Oligonucleotide probes for alpha satellite DNA variants can distinguish homologous chromosomes by FISH
Oligonucleotide probes for alpha satellite DNA variants can distinguish homologous chromosomes by FISHChristine L. O'Keefe1, Peter E. Warburton4 and A. Gregory Matera1,2,3,*
1Department of Genetics, 2Center for Human Genetics and 3Program in Cell Biology, Case Western Reserve University and University Hospitals of Cleveland, 10900 Euclid Avenue, Cleveland, OH 44106-4955, USA and 4Institute of Cell and Molecular Biology, Michael Swann Building, Kings Buildings, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK
Received June 24, 1996;Revised and Accepted August 22, 1996
Chromosomal heteromorphisms have been used extensively to mark individual chromosomes. However, classical banding techniques used to identify these structural variants are imprecise and difficult to quantify. Different chromosomes 17 from the human population are characterized by distinct haplotypes of alpha satellite DNA. We have used these sequence variants to construct oligonucleotide probes for fluorescence in situ hybridization (FISH). These oligomers are the first reported FISH probes that can discriminate between cytogenetically indistinguishable chromosome homologues. They have been used to follow the transmission of a single chromosome 17 through a pedigree, similar to a typical polymorphic marker. Furthermore, extended chromatin fiber techniques reveal the presence of discrete domains of different sequence variants within individual centromeres. Extension of this strategy to create a battery of other variant-specific oligoprobes should provide a powerful diagnostic tool for parent of origin effects in the study of aneuploidy, imprinting and cancer cytogenetics.
Chromosome heteromorphisms are very useful characters for identifying individual homologues. Their use as genetic markers depends on the fact that they are stable, heritable and have a low mutation rate (1 ). In fact, the first assignment of a gene to a human autosome was made using a heteromorphism on chromosome 1 (2 ). Centromeric heteromorphisms are minimally affected by crossing- over and are therefore good markers for tracing chromosomal origin. However, traditional banding techniques are somewhat subjective and thus their utility is limited to only the more extreme variants (3 ,4 ). In addition to being hard to score, heteromorphic chromosomes are relatively infrequent in the population. Thus we wanted to find a more useful method to identify chromosomal homologues. Though physical polymorphisms are rare, an increasing number of sequence polymorphisms within the centromeres of human chromosomes have been reported.
Alpha satellite is a tandemly repeated DNA family found at the centromeres of all primate chromosomes. The basic repeat unit is a 171 base pair (bp) monomer which is tandemly repeated to form chromosome-specific higher-order repeat units (5 -7 ). The human chromosome 17-specific alpha satellite subset consists primarily of a 2.7 kb higher-order repeat unit of 16 monomers (8 ). Variant higher order repeat units of 11-15 monomers can also be found at the centromeres of some chromosomes 17 (9 ,10 ). These variant higher-order repeat units are thought to be derived from the 16mer higher-order repeat unit by precise deletions of an integral number of monomers, most likely by unequal homologous recombination (9 ,11 ).
The presence or absence of variant repeat units within a human chromosome 17 alphoid array defines distinct haplotypes (12 ). These haplotypes are also characterized by several linked sequence polymorphisms (9 ,13 ). Such variation of alpha satellite DNA between centromeric arrays has also been demonstrated for other human chromosomes (14 ,15 ). Polymorphisms may arise within an array of tandemly repeated sequences either by multiple independent mutations within the array or by a single event within one repeat unit followed by gene correction and/or expansion of the variant (16 ). If the first mechanism were to occur, highly divergent variants should be scattered randomly throughout the array and all chromosomes, both homologues and non-homologues, would evolve as independent entities. If the second mechanism were to occur, the alphoid arrays on homologous chromosomes would be more homogenous than the arrays on non-homologues. The existence of chromosome-specific subfamilies of alpha satellite DNA implies that gene correction/conversion occurs more often between homologous centromeric regions. If unequal crossing over were to occur on a local instead of an array-wide manner, the variants should be clustered in distinct domains within the alphoid array (17 ,18 ). Indeed, distinct domains of alpha satellite DNA variants have been shown to exist at the centromeres of several human chromosomes, including chromosome 17 (15 ,19 ,20 ). However, the fundamental repetitive nature of alpha satellite DNA can make the analysis of variant organization extremely difficult. To more fully elucidate the organization of variants within an array of tandemly repeated sequences, we must visualize them in their native arrangement.
