Characterization of an unusual DNA length polymorphism 5'' to the human antithrombin III gene

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.     

A cluster of highly polymorphic dinucleotide repeats in intron 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

A cluster of highly polymorphic dinucleotide repeats has been detected in intron 17b of the CFTR gene, 200 bp downstream from the preceding exon. At least 24 alleles, with sizes ranging from 7 to 56 units of a TA repeat, have been identified in a panel of 92 unrelated carriers of cystic fibrosis (CF). The common ones are those with 7, 30, and 31 dinucleotide units, with frequencies of .22, .19, and .12, respectively, among the non-CF chromosomes. Mendelian, codominant segregation of the alleles has been demonstrated in family studies, as expected. A less polymorphic dinucleotide (CA repeat) cluster has also been detected in a region 167 bp downstream from the TA repeat. The length of the CA repeat cluster varies from 11 to 17 dinucleotide units, and it appears to have an inverse relationship to that of the TA repeats. These dinucleotide repeats should be useful in genetic linkage studies, in counseling for CF families with unknown mutations, and in tracing the origins of the various mutant CF alleles.     

Segments containing alternating purine and pyrimidine dinucleotides: patterns of polymorphism in humans and prevalence throughout phylogeny.

Tandem dinucleotide repeats of GT or AC [(GT)n/(AC)n] where n greater than or equal to 14 are highly polymorphic and other simple repeats such as (CT)n/(AG)n and (A)n(T)n are also polymorphic. The uniformity of these sequences precludes a mechanistic differentiation between recombination or polymerase slippage. Since (GT)n/(AC)n or (CT)n/(AG)n segments of desired size were not available in our gene of interest, we analyzed a 187+ bp segment in the factor IX gene with multiple short dinucleotide repeats. This sequence contains a melody of short dinucleotide repeats which includes a 142+ bp segment of alternating purines and pyrimidines. Amplification of this sequence in 167 individuals of different ethnicity and direct sequencing of 106 individuals (23 kb of sequence) failed to reveal simple variation in the number of tandem dinucleotide repeats. However, polymorphism in the 142+ alternating purine and pyrimidine segment (RY)n was detected due to the insertion of two related repeat units of 24 bp (A) and 26 bp (B). Two previously described alleles (AB, A2B2) and two novel presumptive recombinants were found (A2B, A3B2) for a total of four alleles. An analysis of (RY)n segments in GenBank revealed an extraordinary enrichment in the genome of mammals, invertebrates, and yeast and a marked reduction in bacteria. Rodent (RY)n were larger and substantially more frequent than those in primates. When a second (RY)n was examined in the exon 8 of human factor IX gene, it was polymorphic at short repeats of (GT)n/(AC)n (n = 3-6) in Western Europeans and Koreans. In addition, an (RY)n in the dystrophin gene had four polymorphic alleles involving AT and GT dinucleotides. Thus (RY)n segments appear to be abundant and highly polymorphic. The asymmetric patterns of polymorphism and the absence of simple dinucleotide variation in 23 kb of sequence are compatible with recombination or sister chromatid exchange, but not polymerase slippage. By inference, recombination should underlie the polymorphisms at (GT)n/(AC)n since they are a subset of (RY)n and they commonly occur in the context of longer (RY)n.     

Phylogenetic Analysis of the Friedreich Ataxia GAA Trinucleotide Repeat

Friedreich ataxia is an autosomal recessive neurodegenerative disorder associated with a GAA repeat expansion in the first intron of the gene (FRDA) encoding a novel, highly conserved, 210 amino acid protein known as frataxin. Normal variation in repeat size was determined by analysis of more than 600 DNA samples from seven human populations. This analysis showed that the most frequent allele had nine GAA repeats, and no alleles with fewer than five GAA repeats were found. The European and Syrian populations had the highest percentage of alleles with 10 or more GAA repeats, while the Papua New Guinea population did not have any alleles carrying more than 10 GAA repeats. The distributions of repeat sizes in the European, Syrian, and African American populations were significantly different from those in the Asian and Papua New Guinea populations (p < 0.001). The GAA repeat size was also determined in five nonhuman primates. Samples from 10 chimpanzees, 3 orangutans, 1 gorilla, 1 rhesus macaque, 1 mangabey, and 1 tamarin were analyzed. Among those primates belonging to the Pongidae family, the chimpanzees were found to carry three or four GAA repeats, the orangutans had four or five GAA repeats, and the gorilla carried three GAA repeats. In primates belonging to the Cercopithecidae family, three GAA repeats were found in the mangabey and two in the rhesus macaque. However, an AluY subfamily member inserted in the poly(A) tract preceding the GAA repeat region in the rhesus macaque, making the amplified sequence approximately 300 bp longer. The GAA repeat was also found in the tamarin, suggesting that it arose at least 40 million years ago and remained relatively small throughout the majority of primate evolution, with a punctuated expansion in the human genome. Received: 18 August 2000 / Accepted: 10 November 2000     

