Multilocus Probes Research Articles

Genomic stability and instability in different neuroepithelial tumors. A role for chromosome structure?

Selected childhood and adult neoplasm exemplify fundamental differences in their propensity for genomic change. DNA replication is essential for the formation of neuroepithelial tumors, probably because the genome can be remodeled. Nonetheless, several differentiated and stable childhood neoplasms retain their nuclear controls for differentiation. In contrast, rapidly arising gliomas often show a variety of phenotypic changes. Genomic plasticity and instability allow gliomas to flexibly adapt to new environments. Gene changes (in DNA) can be limited in childhood tumors whereas more widespread genetic changes in malignant gliomas indicate a fundamental alteration in many chromosome regions. Can such regions be defined? We used one repeated DNA sequence (TTAGGG)n, present at the end of all normal human chromosomes, to investigate chromosome termini in more detail. Pulsed-field gel electrophoresis showed this region can be unusually variable, as several other multilocus probes did not reveal comparable changes. Because telomeres form unique chromosomal structures, and are thought to provide essential signals to position chromosomes in the interphase nucleus, it was pertinent to assess these regions by in situ hybridization. Many telomeric domains localized at variable as well as interior nuclear positions in glioma cells. These positions, which are presumably abnormal, may be generated by the DNA variants observed. Such position changes may contribute to the more general 'disorder' observed in glioma nuclei. Other chromosome domains with a unique DNA-protein structure may define additional genomic loci that are preferentially modified in neoplasia. A fundamental understanding of chromosome structure should clarify the problem of multilocus instability in glioblastoma.

Isolation and characterization of hypervariable sequences from tsetse fly genome

A Glossina brevipalpis Newstead genomic library, constructed using a Charomid 9–36 vector, was used to isolate putative clones that hybridize to polymorphic regions of the tsetse genome. Five types of probes, that reveal individual DNA polymorphic in humans and higher animal species, were used to screen 300 tsetse Charomid clones; 15% of the clones hybridized with at least one probe. Twenty four recombinants were further characterized by Southern blotting hybridization using DNA isolated from individual tsetse fly from Glossina morsitans centralis Machado. Two classes of DNA profiles were obtained upon hybridization with the recombinant inserts. The first, termed the “multilocus profile” displayed a complex DNA pattern of multiple components whilst the second, referred to as the “single locus profile” revealed one or two bands in each individual. Of the 24 recombinant inserts tested, 13 were multilocus probes and the remainder were single locus probes, each of which hybridized to a single location when G. m. centralis DNA had been cleaved with Eco RI. These single locus probes revealed a low level of genetic variability among individual flies from an inbred colony. The hybridization profiles using multilocus and single locus probes were also obtained on DNA from individual Glossina palpalis palpalis Robineau-Desvoidy and Glossina palpalis gambiensis Vanderplank and some of the G. brevipalpis recombinant clones also detected multilocus profiles in honey bees and man.

Identification, isolation and characterization of canine minisatellite sequences.

A Charomid ordered-array library containing a 2-16 Kb size fraction of MboI-digested canine genomic DNA has been screened with the Jeffreys multilocus probes, 33.6 and 33.15, to identify and isolate canine minisatellite sequences. Of the 48 positive clones identified, 7 were found to contain polymorphic minisatellites with heterozygosities in the range 20-88%. The majority of the remainder were either monomorphic or dimorphic in the animals tested. Analysis of intrabreed variation in Bedlington Terriers using two polymorphic minisatellites has shown that a significant reduction occurs in the number of alleles seen compared to an agglomerated population sample, correlating with the high level of inbreeding within this breed. Flanking DNA sequence and partial repeat sequence is presented for the most polymorphic minisatellite thus far identified, cCfaMP5. The variable region in this minisatellite is similar to human minisatellites which show a distinct purine or pyrimidine strand bias.

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.

A DNA fingerprinting band associated with the susceptibility to CAE virus-induced arthritis in goats

Caprine arthritis-encephalitis (CAE) virus-induced arthritis can be found in all Swiss goat breeds and it has been shown that there is a genetic pre-disposition for this disease. The 10 Toggenburg, 32 Chamois coloured and 40 Saanen goats, specially selected for this study, were naturally infected with CAE virus. Within each breed there was an equal distribution between clinically healthy animals and animals suffering from arthritis. In the case of the Saanen goats, a DNA fingerprint band generated with the multi-locus probe pV47 was identified associated with susceptibility to CAE virus-induced arthritis. In the Chamois coloured goats, no association was found. Another candidate DNA fingerprint band was present in clinically healthy animals of the Toggenburg breed but their number was insufficient for analysis.

