35-q36, respectively, and speculate that these sites represent particularly large loci homologous to these probes. However, the human minisatellite loci cloned in 33.6 and 33.15 can themselves be assigned by somatic cell hybrid analysis to 1cen-q24 and 7q31.3-qter, respectively, corresponding to the "major loci" of Chimini et al. Furthermore, under their hybridization conditions, both 33.6 and 33.15 act largely as locus-specific minisatellite probes. The "major minisatellite loci" postulated by Chimini et al. do not therefore appear to represent major localized clusters of minisatellites in the human genome, but rather the loci cloned in 33.6 and 33.15.     

Minisatellite DNA markers in the chicken genome

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.     

DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.     

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.     

Chimaeric and constitutive DNA fingerprints in the common marmoset (Callithrix jacchus)

Marmosets normally produce dizygotic twins sharing placental blood vessels and exchanging bone marrow cells. Each individual is therefore likely to be a blood chimaera. To date, marmosets had only been DNA fingerprinted using blood samples and probes 33.6 and 33.15, resulting in highly similar fingerprints among litter mates and little variation between other individuals, thereby limiting this method's use for individual identification and parentage testing. In this study, novel probes were applied to detect greater polymorphism and to produce individual-specific DNA fingerprints. As expected, blood DNA profiles of twins and triplets were virtually identical, confirming chimaerism in this tissue and identifying litter mates. Furthermore, these profiles were sufficiently variable to distinguish between sibs from different litters and between all other individuals. To produce individual-specific DNA fingerprints, the use of DNA extracted from tissues poor in leukocytes was essential. The findings demonstrate that, despite extensive blood chimaerism, marmoset colonies can be effectively DNA fingerprinted for indicidual identification, zygosity testing, and relationship studies.     

The efficiency of multilocus DNA fingerprint probes for individualization and establishment of family relationships, determined from extensive casework.

The properties of human DNA fingerprints detected by multilocus minisatellite probes 33.6 and 33.15 have been investigated in 36 large sibships and in 1,702 Caucasian paternity cases involving the analysis of over 180,000 DNA fingerprint bands. The degree of overlap of minisatellite loci detected by these two probes is shown to be negligible (approximately 1%), and the resulting DNA fingerprints are therefore derived from independent sets of hypervariable loci. The level of allelism and linkage between different hypervariable DNA fragments scored with these probes is also low, implying substantial statistical independence of DNA fragments. Variation between the DNA fingerprints of different individuals indicates that the probability of chance identity is very low (much less than 10(-7) per probe). Empirical observations and theoretical considerations both indicate that genetic heterogeneity between subpopulations is unlikely to affect substantially the statistical evaluation of DNA fingerprints, at least among Caucasians. In paternity analysis, the proportion of nonmaternal DNA fragments in a child which cannot be attributed to the alleged father is shown to be an efficient statistic for distinguishing fathers from nonfathers, even in the presence of minisatellite mutation. Band-sharing estimates between a claimed parent and a child can also distinguish paternity from nonpaternity, though with less efficiency than comparison of a trio of mother, child, and alleged father.     

Increased detectability of somatic changes in the DNA from human tumours after probing with “synthetic” and “genome-derived” hypervariable multilocus probes

Summary DNA fingerprinting with two minisatellite (33.15, M13) and two simple repeat probes [(GACA)₄, (CAC)₄/ (GTG)₅] was performed to screen for somatic changes in the DNA from various solid human tumours in comparison with constitutional DNA from the same patient. Loss of bands or changes in band intensitities were observed. Together the probes 33.15 and (CAC)₅/(GTG)₅ detected deviating fingerprint patterns in 63% of the colorectal carcinomas investigated. In mammary and stomach carcinomas, only 1/11 and 2/11 tumours, respectively, showed differences with either of the three probes, 33.15, (GACA)₄ and (CAC)₅/(GTG)₅.     

DNA fingerprint analysis for the detection of induced mutations in mammalian cells in culture

A mutation assay in cultured mammalian cells based on the direct analysis of minisatellite DNA was developed. Band pattern variations reflect DNA alterations ranging from single base changes to complex rearrangements. By DNA fingerprinting a large number of autosomal loci throughout the human genome can be simultaneously checked, therefore minimizing the size of the samples of cell colonies to be scored in the absence of phenotypic selection. For the mutation assay chinese hamster cells (V79) were treated with Nitrosoguanidine and 14 independent colonies were isolated and expanded. DNA fingerprints were obtained after digestion of the DNA extracted from each clone with bothHinfI andHae III, and hybridisation with both 33.15 and 33.6 probes. Twelve colonies from untreated cells were also analysed. Several differences in the band pattern of treated colonies were observed when compared with untreated cells; digestion withHae III and hybridisation with 33.15 probe allowed the detection of the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic potential of chemical agents directly at the DNA level, without phenotypic selection. Moreover, with the method herein decribed, it is possible to distinguish between true mutations and epimutations, such as those caused by changes in DNA methylation.     