The length polymorphism in the 5′ flanking region of the human β-globin gene with denaturing gradient gel electrophoresis in a Japanese population

Summary The ATTTT repeat polymorphism located approximately 1,400 base pairs (bp) upstream from the -globin structural gene was analyzed by denaturing gradient gel electrophoresis (DGGE) of RNA: DNA duplexes. A study of 81 unrelated Japanese from Hiroshima revealed a sequence heteromorphism in this site. The alleles with five and six repeats of the ATTTT unit, which have been reported, were found in polymorphic proportions. Two unreported alleles were also detected, the first, in two persons, characterized by seven repeats and the other, in a single person, having an A-to-G nucleotide substitution in the fifth repeat.     

Instability of interstitial telomeric sequences in the human genome

The length variability of four human interstitial telomeric sequences (ITs) is described. Three of the ITs contain short telomeric stretches ranging between 53 and 84 bp and are localized in 21q22, 2q31, and 7q36; the fourth IT derives from the subtelomeric domain of chromosome 6p and contains a tract of a few hundred basepairs of exact and degenerate repeats. Using primers flanking the repeats, we amplified the genomic DNA from unrelated individuals and from family members, and we found that all the loci are polymorphic. At the 21q22 IT locus, two equally frequent alleles were found, while the number of alleles at the 2q31, 7q36, and 6pter IT loci was 8, 6, and 4, respectively. Sequence analysis revealed that in the three loci containing short ITs the alleles differ from one another for multiples of the hexanucleotide; it is likely that the mechanism leading to the polymorphism is DNA polymerase slippage. These loci were also unstable in gastric tumor cells characterized by microsatellite instability. At the 6pter IT locus, the four alleles range in length from about 500 to about 700 bp; this variability is probably due to unequal exchange or gene conversion. Our data indicate that stretches of exact internal telomeric repeats can be highly unstable, like microsatellites with shorter units, and that they can be useful polymorphic markers for linkage analysis, for forensic applications, and for the detection of genetic instability in tumors.     

A minisatellite and a microsatellite polymorphism within 1.5 kb at the human muscle glycogen phosphorylase (PYGM) locus can be amplified by PCR and have combined informativeness of PIC 0.95.

We sequenced a genomic clone (pMCMP1), previously reported to detect a VNTR polymorphism at the PYGM locus, and found a dinucleotide repeat segment (CA)14(GA)25 and a complex (AT)-repeat-rich segment containing 63 repeats spanning 160 bp. Resolution of PCR-amplified genomic DNA from the (CA)(GA) repeat region on DNA sequencing gels revealed a highly informative polymorphism with alleles differing by 2-bp intervals and ranging in size from 156 to 190 bp. Among three racial groups, a total of 18 alleles were observed. Fourteen alleles were observed in Caucasians (PIC 0.89), 12 alleles in American Blacks (PIC 0.89), and 9 alleles in Pima Indians (PIC 0.73). PCR amplification of the (AT) repeat region and resolution of the products on DNA sequencing gels revealed a complex variable length polymorphism with alleles distributed in size from 367 to 970 bp. Twenty-eight alleles were found in American Blacks (PIC 0.94), 6 alleles in Pima Indians (PIC 0.70), and 11 alleles in Caucasians (PIC 0.71). Comparison of the previously described VNTR RFLP alleles visualized by Southern hybridization to the PCR products described in this report demonstrated that the polymorphism described in both assays was identical. However, a larger number of alleles could be detected from the PCR-amplified products. Combined informativeness, PIC 0.95, for the two polymorphisms was determined from haplotype analysis of 100 Caucasian chromosomes. Therefore, for genotyping purposes, informativeness is maximized from using both polymorphisms.     

猪PAC克隆的微卫星DNA分离研究

利用(CA)8核心序列,设计锚定引物,采用直接测序法,从单个猪PAC克隆中分离到一个新微卫星DNA。根据该微卫星DNA的侧翼序列,设计了专一引物,在8个猪种40个个体中检测到3个等位基因,片段长度分别为305 bp、307 bp和309 bp。3种等位基因纯合子个体的PCR产物序列分析表明,这3种等位基因分别有12、13和14次CA双核苷酸重复。 Abstract:A novel microsatellite DNA was isolated from a single porcine PAC clone by sequencing the PAC clone directly with (CA)n repeat motif anchored primer.The specific primer pairs flanking the (CA)n repeat region were used to amplify the genomic DNA of 40 individuals from 8 pig breeds,which detected three alleles with the fragment length of 305 bp,307 bp and 309 bp.The PCR product sequencing results of homozygous animals representing three alleles revealed that those three alleles contained 12,13 and 14 CA dinucleotide repeats respectively.     