Unlike cloned probes, oligonucleotides are able to differentiate between closely related sequences that differ at only a few bases and can be designed to target areas of diagnostic potential (21 ). Here we present oligonucleotide probes which are capable of discriminating between cytogenetically indistinguishable homologous chromosomes by FISH. These sequence-specific probes have been used to follow the transmission of a single chromosome 17 through three generation families, similar to a typical polymorphic marker. They specifically hybridize to cloned chromosome 17 alphoid higher-order repeat units which contain the same variant sequence, but not to those which contain similar, closely related sequences. Finally, these oligomers identify discrete domains of chromosome 17 alpha satellite DNA variants on extended chromatin preparations.
Previous work identified multiple sequence polymorphisms among the centromeres of human chromosomes 17 (13 ,22 ). These sequences were derived from either intact chromosomes or cloned individual higher order repeat units and were categorized into three haplotypes. Sequence analysis identified regions in which multiple polymorphic bases are clustered; one such region occurs in monomer 15 of the 16mer higher order repeat unit, while another is in monomer 13 (numbering as in ref. 8 ).
These sequences were used to construct oligonucleotide probes specific for different sequences within each of the haplotypes. Figure 1 A compares the sequences of two of the variant-specific probes with that of the overall alphoid DNA consensus. 17VAR1.1 and 17VAR2.1 (monomer 15, bases 2525-2543) differ from the alphoid consensus at five positions, which render these oligos chromosome 17-specific. However, these oligomers differ from each other at four variant-specific bases. We synthesized two additional oligonucleotide probes, 17VAR1.3 and 17VAR2.4 (monomer 13, bases 2151-2171), which share two base differences from the alphoid consensus, while differing from each other at three variant-specific positions (for sequences, see Materials and Methods). We found that 17VAR1.1 and 17VAR2.1 are better able to discriminate between homologous chromosomes, although both probe sets are variant-specific. Hence, we decided to use them exclusively in the following studies.
Using these probes in a dual-color FISH assay, we hybridized them to metaphase chromosome preparations from 39 unrelated individuals. Hybridization was detected only on chromosome 17, even with amplification. Examples of these experiments are shown in Figure 2 C and D. As can be seen in Table 1 , our screening identified three classes of chromosome 17: (i) those that were positive for 17VAR1.1 only, (ii) those that were positive for 17VAR2.1 only, and (iii) those that were positive for both. We found all possible combinations of chromosomes among the 39 individuals screened. Using these two oligonucleotide probes, 56.4% (22 of 39) individuals scored had distinguishable homologues.
Number and frequency of chromosome 17 alpha satellite DNA genotypes
Genotype
No. of individuals
Observed frequency
Expected frequency
AA
1
0.025
0.004
BB
9
0.231
0.237
AC
2
0.051
0.057
BC
19
0.487
0.437
CC
7
0.179
0.202
AB
1
0.025
0.062
Thirty-nine individuals were scored by FISH using 17VAR1.1 and 17VAR2.1. Genotype A represents chromosomes which were positive for 17VAR1.1 only, genotype B represents chromosomes which were positive for 17VAR2.1 only, and genotype C represents chromosomes positive for both oligonucleotide probes. Of the 78 chromosomes scored, five were genotype A, 38 were genotype B and 35 were genotype C. The expected frequency of genotypes was derived from the Hardy-Weinberg equilibrium for three alleles. Chi-squared analysis shows that the differences between the observed and expected frequencies are not statistically significant ([chi]2 = 5.517 with 5 degrees of freedom).
Willard et al. (10 ) demonstrated that chromosome 17 haplotypes, as defined by differences in restriction patterns, were inherited in a Mendelian fashion and that these centromeric polymorphisms could be followed through three generation families. To test whether our variant-specific probes could also be used in a FISH assay to follow these polymorphisms through a pedigree, we screened the parents of three CEPH families (1331, 1333 and 1345). We then typed the grandparents and the children in two informative families (1333 and 1345). Using 17VAR1.1 and 17VAR2.1, we followed the transmission of individual chromosomes from the grandparental generation through one of the parents to ~50% of the children (Fig. 3 ).
One intriguing question raised by our data concerns how haplotype I- and haplotype II-specific sequences are organized within the centromere of a single chromosome 17. Evidence for the existence of distinct domains of alpha satellite DNA variants has been presented for several chromosomes, including chromosome 17 (20 ). However, traditional analytic techniques, such as restriction enzyme digestion and 2D gel electrophoresis, are unable to elucidate the precise organization of centromeric DNA. In order for such analyses to work, the variants must be defined by differences in restriction patterns. If the actual variants are defined by sequence changes that do not create or abolish restriction enzyme sites, then the organization of the variants cannot be explored by such methods. Furthermore, if a tandem repeat is digested down to its constituent building blocks, the organization of those building blocks with respect to one another will be lost. Only when variant repeat units exist as part of a larger domain, defined by a different restriction enzyme, can their presence be unequivocally demonstrated (23 ).
