Limited genetic diversity in polynesians reflected in the highly polymorphic 3'HVR alpha-globin gene marker.

Polynesians from five distinct island groups were studied with DNA probe alpha 3'HVR, a highly polymorphic VNTR (variable number of tandem repeats) minisatellite region associated with the alpha-globin gene cluster. Results showed a paucity of genetic heterozygosity together with clustering of alpha 3'HVR alleles with alpha-globin DNA haplotypes and alpha-globin gene rearrangements. This restricted diversity is consistent with population bottlenecks in the colonization of Polynesia.     

VNTR alleles associated with the alpha-globin locus are haplotype and population related.

The human alpha-globin complex contains several polymorphic restriction-enzyme sites (i.e., RFLPs) linked to form haplotypes and is flanked by two hypervariable VNTR loci, the 5' hypervariable region (HVR) and the more highly polymorphic 3'HVR. Using a combination of RFLP analysis and PCR, we have characterized the 5'HVR and 3'HVR alleles associated with the alpha-globin haplotypes of 133 chromosomes, and we here show that specific alpha-globin haplotypes are each associated with discrete subsets of the alleles observed at these two VNTR loci. This statistically highly significant association is observed over a region spanning approximately 100 kb. With the exception of closely related haplotypes, different haplotypes do not share identically sized 3'HVR alleles. Earlier studies have shown that alpha-globin haplotype distributions differ between populations; our current findings also reveal extensive population substructure in the repertoire of alpha-globin VNTRs. If similar features are characteristic of other VNTR loci, this will have important implications for forensic and anthropological studies.     

Molecular characterisation of a hypervariable region downstream of the human alpha-globin gene cluster.

We have characterised an unusual, highly polymorphic region of DNA located 8-kb downstream of the human alpha-globin gene complex. This hypervariable region (alpha-globin 3' HVR) is composed of an array of 17-bp tandem repeats, the number of which differs considerably (70-450) from one allele to another. The sequence of the 17-bp repeats is highly conserved within and between alleles. Furthermore, this sequence identifies a core oligonucleotide [5'-GNGGGG(N)ACAG-3'] that is common to three previously characterised hypervariable regions. At reduced stringency, a probe to the 3' HVR detects a new family of multiallelic loci that will be of value in the study of human genetics.     

Genome-wide prediction of human VNTRs

Polymorphic minisatellites, also known as variable number of tandem repeats (VNTRs), are tandem repeat regions that show variation in the number of repeat units among chromosomes in a population. Currently, there are no general methods for predicting which minisatellites have a high probability of being polymorphic, given their sequence characteristics. An earlier approach has focused on potentially highly polymorphic and hypervariable minisatellites, which make up only a small fraction of all minisatellites in the human genome. We have developed a model, based on available minisatellite and VNTR sequence data, that predicts the probability that a minisatellite (unit size > or = 6 bp) identified by the computer program Tandem Repeats Finder is polymorphic (VNTR). According to the model, minisatellites with high copy number and high degree of sequence similarity are most likely to be VNTRs. This approach was used to scan the draft sequence of the human genome for VNTRs. A total of 157,549 minisatellite repeats were found, of which 29,224 are predicted to be VNTRs. Contrary to previous results, VNTRs appear to be widespread and abundant throughout the human genome, with an estimated density of 9.1 VNTRs/Mb.     

DNA fingerprinting in three species of neotropical primates

DNA fingerprinting allows the simultaneous detection of a large number of hypervariable loci consisting of highly polymorphic tandem repeat units that are extensively dispersed in the genome. With the 33.6 human minisatellite probe, hypervariable fragments were detected, for the first time, in the genome of three different species of wild-caught neotropical primates: Aotus infulatus, Aotus azarae, and Cebus apella. As in the human, these species were highly polymorphic, showing distinctive, individual-specific patterns. Estimates of relatedness within each group were calculated from interspecific comparisons based on the number of shared fragments between individuals. This work shows that the 33.6 human minisatellite probe can be very useful for increasing our understanding of population dynamics and behavior of these species in their natural habitat. © 1996 Wiley-Liss, Inc.     

Stable minihairpin structures forming at minisatellite DNA isolated from yellow fin sea bream Acanthopagrus latus

The lengths of simple repeat sequences are generally unstable or polymorphic (highly variable with respect to the numbers of tandem repeats). Previously we have isolated a family of minisatellite DNA (GenBank accession AF422186) that appears specifically and abundantly in the genome of yellow fin sea bream Acanthopagrus latus but not in closely-related red sea bream Pagrus major, and found that the numbers of tandem arrays in the homologous loci are polymorphic. This means that the minisatellite sequence has appeared and propagated in A. latus genome after speciation. In order to understand what makes the minisatellite widespread within the A. latus genome and what causes the polymorphic nature of the number of tandem repeats, the structural features of single-stranded polynucleotides were analyzed by electrophoresis, chemical modification, circular dichroism (CD), differential scanning calorimetry (DSC) and electron microscopy. The results suggest that a portion of the repeat unit forms a stable minihairpin structure, and it can cause polymerase pausing within the minisatellite DNA.     