中国人端粒酶催化亚基(hTERT)基因第六内含子VNTR多态性研究

端粒酶的激活在肿瘤发生、衰老以及细胞永生化过程中起重要作用,端粒酶催化亚基(hTERT)的表达是诱导端粒酶活性的限制性步骤,hTERT的表达受到复杂的调控。hTERT基因组全长40kh,包含16个外显子及15个内含子,其中,在第2和第6内含子中各存在2个可变数目串联重复(VNTR)多态性序列(VNTR2—1st、VNTR2—2nd以及VNTR6—1st和VNTR6—2nd),在第12内含子存在另一个单态性串联重复序列。通过对北京地区210例汉族健康人群hTERT基因第6内含子中36-bpVNTR6—1st的多态性进行调查研究．结果在调查人群中观察到18、20、21、22、23、26和35次重复共7种等位基因型以及14种基因型。基因型频率分布符合Hardy—Weinberg平衡。与韩国浦山地区调查人群类似,20、22及35次重复为最常见的等位基因型,这3种等位基因型频率占调查人群总数的94．76％。除了35次重复等位基因型频率与浦山地区人群存在差异外,其他基因型频率差异不显著。此外,除了在部分重复22次的等位基因型中VNTR5′端与浦山地区人群相同外,其他等位基因型中,北京地区汉族人VNTR6—1st5′端均存在53bD插入片段,显示出不同人种vNTR6—1st周边序列的差异性。北京地区汉族正常人群hTERT VNTR基因多态性资料为研究多态性与肿瘤或衰老的相关性提供了资料。     

Frequencies of promoter pentanucleotide (TTTTA)n of CYP11A gene in European and North African populations

The present work attempts to determine the distribution of CYP11A (TTTTA)n genotype and allele frequencies in 10 European and North African populations. This polymorphism has been associated with hyperandrogenism by several association studies. To our knowledge, this is the first study investigating the ethnic variation of this polymorphism. DNA was extracted from 868 whole-blood samples with the standard phenol-chloroform technique, and PCR reactions were carried out using fluorescent primers as described previously. PCR products were analyzed by an ABI 3,730 DNA Analyzer. A total of six alleles were identified, ranging from 220 bp (4 repeats [4R]) to 250 bp (10R). The most frequent allelic fragment size in all populations was 4R, with frequencies ranging from 47.9% (Sicily) to 62.8% (Tuscany and Germany). Allelic frequencies showed high heterogeneity between analyzed populations. We detected a significant gradient for alleles 4R and 8R. In this study, we report the allele frequency distribution of CYP11A (TTTTA)n showing a north-south geographic gradient. This result could be useful for epidemiological studies about hyperandrogenism.     

Characterization of a highly polymorphic region 5′ to JH in the human immunoglobulin heavy chain

A cloned DNA segment 1.25 kilobases (kb) upstream from the joining segments of the human heavy chain immunoglobulin gene revealed extensive polymorphic variation at this locus, and the polymorphic pattern was stably transmitted to the next generation. Genomic restriction analysis showed that the polymorphism was caused by insertions/deletions within an MspI/BamHI fragment. Sequencing of one allele, 848 base pairs (bp) long, revealed eleven 50-base-pair tandem repeats. A second allele, 648 bp long, was cloned from a human genomic cosmid library, sequenced, and found to contain four fewer repeats than the first allele. A survey of 186 chromosomes from unrelated individuals of primarily northern European descent revealed at least six alleles.     