Our sequence-specific oligoprobes allow us to investigate the organization of sequences on spread chromatin in situ, thereby preserving the organization of alphoid repeat units in relation to each other. We therefore used several different extended chromatin techniques, including nuclear spreads (24 ). The level of stretching of nuclear spreads varied widely both between slides and on a single slide. We found that euchromatic sequences separated by as little as 5 kb are resolvable on some nuclear spreads using cosmid clones (data not shown), suggesting that the level of resolution of such a preparation approaches that of other extended chromatin preparations which use a harsher extraction to remove chromosomal proteins (25 -27 ). However, for the visualization of the entire alphoid array, cells with a lesser degree of spreading were most informative.
We hybridized oligoprobes 17VAR1.1 and 17VAR2.1 to nuclear spreads from cell line L65-14A, which contains sequences complementary to both oligoprobes. We found that distinct domains of a single chromosome 17 alpha satellite DNA variant were visible on these chromosomes (Fig. 4 A). In the spread, a 17VAR1.1-specific domain (red) is visible at one end of the spread array, while a 17VAR2.1-specific domain (green) is visible at the other end of the array. Some smaller, internal domains also appear. However, the order of the internal domains was not consistent from spread to spread, which may be a reflection of differential chromatin packaging (see Discussion).
To demonstrate that our oligoprobes each detect a subset of the entire chromosome 17 alpha satellite DNA array, we hybridized either 17VAR1.1 or 17VAR2.1 to L65-14A nuclear spreads simultaneously with p17H8, a cloned chromosome 17 alpha satellite DNA higher order repeat unit. The p17H8 probe marks the entire chromosome 17 alphoid array and was nick translated to a size compatible with our oligo-FISH detection protocol. When used singly, each oligoprobe detects a subset of the alphoid array, as described above (data not shown). It was possible that the large gaps seen in the experiments outlined above may have been due to artefactual failure of hybridization of our oligoprobes to sequences within the alphoid array, and may not reflect the actual organization of chromosome 17 alpha satellite DNA variants. In order to demonstrate that the gaps detected by our variant-specific oligoprobes were not simply due to a failure of hybridization, we hybridized 17VAR1.1, 17VAR2.1 and p17H8 (Fig. 4 B) to nuclear spreads from cell line L65-14A. The signal from both oligoprobes is entirely coincident with the signal from p17H8. Furthermore, no gaps are detected in the combined oligoprobe image as compared with the entire alphoid array, suggesting that our probes hybridize throughout the array. These findings suggest that the gaps seen in the single oligoprobe/p17H8 hybridizations (described above) are authentic and reflect the organization of these variants within the alphoid array.
In this report, we have described oligonucleotide probes specific for chromosome 17 alpha satellite DNA variants. These probes not only provide a means to explore the organization of alpha satellite DNA within the centromere, but are capable of distinguishing between `normal' homologous chromosomes in a FISH assay. Unlike typical chromosomal heteromorphisms, these sequence-specific oligonucleotide probes discriminate on the basis of sequence composition, not gross structural properties.
Tandemly repeated DNA families tend to evolve in concert. Hence, paralogous repeat sequences within a species are often more similar than orthologous sequences from closely related species (11 ,28 ). The higher order repeat unit of alpha satellite DNA is thought to be the conserved unit of sequence amplification (8 ). Individual monomers within a higher order repeat unit can be highly diverged, with as little as 65% sequence similarity. In contrast, the sequences of the same monomer in different higher order repeat units show a level of similarity approaching 99% (6 ,29 ). Our probes, designed to target polymorphic sequences in the same monomer in different higher order repeat units, are capable of distinguishing between sequences that differ at only a few base positions. During the course of our experiments, we identified three classes of chromosome 17, and found all possible combinations of these chromosomes in a screen of 39 unrelated individuals (Fig. 2 ; Table 1 ).
The ability to rapidly distinguish homologous chromosomes will have many other applications in biomedical science. For example we are presently using these variant-specific probes to study non-disjunction of chromosome 17 during male meiosis (Griffin et al., manuscript in preparation). If non-disjunction occurs during meiosis I in an individual who is heterozygous at a particular locus, the locus will remain heterozygous in the aneuploid gamete. If non-disjunction occurs during meiosis II, the locus will be reduced to homozygosity (32 ). Using oligonucleotide probes specific for chromosome 17 alpha satellite DNA variants on sperm preparations from heterozygous individuals, we can rapidly score a large number of meiotic events and determine not only the rate of overall non-disjunction, but also the relative rates of meiosis I versus meiosis II errors. In principle, one can extend our analyses to the centromeres of other human chromosomes by creating oligomers specific to other alphoid sub-types.