Restriction enzyme/multi-locus probe combinations useful for DNA fingerprinting of the striped bass, white bass and their F1 hybrid

DNA fingerprints were produced in striped bass ( Morone saxatilis), white bass ( Morone chrysops) and their F1 hybrid to identify useful restriction enzyme/probe combinations, determine within- and between-genetic group bandsharing and identify allelic and linkage relationships among fingerprint bands. Five restriction enzymes ( AluI, HaeIII, HinfI, Sau3A and DraI) and three multi-locus probes (33.6, 33.15 and R18.1) were screened. AluI/33.15, AluI/R18.1, HinfI/33.15 and HinfI/R18.1 were identified as the best enzyme/probe combinations. Within-group bandsharings calculated using these combinations ranged from 0.19 to 0.35 for striped bass, 0.39 to 0.65 for white bass and 0.34 to 0.45 for their hybrid. Between-group bandsharing ranged from 0.12 to 0.26 for striped bass vs. white bass, 0.27 to 0.47 for striped bass vs. hybrid and 0.24 to 0.46 for white bass vs. hybrid. Pedigree analysis of a full-sib, F1 hybrid family indicated no bands were linked, allelic pairs were present in the dam (striped bass) and no allelic pairs were present in the sire (white bass). Potential uses of DNA fingerprinting in Morone breeding programs are discussed.

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.

Patterns of reproductive success determined by DNA fingerprinting in a communally breeding oceanic bird

Using DNA fingerprinting we estimated the reproductive success of 49 adult birds belonging to 16 breeding groups of the sexually monomorphic brown skua ( Catharacta lonnbergi) from the Chatham Islands (New Zealand). This population has a variable mating system, breeding in both monogamous and polyandrous groups. The parentage of 45 chicks produced over three breeding seasons was unequivocally determined using the multilocus probes 33.15 and 33.6. We found no evidence of either extra-pair or extra-group fertilization and there was no evidence to suggest egg dumping by females in any breeding group. Consequently, in the case of pairs, parentage of all chicks was assigned to the resident adult birds. In addition, band sharing analysis indicated that members of communal groups were not close relatives. In the 10 communally breeding groups examined, multiple paternity within a clutch was recorded on two of the 12 occasions in which two chicks were reared. Analysis of the parentage of offspring belonging to different groups, from different years, demonstrated that the number of chicks produced by some adult males varied considerably between seasons. In contrast, the reproductive success of other individuals was constant; for example, one male produced two chicks in each of the three seasons it was studied, while other males in communal groups did not produce any chicks during the course of this study. Fitness is a lifetime parameter, and any assessment of it requires studies over at least the average lifetime of an individual. The findings presented in this study suggest that, for brown skuas, there are significant differences in the reproductive success of some adult males in different breeding seasons. These results indicate that estimates of reproductive fitness based on only a single breeding season's data can be seriously inaccurate. Should temporal changes in paternity (and/or maternity) be shown to be common phenomena in other species, then such results would have major implications for the interpretation of parentage studies.

Patterns of reproductive success determined by DNA fingerprinting in a communally breeding oceanic bird

Using DNA fingerprinting we estimated the reproductive success of 49 adult birds belonging to 16 breeding groups of the sexually monomorphic brown skua (Catharacta lonnbergi) from the Chatham Islands (New Zealand). This population has a variable mating system, breeding in both monogamous and polyandrous groups. The parentage of 45 chicks produced over three breeding seasons was unequivocally determined using the multilocus probes 33.15 and 33.6. We found no evidence of either extra-pair or extra-group fertilization and there was no evidence to suggest egg dumping by females in any breeding group. Consequently, in the case of pairs, parentage of all chicks was assigned to the resident adult birds. In addition, band sharing analysis indicated that members of communal groups were not close relatives. In the 10 communally breeding groups examined, multiple paternity within a clutch was recorded on two of the 12 occasions in which two chicks were reared. Analysis of the parentage of offspring belonging to different groups, from different years, demonstrated that the number of chicks produced by some adult males varied considerably between seasons. In contrast, the reproductive success of other individuals was constant; for example, one male produced two chicks in each of the three seasons it was studied, while other males in communal groups did not produce any chicks during the course of this study. Fitness is a lifetime parameter, and any assessment of it requires studies over at least the average lifetime of an individual. The findings presented in this study suggest that, for brown skuas, there are significant differences in the reproductive success of some adult males in different breeding seasons. These results indicate that estimates of reproductive fitness based on only a single breeding season's data can be seriously inaccurate. Should temporal changes in paternity (and/or maternity) be shown to be common phenomena in other species, then such results would have major implications for the interpretation of parentage studies.