An unstable trinucleotide-repeat region on chromosome 13 implicated in spinocerebellar ataxia: a common expansion locus

Larger CAG/CTG trinucleotide-repeat tracts in individuals affected with schizophrenia (SCZ) and bipolar affective disorder (BPAD) in comparison with control individuals have previously been reported, implying a possible etiological role for trinucleotide repeats in these diseases. Two unstable CAG/CTG repeats, SEF2-1B and ERDA1, have recently been cloned, and studies indicate that the majority of individuals with large repeats as detected by repeat-expansion detection (RED) have large repeat alleles at these loci. These repeats do not show association of large alleles with either BPAD or SCZ. Using RED, we have identified a BPAD individual with a very large CAG/CTG repeat that is not due to expansion at SEF2-1B or ERDA1. From this individual's DNA, we have cloned a highly polymorphic trinucleotide repeat consisting of (CTA)n (CTG)n, which is very long ( approximately 1,800 bp) in this patient. The repeat region localizes to chromosome 13q21, within 1.2 cM of fragile site FRA13C. Repeat alleles in our sample were unstable in 13 (5.6%) of 231 meioses. Large alleles (>100 repeats) were observed in 14 (1. 25%) of 1,120 patients with psychosis, borderline personality disorder, or juvenile-onset depression and in 5 (.7%) of 710 healthy controls. Very large alleles were also detected for Centre d'Etude Polymorphisme Humaine (CEPH) reference family 1334. This triplet expansion has recently been reported to be the cause of spinocerebellar ataxia type 8 (SCA8); however, none of our large alleles above the disease threshold occurred in individuals either affected by SCA or with known family history of SCA. The high frequency of large alleles at this locus is inconsistent with the much rarer occurrence of SCA8. Thus, it seems unlikely that expansion alone causes SCA8; other genetic mechanisms may be necessary to explain SCA8 etiology.     

Cloning of highly polymorphic microsatellites in the horse

We have isolated equine microsatellites by screening a genomic library with (TG)n and (TC)n probes. TG microsatellites were found to be more abundant than TC repeats, with an estimated frequency of one per 100,000bp. Sequence analysis of eight TG-positive clones revealed varying structures of the repeat regions; perfect stretches of TG repeats, imperfect stretches of TG repeats and compound regions of TG and TC repeats. Five loci were analysed by PCR and showed extensive polymorphism; three to seven alleles and heterozygosities of 0.40-0.76 were observed when screening 20-30 unrelated individuals. The high degree of polymorphism, their abundance and the possibility of automating the typing procedure make these loci ideal for standardized paternity testing in the horse. Furthermore, we demonstrate that single hairs can be used as starting material for the PCR analysis.     

Association of A/T rich microsatellites with responses to artificial selection for larval developmental duration in the silkworm Bombyx mori

Simple sequence repeats (SSRs) and interSSR (ISSR) marker systems were used in this study to reveal genetic changes induced by artificial selection for short/long larval duration in the tropical strain Nistari of the silkworm Bombyx mori. Artificial selection separated longer larval duration (LLD) (29.428 (+/-) 0.723 days) and shorter larval duration (SLD) (22.573 (+/-) 0.839 days) lines from a base, inbred population of Nistari (larval span of 23.143 (+/-) 0.35 days). SSR polymorphism was observed between the LLD and SLD lines at one microsatellite locus, Bmsat106 (CA7) and at two loci of 1074 bp and 823 bp generated with the ISSR primer UBC873. Each of these loci was present only in the LLD line. The loci segregated in the third generation of selection and were fixed in opposite directions. In the F2 generation of the LLD x SLD lines, the alleles of Bmsat106 and UBC8731074bp segregated in a 1:1 ratio and the loci were present only in the LLD individuals. UBC873823bp was homozygous. Single factor ANOVA showed a significant association between the segregating loci and longer larval duration. Together, the two alleles contributed to an 18% increase in larval duration. The nucleotide sequences of the UBC8731074bp and UBC873823bp loci had 67% A/T content and consisted of direct, reverse, complementary and palindromic repeats. The repeats appeared to be "nested" (59%) in larger repeats or as clustered elements adjacent to other repeats. Of 203 microsatellites identified, dinucleotides (67.8%) predominated and were rich in A/T and T/A motifs. The sequences of the UBC8731074bp and UBC873823bp loci showed similarity (E = 0.0) to contigs located in Scaffold 010774 and Scaffold 000139, respectively, of the B. mori genome. BLASTN analysis of the UBC8731074bp sequence showed significant homology of (nt.) 45-122 with upstream region of three exons from Bombyx. The complete sequence of this locus showed approximately 49% nucleotide conservation with transposon 412 of Drosophila melanogaster and the Ikirara insertions of Anopheles gambiae. The A + T richness and lack of coding potential of these small loci, and their absence in the SLD line, reflect the active process of genetic change associated with the switch to short larval duration as an adaptation to the tropics.     

Identification of internal variation in the pseudoautosomal VNTR DXYS17, with nonrandom distribution of the alleles on the X and the Y chromosomes.