Multilocus DNA fingerprints in the plant Cynoglossum officinale L. and their use in the estimation of selfing

We have developed a nonradioactive oligonucleotide multilocus DNA fingerprinting method for Cynoglossum officinale . Of the 19 probes tested, six probes yielded banding patterns for all restriction enzymes used. All but one of the informative probes are repeats with a four-base motif. Approximately 60% of the loci appeared to be polymorphic. The sensitivity of the nonradioactive method was equal to that of the radioactive method. In addition, a new simple calculation method is presented to estimate selfing rates and approximate 95% confidence limits from the DNA fingerprint profiles avoiding 'between-gel' comparisons. The selfing rates differed significantly (as determined from 95% confidence intervals) between naturally pollinated individuals of C. officinale within the experimental population. The estimates ranged from 0 to 70% selfing.     

Use of DNA fingerprinting for human population genetic studies

DNA fingerprinting techniques have been used in population genetic studies on many different kinds of organisms. Here, we present new applications for multilocus DNA fingerprint probes in population studies and demonstrate the applicability of DNA fingerprinting to human population genetics, using M13 phage DNA as a probe. The new approach, which is based on a factor method of numerical coding of non-quantitative data (factor correspondence analysis-FCA), shows good agreement between population position, as indicated by the three principal factors, and ethnogenetic proximity.     

Characterization of tomato DNA clones with sequence similarity to human minisatellites 33.6 and 33.15

A tomato lambda genomic library was screened with the human minisatellites 33.6 and 33.15. Similar tomato sequences are estimated to occur on average every 4000 kb. In thirteen hybridizing clones characterized, the size of minisatellite arrays varied between 100 bp and 3 kb. The structure of the repetitive elements is complex as the human core sequence is interspersed with other elements. In three cases, sequences similar to the human minisatellites were part of a higher-order tandem repeat. The chromosomal position of these sequences was established by ascertaining linkage to previously mapped RFLP markers. In contrast to the human genome, no clustering of minisatellite loci was observed in tomato. The fingerprints generated by hybridizing tomato minisatellites to genomic DNA of a set of cultivars were, in two cases, more variable than those obtained with 33.6 or 33.15. Two of the characterized probes detected 4–8 alleles of a single locus, which displayed 10–15 times more polymorphism than random RFLP clones. Some minisatellites contain di- and tri-nucleotide microsatellite repeated motifs which may account for the high level of polymorphism detected with these clones.     

In situ hybridization and pulsed-field gel analysis define two major minisatellite loci: 1q23 for minisatellite 33.6 and 7q35-q36 for minisatellite 33.15

The two classical minisatellite probes, 33.6 and 33.15, were used for in situ hybridization to human metaphase chromosomes. Surprisingly, a single major hybridization peak was observed with each probe, respectively at 1q23 for 33.6 and 7q35-q36 for 33.15. Hybridization to human DNA cleaved with "rare-cutter" enzymes and fractionated on pulsed-field gels also showed a fairly simple, largely monomorphic pattern which allows chromosomal assignment using somatic cell hybrids. Differences in hybridization stringency and degree of resolution account for most of the discrepancy between these results and the accepted view of minisatellites, i.e., a large number of unlinked loci spread over the genome. Our results nevertheless indicate the existence of particularly large and homologous loci on a particular chromosome for each of these probes.     

Identification of hypervariable single locus minisatellite DNA probes in the blue tit Parus caeruleus

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.     

Estimation of genetic distance and coefficient of gene diversity from single-probe multilocus DNA fingerprinting data

DNA fingerprinting exhibits multilocus genotypes of individuals, detected by the use of a single multilocus probe. Consequently, population data on DNA fingerprinting do not provide a complete characterization of the genetic variation in terms of allele-frequency distributions, since neither the number of loci nor the locus affiliation of alleles is directly observable. Yet DNA fingerprinting has been proved to be a cost-effective method of detecting hypervariable polymorphisms in several organisms, where the traditional loci fail to detect enough variation for microevolutionary studies. In the present paper we demonstrate that the above-mentioned features of DNA fingerprinting data do not cause any serious problem when they are used in evolutionary studies. Bias-corrected estimators of Nei's standard and minimum genetic distances are derived, and, by an application of this theory to data on seven short tandem repeat loci in three major human populations, it is shown that these modified measures of genetic distances based on DNA fingerprint patterns are quite close to Nei's distances based on locus-specific allele frequencies. Empirical as well as theoretical support of the adequacy of such genetic distances from DNA fingerprinting data is also discussed, and it indicates that the technical limitations of DNA fingerprinting should not deter the use of the method for short-term evolutionary studies.      

Hypervariable minisatellite DNA sequences in the Indian peafowl Pavo cristatus.