DNA fingerprinting in roe deer using the digoxigenated probe (GTG)5

The digoxigenin-labelled oligonucleotide (GTG)5 was used as a multilocus probe to detect hypervariable microsatellites in roe deer DNA digested with HaeIII. The resulting fingerprints of 24 animals belonging to four subpopulations were characterized with regard to within-subpopulation as well as between-subpopulation similarity. The mean number of polymorphic fragments was 20 and the average band-sharing rate for unrelated animals 0.27. A mean probability of 91.5% for a fragment to be present in the heterozygous state was evaluated and the probabilities of identical band patterns in unrelated individuals were estimated to be in the range 1.3 x 10(-16)-2.5 x 10(-18). Though band-sharing rates of animals belonging to different subpopulations (range 0.18-0.24) were lower than those of within-subpopulations, several measures of population subdivision and the genetic distance do not reveal a striking differentiation of the subpopulations studied.

Patterns of Paternity in Relation To Male Social Rank in the Stumptailed Macaque, Macaca Arctoides

Abstract The macaques have been a central focus of research designed to illuminate the interrelationship of behaviour and patterns of genetic transmission in non-human primates. Due to unique aspects of the socio-sexual behaviour and reproductive biology of stumptailed macaques (Macaca arctoides), we hypothesized a stronger relationship between male status and reproductive success than has been documented in other macaque species. The relationship between male social rank and success in siring offspring was examined for a sequence of 27 infants born over an 8-year period (1984-1992) in a large captive social group. Paternity of 25 of the 27 infants (all those still living at the time of testing) was confirmed with the use of "DNA profile tests" using mini-satellite, multi-locus probes in collaboration with Drs. CASNA and GERGITS of Therion Corp. Results showed that each of three consecutive alpha males was effective in achieving a virtual monopoly in siring offspring during his tenure, with one exception possibly related to inbreeding avoidance. Although patterns of sexual behaviour are not emphasized in this report, behavioural observations over the past six years support the findings of other researchers regarding the

Genetic markers for the Booroola fecundity (Fec) gene in sheep

Animals from the Booroola line of Australian Merino sheep are characterized by a high ovulation rate that can be attributed to the presence of a codominant allele (FecB). The specific function of the gene has not been identified. Effective use of the trait within the sheep breeding industry requires one or more genetic markers that can distinguish between alternative alleles at the locus Fec. With a combination of DNA minisatellite markers and polymorphic protein markers, a cluster of seven minisatellite fragments has been identified as being linked to the Fec gene and to the ovine A blood group locus. The minisatellite fragments have been derived from multilocus probes and hence cannot be used to define the chromosomal location of the Fec gene or to serve as diagnostic markers for Fec. The derivation of cloned single locus markers from the minisatellite fragments will enable finer scale mapping of the Fec and the A blood group locus in sheep.

Calculation of paternity probabilities from multilocus DNA profiles.

We describe a procedure for evaluation of paternity evidence from multi-locus DNA probe patterns. A computer program abstracts a "+/-" notation description from the multilocus profile and then calculates a paternity index based on observed phenotypic fragment frequencies. The biostatistical evaluation considers only bands found in the child and missing from the mother--a simplified approach that is at once robust and conservative. Mutations are of course taken into account. Particular features lending objectivity to the interpretation include computer reading and matching decisions, and specific recognition and statistical compensation for ambiguities ("faint orphans").

Phylogenetic relationships of Manihot species revealed by restriction fragment length polymorphism

Two cloned cassava genes (pCAS6, pHNL) and a barley gene (pBLT63) which is highly conserved in higher plants, were evaluated as potential multilocus probes for RFLP analysis of phylogenetic relationships of species within the genus Manihot. For M. rubricaulis, M. chlorosticta and M. esculenta subsp. flabellifolia dendrograms based on 13 probe-restriction enzyme combinations show very little variation between accessions of the same species. A close relationship is demonstrated between the Mexican species M. chlorosticta and the M. esculenta subsp. flabellifolia accessions, which does not support the classification of flabellifolia as a true South American wild species.

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.