In addition, variant-specific probes should prove useful in studies of parent-of-origin effects in cancer cytogenetics and genomic imprinting. At least one disease locus, NF1 (neurofibromatosis), has been linked to centromeric polymorphisms that can be followed through a pedigree (10 ,33 ,34 ). However, the fact that we can now perform these analyses by FISH (Fig. 2 ) will allow for more rapid diagnosis of informative families.
One of the ultimate goals of oligo-FISH is to detect specific alleles. We have shown that oligonucleotide probes have the ability to discriminate between highly homologous sequences in situ. Having resolved the specificity issue, the next step in the evolution of allele-specific detection methods will be to increase the sensitivity of the approach in order to detect single-copy sequences.
The oligonucleotides used in this study are: 17VAR1.1 (5'-AT*CATT*GCACTCTT*TGAGGAGT*ACCG-3'); 17 VAR- 2.1 (5'-ATAAT*TGCACTT*CTTT GAGGCCT* ACCG-3'); 17VAR1.3 (5'-ATAGT*TCAGGTTT*GAAACGGT*C-3'); and 17VAR2.4 (5'-ATAGT*GCAGCTT*TGAAACACT*C-3'). Primary amine groups were introduced at specific T residues (marked with asterisks). The oligonucleotides were labeled at these sites with either biotin or digoxigenin as described previously (35 ).
Clone p17H8 (8 ) was used as a chromosome 17 alpha satellite DNA-specific probe. Plasmid DNA (~2 [mu]g) was nick-translated with either biotin-16-dUTP, digoxigenin-11-dUTP (Boehringer Mannheim) or Cy5-AP3-dUTP (Amersham Life Science) as previously described (36 ), with the following modifications: 200 ng of DNase was used in a 100 [mu]l nick translation reaction, and the reaction was run for 2.5 h at 15oC. The high concentration of DNase in the reaction nicked the plasmid DNA down to a size (~50-75 bp) that was compatible with the FISH hybridization and detection stringencies used.
Plasmid clones 60 and 74 (from chromosomes of haplotype II and haplotype I, respectively) were generous gifts of H. Willard (13 ). The clones were digested with PstI to free the insert and transferred to nitrocellulose membranes by standard procedures (37 ). Oligonucleotides 17VAR1.1 and 17VAR2.1 were 5'-end labeled with 32P using T4 polynucleotide kinase (New England Biolabs). Hybridization of the oligonucleotides was performed in 20% formamide/2* SSC/10% dextran sulfate at 37oC for 2 h. Stringency washes (3* 5 min) of 20% formamide/2* SSC at 37oC were followed by 3* 5 min washes in 0.1% Tween/4* SSC at 37oC.
Metaphase chromosome spreads from normal human peripheral blood lymphocytes, normal human lymphoblast cell lines (CEPH families) and mouse/human somatic cell hybrid cell lines were prepared by standard techniques. Nuclear spreads from mouse/human somatic cell hybrid cell lines were prepared as described previously (24 ), except that the protease inhibitors were omitted from the solution.
After fixation, the preparations were denatured in 70% formamide/2* SSC at 70oC for 2 min and immediately dehydrated in a series of 70%, 90%, 100% ice-cold ethanol washes for 3 min each and air dried. Labeled oligonucleotides (~2 ng/[mu]l) along with unlabeled competitor DNA (~600 ng/[mu]l) were hybridized for 1-2 h at 37oC in 20% formamide/2* SSC/10% dextran sulfate. Two sets of washes (3* 4 min) were performed as follows: 20% formamide/2* SSC at 37oC, followed by 0.1% Tween/4* SSC at 37oC. Detection was performed with appropriately-conjugated avidin or anti-digoxigenin Fab fragments. Although the signals were readily visible by eye on metaphase chromosome preparations, one round of amplification of the oligonucleotide probe signals was used to more easily visualize the extended chromatin preparations. Image acquisition was performed as described previously (38 ).
We thank the Willard laboratory for supplying us with the various mouse/human hybrid lines as well as the variant-specific plasmids and S. Schwartz for providing cell pellets of the unrelated individuals. We also thank H. Willard, T. Hassold and T. Haaf for helpful discussions and members of the Matera laboratory for critical reading of the manuscript. This work was supported by a Basil O'Connor Scholar award to A.G.M. (#5-FY96-0554) from the March of Dimes Birth Defects Foundation. C.L.O. was supported in part by an NIH predoctoral traineeship in Cell and Molecular Biology. A.G.M. was also supported by a Junior Faculty Research Award (JFRA-570) from the American Cancer Society.
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*To whom correspondence should be addressed at: Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955, USA
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