Informativeness of human (dC-dA)n.(dG-dT)n polymorphisms

Abundant human interspersed repetitive DNA sequences of the form (dC-dA)n.(dG-dT)n have been shown to exhibit length polymorphisms. Examination of over 100 human (dC-dA)n.(dG-dT)n sequences revealed that the sequences differed from each other both in numbers of repeats and in repeat sequence type. Using a set of precise classification rules, the sequences were divided into three categories: perfect repeat sequences without interruptions in the runs of CA or GT dinucleotides (64% of total), imperfect repeat sequences with one or more interruptions in the run of repeats (25%), and compound repeat sequences with adjacent tandem simple repeats of a different sequence (11%). Informativeness of (dC-dA)n.(dG-dT)n markers in the perfect sequence category was found to increase with increasing average numbers of repeats. PIC values ranged from 0 at about 10 or fewer repeats to above 0.8 for sequences with about 24 or more repeats. (dC-dA)n.(dG-dT)n polymorphisms in the imperfect sequence category showed lower informativeness than expected on the basis of the total numbers of repeats. The longest run of uninterrupted CA or GT repeats was found to be the best predictor of informativeness of (dC-dA)n.(dG-dT)n polymorphisms regardless of the repeat sequence category.     

Mapping of human chromosome 5 microsatellite DNA polymorphisms.

Thirteen moderately to highly informative microsatellite DNA polymorphisms based on (dC-dA)n.(dG-dT)n repeats were mapped to segments of human chromosome 5 using both linkage analysis and a panel of somatic cell hybrids which contained rearranged chromosomes. The markers were distributed throughout most of the length of the chromosome from the regions p15.3-p15.1 to q33.3-qter. Maps of the sites of meiotic recombination within the reference families proved particularly useful for the purpose of integrating new polymorphisms into the existing linkage map.     

Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms.

To improve carrier detection and prenatal diagnosis for Duchenne and Becker muscular dystrophy families, we determined allele frequencies and measures of variation for four (dC-dA)n.(dG-dT)n loci identified within a deletion-prone region of the human dystrophin gene. The loci are highly polymorphic, with predicted heterozygosities of 71.6%-93.3%. Direct DNA sequence analysis of the (dC-dA)n.(dG-dT)n locus in intron 49 revealed an additional length polymorphism which varies by single-basepair increments, is adjacent to the dinucleotide repeat block, and enhances the polymorphic content of this marker. The four (dC-dA)n.(dG-dT)n loci are each easily amplified by PCR in two diplex reactions. The variability of allele lengths at these loci makes them ideal for carrier detection and prenatal diagnosis, often providing diagnostic information when RFLP analysis is uninformative. These markers have aided in identification of deletion mutations, exclusion of maternal cell contamination of chorionic villus samples, confirmation of paternity, and mapping of gene recombinations. The allele identification of these loci can be performed either with a radiolabel or with an automated, nonradioactive, fluorescent gel detection system.     

(dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function.

In situ hybridization of (dC-dA)n.(dG-dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non-random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC-dA)n.(dG-dT)n. Another striking feature is the lack of hybridization of the beta-heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC-dA)n.(dG-dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC-dA)n.(dG-dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC-dA)n.(dG-dT)n in beta-heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination.     

Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n.d(G-T)n microsatellite repeats.

The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors. An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n x (dG-dT)n microsatellites in the polymerase chain reaction (PCR). These products differ from the primary product by multiples of 2 nucleotides. We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n x (dG-dT)n frameshifting during the PCR. Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification. However, 3'-->5' exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3' terminal nucleotides. Most interestingly, extension temperatures of 37 degrees C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3'-->5' exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products. This method can improve the resolution of heterozygous or mutant (dC-dA)n x (dG-dT)n alleles differing in size by one or two repeat units.     

Variable (dG-dT)n.(dC-dA)n sequences in the porcine genome.