Preparation of synthetic tandem-repetitive probes for DNA fingerprinting

DNA fingerprints are generated using probes that hybridize to hypervariable minisatellites, also known as variable number tandem repeat loci. Cloned minisatellites have served as the predominant source of DNA fingerprinting probes. A short segment within the repeat units of minisatellites, called the "core" sequence, is highly conserved within a family of related minisatellites, thereby allowing a single-cloned minisatellite to cross-hybridize to 20 to 40 other minisatellites. In this article, we describe a method for the synthetic preparation of polymeric core sequence probes for DNA fingerprinting. Unlike "monomeric" oligonucleotide probes, the polymeric probes mimic the tandem-repetitive structure of minisatellites, and thus each probe molecule can potentially form many sites of hybridization with a target minisatellite. The synthetic probes are cloned into plasmid DNA to provide a perpetual source of probe material.     

Conservation of intronic minisatellite polymorphisms in the SCK1/SHC2 gene of Hominidae

The neuronally expressed Shc adaptor homolog SCK1/SHC2 gene contains an unusually high number of minisatellites. In humans, twelve different minisatellite sequences are located in introns of SCK1/SHC2 and ten of them are highly polymorphic. Here we used primers developed for humans to screen ten intronic loci of SCK1/SHC2 in chimpanzee and gorilla, and undertook a comprehensive analysis of the genomic sequence to address the evolutionary events driving these variable repeats. All ten loci amplified in chimpanzee and gorilla contained hypervariable and low-variability minisatellites. The human polymorphic locus TR1 was monomorphic in chimpanzee and gorilla, but we detected polymorphic alleles in these apes for the human monomorphic TR7 locus. When we examined the repeat size among these hominoids, there was no consistent variation by length from humans to great apes. In spite of the inconsistent evolutionary dynamics in repeat length variation, exon 16 was highly conserved between humans and great apes. These results suggest that non-coding intronic minisatellites do not show a consistent evolutionary paradigm but evolved with different patterns among each minisatellite locus. These findings provide important insight for minisatellite conservation during hominoid evolution.     

Evolutionary transience of hypervariable minisatellites in man and the primates.

Using PCR, two minisatellite loci showing extreme repeat-unit copy-number variation in humans have been characterized in great apes and monkeys. In contrast to humans, minisatellite locus MS32 is monomorphic with only 3-4 diverged repeat units in great apes, Old World and New World monkeys, this organization presumably representing the relatively stable ancestral precursor state of the human hypervariable locus. Similarly, minisatellite MS1 shows extreme repeat-copy-number variability in man compared with low copy number and minimal variability in great apes. Analysis of variant repeat units shows that the 5' and 3' regions of MS1 are relatively stable in great apes and man, and that variability in man is confined to the central region of the minisatellite. In contrast to the great apes, MS1 is highly variable in Old World monkeys. These results, as well as computer simulations of minisatellite evolution based on known mutation rates, show that short minisatellites are stable within the genome, and that the degree of polymorphism at a given locus can change dramatically over a short period of evolutionary time. The ability of hypervariable minisatellites to detect highly informative loci by cross-species hybridization is therefore largely unpredictable.     

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.     

A GGCAGG motif in minisatellites affecting their germline instability

Mouse and human genomes contain hypervariable DNA regions consisting of tandem repeats of a short sequence referred to as minisatellites. This variation is thought to arise through processes such as unequal crossover or replication slippage. A mo-1 minisatellite probe comprising a 14-base pair repeat sequence reveals many polymorphic fragments even in DNA of BALB/c sublines. Oligonucleotide probes with single base substitution in the mo-1 have been synthesized and used for assessing sequence involved in generation of polymorphisms. The results indicate that the loci containing mo-1 homologues with mutation in the GGCAGG sequence are monomorphic despite the other mutants showing polymorphism. Reciprocally, locus-specific polymorphic clones, Pc-1 and Pc-2, have been isolated with hybridization to mo-1, and both are shown to contain repeated sequence comprising the GGCAGG sequence. They reveal high mutation rates of 8.8% and 3.3% per gamete, respectively. These results strongly suggest that the motif contributes to the germline instability of minisatellites.     

Association of two DNA polymorphisms in the alpha-globin gene cluster: implications for genetic analysis.

A new polymorphic BgI II restriction endonuclease site in the alpha-globin gene complex has been found in Cypriot, Sardinian, and Greek populations. In all cases, this polymorphism is linked to a particular hypervariable region between the zeta 2 and zeta 1 genes. This suggests that these hypervariable regions are stable and will be useful for genetic analysis.     

Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells.

Hypervariable minisatellites can be amplified from human DNA by the polymerase chain reaction, using primers from DNA flanking the minisatellite to amplify the entire block of tandem repeat units. Minisatellite alleles up to 5-10 kb long can be faithfully amplified. At least six minisatellite loci can be co-amplified from the same DNA sample and simultaneously detected to provide a reproducible and highly variable DNA fingerprint which can be obtained from nanogram quantities of human DNA. The polymerase chain reaction can also be used to analyse single target minisatellite molecules and single human cells, despite the appearance of spurious PCR products from some hypervariable loci. DNA fingerprinting at the level of one or a few cells therefore appears possible.     

Nucleotide sequence and genomic organization of bird minisatellites.

Two minisatellite loci from a Eurasian songbird, the willow warbler (Phylloscopus trochilus) were isolated, sequenced and used as probes to detect more than 20 related hypervariable loci. In addition, a sequence flanking one of the minisatellite loci was isolated, and used to study a VNTR locus. The bird minisatellites have a repeat unit of either 12 (AGGGAAGGGCTC) or 17 bp (GGGGACAGGGGACACCC), repeated in tandem 40-100 times per locus, and shows partial similarity to the sequence motifs of human minisatellites. These sequences are among the most variable minisatellites known, with the incidence per gamete of new length alleles estimated from family studies of warblers to about 5.6% per locus. The bird minisatellite alleles show mendelian inheritance and segregation analysis indicates that they are derived from families of sequences with members on several autosomal linkage groups. Some of the warbler core sequences cross-hybridize to hypervariable loci in other species of birds, mammals and fishes.     

The human factor VII gene is polymorphic due to variation in repeat copy number in a minisatellite

The gene coding for human factor VII, a vitamin K-dependent coagulation factor, contains five minisatellite imperfect tandem repeats with monomer element lengths ranging from 14 to 37 bp, and copy numbers ranging from 6 to 52. Three of these repeats are entirely within introns, one is entirely in an untranslated portion of an exon, and one spans an exon-intron border and contains coding sequence. A consensus sequence derived from a comparison of the monomers is similar to a core sequence found in other minisatellites. All of the minisatellites display higher-order periodicities. At least one of these minisatellites is polymorphic. A variation in repeat copy number has been observed in a tandem-repeat region in the seventh factor-VII intron.     

How is it that microsatellites and random oligonucleotides uncover DNA fingerprint patterns?

Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspered tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment. The largest fragments would be those that contained one or more long minisatellite tracts. Some of these large fragments would be highly polymorphic by virtue of the included minisatellite sequences; by virtue of their complex structure, all would be capable of hybridizing to a wide variety of probes, uncovering a DNA fingerprint pattern.     

A highly polymorphic region 3'' to the human type II collagen gene.

We have characterised a highly polymorphic region 1.3kb downstream of the human Type II collagen gene. It consists of a highly AT-rich tandem repetitive region (minisatellite) approximately 650bp long. Two alleles had been observed previously, differing in size by approximately 300bp. When this region was cloned from four unrelated individuals carrying the larger allele, DNA sequence data identified three alleles, suggesting far higher polymorphism than was originally supposed. This minisatellite was shown to be present in a single copy in the human genome, and to have arisen after the divergence of Old and New World monkeys.     

Comparison of minisatellites.

In the class of repeated sequences that occur in DNA, minisatellites have been found polymorphic and became useful tools in genetic mapping and forensic studies. They consist of a heterogeneous tandem array of a short repeat unit. The slightly different units along the array are called variants. Minisatellites evolve mainly through tandem duplications and tandem deletions of variants. Jeffreys et al. (1997) devised a method to obtain the sequence of variants along the array in a digital code and called such sequences maps. Minisatellite maps give access to the detail of mutation processes at work on such loci. In this paper, we design an algorithm to compare two maps under an evolutionary model that includes deletion, insertion, mutation, tandem duplication, and tandem deletion of a variant. Our method computes an optimal alignment in reasonable time; and the alignment score, i.e., the weighted sum of its elementary operations, is a distance metric between maps. The main difficulty is that the optimal sequence of operations depends on the order in which they are applied to the map. Taking the maps of the minisatellite MSY1 of 609 men, we computed all pairwise distances and reconstructed an evolutionary tree of these individuals. MSY1 (DYF155S1) is a hypervariable locus on the Y chromosome. In our tree, the populations of some haplogroups are monophyletic, showing that one can decipher a microevolutionary signal using minisatellite maps comparison.     

Identification, isolation and characterization of canine minisatellite sequences

A Charomid ordered-array library containing a 2–16 Kb size fraction of MbeoI-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 mini-satellites 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 mini-satellite is similar to human minisatellites which show a distinct purine or pyrimidine strand bias.