Subordinate reproduction in dwarf mongooses

Abstract. Dwarf mongooses, Helogale parvula, are group-living carnivores in which the oldest male and female dominate reproduction, while subordinates tolerate reproductive suppression and provide care for the offspring of the oldest pair. Subordinates of both sexes, however, mate and subordinate females occasionally become pregnant. In addition, a model of 'power-sharing' in social groups (Vehrencamp 1983, Anim. Behav., 31, 667-682) predicts that dominant individuals should cede some fitness to subordinates, particularly older, higher-ranking individuals not closely related to the dominants, in order to retain them within the group. Human multilocus probes (Jeffreys' 33.15 and 33.6) were used to 'fingerprint' dwarf mongooses from nine different packs in the Serengeti National Park, Tanzania. The results demonstrate that subordinates of both sexes obtain direct fitness: 24% of young had subordinate fathers, while 15% had subordinate mothers. Multiple paternity can occur within a single female's litter. Those subordinates that reproduced were of high social rank and tended to be distantly related to the same-sex dominant, as predicted by Vehrencamp's model. The number of yearlings produced by subordinate females was similar to that predicted by the model. Subordinate males father a relatively high proportion of offspring given their frequency of mating, while subordinate females mate relatively more often than they produce offspring, consistent with previously inferred differences between the mechanisms of male and female reproductive suppression (Creel et al. 1992, Anim. Behav., 43, 231-245). Even though DNA analyses indicate that subordinates produced a substantial proportion of young born in this population, the magnitude of direct fitness obtained by subordinates was still low compared with the magnitude of indirect fitness obtained by helping.

Paternity testing with oligonucleotide multilocus probe (CAC) 5/(GTG) 5: A multicenter study

The statistical analysis is reported of 256 paternity cases referred to seven different German laboratories for multilocus DNA fingerprinting with oligonucleotide probe (CAC) 5/(GTG) 5 and restriction enzyme HinfI. All parameters characteristic of multilocus DNA fingerprints were found to differ significantly between the contributing centres: the number of analyzed gel positions, the number of bands scored per individual, the probability of occurrence of a band at a particular position, and the band-sharing probabilities between the mother and both child and alleged father. Despite these differences, paternity cases could be divided clearly into two distinct subgroups on the basis of (i) offspring bands that could not be assigned to either the mother or the alleged father and (ii) the extent of band-sharing between child and alleged father. This partitioning, which is likely to correspond to true and false paternity, confirms previous findings for other multilocus probes. A goodness-of-fit test on the normalized number of bands scored per individual revealed no systematic deviations from commonly adopted analytical models regarding electrophoretic bands as independent entities. Log 10-likelihood ratios of paternity vs. non-paternity were calculated utilizing one of these models, and a clear-cut partitioning was again obtained which coincides with that mentioned before. Only one case could not be decided unambiguously, and was either due to two independent mutations or to a close relative of the alleged fater being the true father.

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.

DNA fingerprinting of the roe deer, Capreolus capreolus L.

1. 1. After digestion of roe deer DNA with the restriction enzyme HaeIII, microsatellite sequences cross-hybridize with the digoxigenin-labeled multilocus probe (GTG) 5 yielding non-radioactive DNA fingerprints. 2. 2. Since different organs of the same individual exhibit identical fingerprints, somatic stability is confirmed. 3. 3. The method discloses a high potential of individualization. The probability that two unrelated roe deer have identical fingerprints was found to be 10 −7. 4. 4. It is shown that band-similarity indices can be used to estimate the degree of relationship within groups of individuals.

The 'individualization' of large North American mammals.

The enforcement of wildlife laws and the captive breeding of threatened/endangered species requires the ability to identify individual animals. DNA profiles of a variety of large North American mammals, birds, and fish were generated using ten different oligonucleotide probes. The probes tested were four multilocus probes [33.6, 33.15, JE46, and (TGTC)5] and six 'human unilocus' probes [MS1 (D1S7), CMM101 (D14S13), YNH24 (D2S44), EFD52 (D17S26), TBQ7 (D10S28), and MS43 (D12S11). Each of the probes was chemically synthesized, and labeled by the attachment of alkaline phosphatase; after hybridization, the probes were detected by chemiluminescence catalyzed by the enzyme. Initial screening against zoo blots including samples of bear, wolf, large cat, wild sheep, deer, birds, marine mammals, and fish indicated that three multilocus probes [33.15, 33.6, (TGTC)5] gave informative patterns containing 15-40 bands for most or all of the animals tested, as did two of the 'human unilocus' probes (MS1 and CMM101). The other five probes appeared informative only in some species (for example, YNH24 against canids). Subsequent screenings of populations within species were used to determine genetic diversity by analysis of observed bandsharing (S). Large heterologous populations, such as white-tailed deer, exhibited highly diverse band patterns (S < or = 0.2). Geographically isolated and/or genetically constricted animals, such as endangered Mexican wolves, Tule elk, and Columbian white-tailed deer, exhibited much higher frequencies of bandsharing (0.6 < or = S < or = 0.95).(ABSTRACT TRUNCATED AT 250 WORDS)