One of the more widely studied simple repeat sequences in the mammalian genome is the (dG-dT)n.(dC-dA)n dinucleotide repeat sequence. As these repeats are highly polymorphic and fairly evenly distributed in diverse mammalian genomes, they constitute a very powerful tool for genetic mapping in a wide variety of species. So far, the knowledge about repeat sequences in the porcine genome is sparse and only a few areas of this genome have been sequenced. We have isolated and characterized 108 porcine (dG-dT)n.(dC-dA)n sequences and studied the distribution of these, both by investigating random clones and by performing in situ hybridization. A remarkable correlation between humans and pigs was found with respect to the structure, to the number of repeat blocks, and to the chromosomal distribution.     

Informativeness of human (dC-dA)n · (dG-dT)n polymorphisms

Abundant human interspersed repetitive DNA sequences of the form (dC-dA)_n · (dG-dT)_n have been shown to exhibit length polymorphisms. Examination of over 100 human (dC-dA)_n · (dG-dT)_n sequences revealed that the sequences differed from each other both in numbers of repeats and in repeat sequence type. Using a set of precise classification rules, the sequences were divided into three categories: perfect repeat sequences without interruptions in the runs of CA or GT dinucleotides (64% of total), imperfect repeat sequences with one or more interruptions in the run of repeats (25%), and compound repeat sequences with adjacent tandem simple repeats of a different sequence (11%). Informativeness of (dC-dA)_n · (dG-dT)_n markers in the perfect sequence category was found to increase with increasing average numbers of repeats. PIC values ranged from 0 at about 10 or fewer repeats to above 0.8 for sequences with about 24 or more repeats. (dC-dA)_n · (dG-dT)_n polymorphisms in the imperfect sequence category showed lower informativeness than expected on the basis of the total numbers of repeats. The longest run of uninterrupted CA or GT repeats was found to be the best predictor of informativeness of (dC-dA)_n · (dG-dT)_n polymorphisms regardless of the repeat sequence category.     

Isolation and characterization of microsatellites from the canine genome

Microsatellite sequences comprising (dC-dA)_n.(dG-dT)_n repeats have been isolated from canine libraries and sequenced. Oligonucleotide primers have been synthesized to the micro-satellite flanking sequences and used in the polymerase chain reaction to amplify those loci from genomic DNA. The degree of polymorphism of each microsatellite was estimated in a set of unrelated dogs. It is concluded that of the 10 loci studied, nine are sufficiently polymorphic to be useful in genetic studies.     

A rapidly evolving region in the immunoglobulin heavy chain loci of rat and mouse: postulated role of (dC-dA)n.(dG-dT)n sequences

The nucleotide sequences of the introns that are located between the C4 exon and the first membrane exon of mouse and rat immunoglobulin epsilon-chain genes have been determined. The rat intron sequence was found to contain four separate clusters of repetitive sequences all of which consisted of (dC-dA)n.(dG-dT)n dinucleotide repeats. A comparison between this chromosomal region in mouse and rat revealed four deletions or duplications, three of which have occurred inside or at the borders of the CA clusters. Rearrangements have occurred inside or at the borders of all four repeats after the evolutionary separation of mouse and rat. The sequence comparison reveals in addition a duplication, connected to the CA repeats, which has occurred early in evolution, before the evolutionary divergence of mouse and rat. These findings suggest that (dC-dA)n.(dG-dT)n sequences are potential targets for recombination events.     

Linkage disequilibrium between two highly polymorphic microsatellites.

The PCR was used to amplify genomic DNA from two microsatellite (dC-dA)n.(dG-dT)n sequences found to be present in the same chromosome 5 genomic clone. Analysis of the haplotype frequencies of these two interspersed repeat sequences in individuals showed strong allelic association or linkage disequilibrium. Six alleles were found for p599 (CA)n with a PIC value of 0.71 and 8 alleles were seen for lambda 599 (CA)n with a PIC value of 0.74. The two microsatellites are separated by approximately 7 kb. Analysis of the length variations for the two microsatellites showed that they were positively correlated, a finding that has no obvious explanation. The strong linkage disequilibrium found demonstrates stability during evolution for these novel markers. Therefore they should be powerful new tools for studying genetic drift and admixture of populations. Furthermore, disequilibrium data from microsatellites can be used in the fine mapping and cloning of disease genes.     