We report here for the first time the large-scale isolation of hypervariable minisatellite DNA sequences from a non-human species, the Indian peafowl (Pavo cristatus). A size-selected genomic DNA fraction, rich in hypervariable minisatellites, was cloned into Charomid 9-36. This library was screened using two multilocus hypervariable probes, 33.6 and 33.15 and also, in a "probe-walking" approach, with five of the peafowl minisatellites initially isolated. Forty-eight positively hybridizing clones were characterized and found to originate from 30 different loci, 18 of which were polymorphic. Five of these variable minisatellite loci were studied further. They all showed Mendelian inheritance. The heterozygosities of these loci were relatively low (range 22-78%) in comparison with those of previously cloned human loci, as expected in view of inbreeding in our semicaptive study population. No new length allele mutations were observed in families and the mean mutation rate per locus is low (less than 0.004, 95% confidence maximum). These loci were also investigated by cross-species hybridization in related taxa. The ability of the probes to detect hypervariable sequences in other species within the same avian family was found to vary, from those probes that are species-specific to those that are apparently general to the family. We also illustrate the potential usefulness of these probes for paternity analysis in a study of sexual selection, and discuss the general application of specific hypervariable probes in behavioral and evolutionary studies.     

Minisatellite DNA fingerprints of Atlantic cod (Gadus morhua)

The human minisatellite probes 33.6 and 33.15 cross–hybridized to Hae III and Hinf I digested cod DNA, revealing complex fragment patterns in both Arctic and coastal cod. The DNA fingerprints were highly polymorphic, individual specific and stable in the germline. The potential applications of multi locus probes in cod research are discussed.     

Biomolecular analysis of a defective nontransforming Epstein-Barr virus (EBV) from a patient with chronic active EBV infection.

A virus recovered from the saliva of a child with chronic active Epstein-Barr virus (EBV) infection for 8 years was shown to induce EBV early antigen (EBV-EA) in Raji cells and to be expressed into EBV-EA in fresh EBV-negative peripheral blood leukocytes. However, it did not replicate its DNA. Oropharyngeal epithelial cells scraped from recurrent mouth lesions were similarly positive for EBV-EA. DNA extracted from these cells and digested with BamHI contained a 6-kilobase-pair fragment homologous to BamHI fragment V and B1 EBV DNA probes. Furthermore, Southern blots of the BamHI and EcoRI digests of the DNA extracted from the cell lines of the patient (transformed with EBV strain B95-8) and of her mother (spontaneous) revealed, in addition to the expected BamHI G, H, H2, and B1 fragments used as probes, additional shorter ones of a presumably endogenous defective virus.     

Ig repertoire of human polyspecific antibodies and B cell ontogeny.

A total of 463 EBV Ig-secreting clones were derived from embryonic tissues, cord blood, and adult peripheral blood. Subcloning and analysis of the H and K loci (germline vs rearranged DNA status) of 44 primary clones insured clonality in at least 92% of cases. Whatever the cell origin, a somewhat constant proportion of clones (i.e., 11 to 16%) expressed polyspecific antibodies when tested on a panel of nine Ag, including self-Ag. The VH and VK repertoires have been studied using VH1-VH6 and VK1-VK4 family-specific probes. For all EBV clones the VH and VK utilization was similar to that of the normal untransformed population. A correlation was observed between the level of expression and the gene number for VH, whereas a clear distortion appeared for VK. Moreover, the usage pattern of VH and VK families of the polyspecific clones did not significantly differ from that of clones of unknown specificity, suggesting that polyspecificity was not linked to a restricted repertoire.     

DNA fingerprinting—a valuable new technique for the characterisation of cell lines

DNA fingerprinting is an important new development for the authentication of cell lines. Multilocus methods such as those developed by Alec Jeffreys provide information on a wide range of genetic loci throughout the human genome and thus give a useful genetic “snap-shot” of a cell culture. Our work has shown that Jeffreys multilocus fingerprinting method can be applied to cell lines from a wide range of animals including reptiles, birds, fish and diverse mammals. It can also differentiate very closely related cell lines including those from the same mouse strain. Routine fingerprint analysis has enabled an unprecedented level of confidence in the consistency of cell stocks. Our results demonstrate that this straightforward method represents a powerful and readily interpreted system for cell authentication and exclusion of cross-contamination.     

A suite of falcon single-locus minisatellite probes: a powerful alternative to DNA fingerprinting

Nine falcon single-locus minisatellite probes have been cloned, characterized and shown to provide a powerful alternative to multilocus DNA fingerprinting for determining the parentage of broods of young. Eight clones derived from size-selected peregrine Falco peregrinus and merlin F. columbarius charomid libraries were found to detect single minisatellite loci in all tested members of the genus Falco . A further clone (c Fti 1) randomly selected from a kestrel F. tinnunculus library identified a single locus in other members of the genus and certain species of the Accipitridae. The nine loci display a mean heterozygosity of 88% and considerable allelic diversity in the peregrine and merlin. Pedigree analysis provides evidence consistent with the clustering of minisatellites in linkage groups that are conserved between species. Mutations were observed at five loci among families of peregrines and kestrels. Composite profiles produced with this suite of probes provide an excellent means of confirming identity and parentage. The use of profiling for investigating familial relationships, population dynamics and combating the illegal trade in wild falcons is discussed.