Left-handed Z-DNA helices in polymers, restriction fragments, and recombinant plasmids

Studies on DNA polymers, restriction fragments, and recombinant plasmids have revealed the following: A) A family of left-handed DNA conformations exists for (dC-dG)n.(dC-dG)n. The observation of a particular conformation is dependent on the salt, the salt concentration and dehydrating agent. B) In sodium acetate solutions, (dC-dG)n.(dC-dG)n forms left-handed, psi(+)-condensed structures as detected by Raman spectroscopy and circular dichroism. C) (dT-dG)n.(dC-dA)n undergoes a right-to-left-handed transition only when reacted with AAF and at high salt concentrations. D) Transitions observed for polymer DNAs also are observed for restriction fragments containing both (dC-dG).(dC-dG) and (dT-dG).(dC-dA) sequences, but the transitions in the fragments generally require higher salt concentrations than observed for the polymers. E) Studies with recombinant plasmids containing (dC-dG) sequences from 10 to 58 bp in length demonstrate that left-handed Z-DNA segments can exist contiguous to B-DNA segments. F) Negative supercoil density (sigma less than or equal to -0.072) is sufficient to convert the (dC-dG) regions in those plasmids into left-handed structures under physiological ionic conditions (200 mM NaCl). G) The favorable free energy contribution of methylation in stabilizing the Z form in fragments and plasmids is approximately offset by the unfavorable free energy contributions of the B/Z junctions. H) Sl and BAL 31 nucleases recognize aberrant structural features at the confluence of the B and Z regions. I) Detailed mapping of Sl nuclease cleavage on supercoiled plasmids shows that the nuclease sensitive regions extend over at least five to ten bp. J) Even though the (dT-dG)n.(dC-dA)n polymer requires base modification and high salt conditions to undergo the R----L transition, supercoiling (sigma less than or equal to -0.07) can supply enough energy to allow a plasmid containing the intervening sequence of a human fetal globin gene with (dT-dG).(dC-dA) sequences to undergo a R----L transition.     

The bovine genome contains polymorphic microsatellites

Dinucleotide repeats constitute so-called microsatellites of the human and other eukaryotic genomes. Microsatellite polymorphisms can be identified through the amplification of the microsatellite DNA using the polymerase chain reaction (PCR), followed by resolution of the amplified DNA fragments on a polyacrylamide sequencing gel. We performed a preliminary sequence database search to identify bovine sequences containing (CA)n, (AC)n, (GT)n, or (TG)n blocks, with n greater than or equal to 6. This search yielded 10 sequences containing one or two of the specified repeat blocks and often additional dinucleotide repeat blocks. One of the microsatellite-containing regions has been sequenced twice from independent clones and the reported sequences showed variation in the number of repeats. PCR-amplified fragments of another sequence, the gene for steroid 21-hydroxylase, ranged from 186 to 216 nucleotides in 43 unrelated animals. The database search, as well as the hypervariable microsatellite in the bovine steroid 21-hydroxylase gene, indicates that dinucleotide blocks may be an abundant source of DNA polymorphism in cattle.     

A microsatellite polymorphism associated with the PLC1 (phospholipase C) locus: identification, mapping, and linkage to the MODY locus on chromosome 20.

A highly polymorphic (dC-dA)n.(dG-dT)n dinucleotide repeat at the PLC1 locus on human chromosome 20 has been identified. Primers flanking the dinucleotide repeat were used for PCR amplification of the repeat region in 37 informative kindreds from the Centre d'Etude du Polymorphisme Humain. Two-point linkage analysis indicates that PLC1 is closely linked to several chromosome 20 markers, including D20S16 (Zmax = 41.25; theta = 0.07), D20S17 (Zmax = 42.81; theta = 0.09), and ADA (Zmax = 57.24; theta = 0.05). Multipoint linkage analysis places the PLC1 locus between D20S18 and D20S17, 11.2 and 6.6 cM, respectively, from these loci (sex-averaged distances). In addition, the PLC1 gene shows linkage to the maturity-onset diabetes of the young (MODY) locus on chromosome 20 with a lod score of 4.57 at theta = 0.089.     

In vitro synthesis of short DNA pieces by DNA polymerase alpha from mouse myeloma

The DNA chain elongation mechanisms of mouse DNA polymerases alpha and beta have been analyzed by using denatured DNA with a (dT)n block at the 3'-end as a template in combination with RNA ((rA)12-20)primer. The (rA)12-20-primed DNA product synthesized by DNA polymerase alpha was 3-5 s in size even after prolonged reaction; instead of a size increase, the number of 3-5 s molecules increased with the reaction time. The size of products was not affected by differences in 3H-labeled substrate (dATP or dTTP), enzyme amount, KCl concentration, or the length of 3'-(dT)n blocks. On the other hand, DNA polymerase beta synthesized long DNA products by a highly distributive reaction mechanism. 3-5 sDNA pieces synthesized by DNA polymerase alpha were not elongated any further by DNA polymerase alpha, but were converted into long DNA chains by DNA polymerase beta. The results imply that DNA polymerase alpha recognizes the size of the product DNA, and shuts off further elongation.     

Organization of simple sequences in the Drosophilia melanoga ter genome

Fragments of Drosophila melanogaster DNA that intensively hybridize with simple sequences poly[(dG-dT).(dC-dA)], poly[(dA).(dT)] and poly[(dG-dA).(dC-dT)] were cloned. The first two types of simple sequences are organized in these clones as separated stretches of moderate length, repeated many times within 12-15 kb. Each cluster contains only one type of the simple sequences and originates from a unique in the genome. In contrast, poly[(dG-dA).(dC-dT)] occurs in the genome as several isolated motifs.     

Detection of a new polymorphism of the human prothrombin (F2) gene by combination of PASA and mutated primer-mediated PCR-RFLP

A new polymorphism of the human prothrombin (F2) gene was detected by a combination of polymerase chain reaction (PCR) amplification of specific alleles (PASA) and mutated primer-mediated PCR restriction fragment length polymorphism (PCR-RFLP). The method is simple and useful for detecting polymorphisms and mutations. The new polymorphism of C1 and C2 examined by this method is highly heterozygous and serves as a good human DNA marker.     

Distribution of Drosophila melanogaster mobile genetic elements along X-chromosome and autosomes

The distribution along Drosophila melanogaster polytene X-chromosome and autosomes of 10911 in situ hybridization sites of a broad spectrum of copialike mobile elements is investigated. It is shown that against DNA content X-chromsomal cytological sections 14 + 15 and 16 + 17 contain much less mobile elements than other chromosomal regions. These X-chromosomal regions are also characterized both by significant decrease in the meiotic recombination frequencies and the amount of poly(dC-dA).poly(dG-dT) sequences which are capable to generate the Z form of DNA.     

A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences

We describe a new class of DNA length polymorphism that is due to a variation in the number of tandem repeats associated with Alu sequences (Alu sequence-related polymorphisms). The polymerase chain reaction was used to selectively amplify a (TTA)n repeat identified in the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from genomic DNA of 41 human subjects, and the size of the amplified products was determined by gel electrophoresis. Seven alleles were found that differed in size by integrals of three nucleotides. The allele frequencies ranged from 1.5% to 52%, and the overall heterozygosity index was 62%. The polymorphic TTA repeat was located adjacent to a repetitive sequence of the Alu family. A homology search of human genomic DNA sequences for the trinucleotide TTA (at least five members in length) revealed tandem repeats in six other genes. Three of the six (TTA)n repeats were located adjacent to Alu sequences, and two of the three (in the genes for beta-tubulin and interleukin-1 alpha) were found to be polymorphic in length. Tandemly repetitive sequences found in association with Alu sequences may be frequent sites of length polymorphism that can be used as genetic markers for gene mapping or linkage analysis.