The PCR technique was used to analyze the DXYS17 locus in the pseudoautosomal region of the X and the Y chromosomes. Analysis on an automated DNA sequencer allowed for sensitive and highly accurate typing of 16 different alleles with a size between 480 and 1,100 bp. Two DXYS17 alleles migrated with the same size on agarose or denaturing polyacrylamide gels but with different mobilities on nondenaturing polyacrylamide gels. Sequence analysis showed that, while an identical number of repeats were present in both alleles, differences in the composition of the units were observed. The origin of these differences was found in the 28- and 33-bp units, which only had a specific repeat pattern at the 5' and 3' ends of the region. The genotype distribution for DXYS17 in a Caucasian population did not deviate from the values expected under Hardy-Weinberg equilibrium. However, the frequency of one allele and one genotype was significantly different between males and females. Segregation analysis showed that this difference was the result of a nonrandom distribution of certain alleles on the sex chromosomes in males.     

Genetic polymorphism of MJD1 alleles and molecular analysis of SCA3 patients from Rio de Janeiro, Brazil

Spinocerebellar ataxia type 3 is the most common form of autosomal dominant cerebellar ataxia. It is a severe progressive neurological disorder caused by an expansion of an exonic CAG repeat of the MJD1 gene. The repeated sequence is polymorphic among both normal individuals and patients. In general, expanded alleles are paternally inherited and the disorder exhibits anticipation. We performed a PCR-based study to determine polymorphisms of the number of CAG repeats of the MJD1 gene in an anonymous sample of normal Brazilian individuals. We also analyzed DNA samples from 9 patients with ataxia. We identified 29 different allele sizes ranging from 12 to 40 CAG repeats, with heterozygosity of 79%. The distribution of allele sizes showed two major peaks of 16 (7%) and 26 (10.1%) CAG repeats. When grouping normal alleles by size, we observed that the distribution varies between males and females, and a significant deviation from the Hardy-Weinberg equilibrium was observed with an excess of normal large alleles among males. We also detected expanded alleles with 68-73 CAG repeats in 3 out of 9 ataxic patients.     

Drosophila melanogaster is polymorphic for a specific repeated (CATA) sequence in the regulatory region of hsp23.

To identify sequence variation associated with a selection response for heat tolerance in Drosophila melanogaster, we sequenced 1400bp of the heat shock protein 23 gene (hsp23) promoter region in four heat-selected and two control lines. The region was found to be variable for a specific (CATA) repeated sequence, and the sequence CTT seems to be a hot spot for mutation. The repeated tetranucleotide sequence was located in several short repeats scattered throughout the entire region. Similar variable repeats are also located downstream the of hsp23 gene in the intergenic region between hsp23 and hsp27. We detected nine different hsp23 alleles. Their frequencies in the selection and control lines seemed to be mainly determined by genetic drift. The function of the CATA repeats is not yet known, though these regions have homology to SAR elements located in the intergenic region between two hsp70 genes, suggesting a similar function.     

Deletions in Plasmid Pbr322: Replication Slippage Involving Leading and Lagging Strands

We test here whether a class of deletions likely to result from errors during DNA replication arise preferentially during synthesis of either the leading or the lagging DNA strand. Deletions were obtained by reversion of particular insertion mutant alleles of the pBR322 amp gene. The alleles contain insertions of palindromic DNAs bracketed by 9-bp direct repeats of amp sequence; in addition, bp 2 to 5 in one arm of the palindrome form a direct repeat with 4 bp of adjoining amp sequence. Prior work had shown that reversion to Ampr results from deletions with endpoints in the 8- or 4-bp repeat, and that the 4-bp repeats are used preferentially because one of them is in the palindrome. To test the role of leading and lagging strand synthesis in deletion formation, we reversed the direction of replication of the amp gene by inverting the pBR322 replication origin, and also constructed new mutant alleles with a 4-bp repeat starting counterclockwise rather than clockwise of the insertion. In both cases the 4-bp repeats were used preferentially as deletion endpoints. A model is presented in which deletions arise during elongation of the strand that copies the palindrome before the adjoining 4-bp repeat, and in which preferential use of the 4-bp repeats independent of the overall direction of replication implies that deletions arise during syntheses of both leading and lagging strands.     

Population and Family Studies of the CAG Repeats in the IT-15Gene

A simple and effective method for typing of CAG repeats in the IT-15gene has been suggested. This method was applied for examination of the CAG allele distribution in Huntington's disease (HD) patients in five different populations from the Commonwealth of Independent States. A total of 21 normal alleles with the sizes ranging from 9 to 32 triplet repeats units were revealed. Moreover, alleles with the sizes ranging from 16 to 20 repeats predominated constituting from 54.4 to 74.6% of all alleles in different populations. The number of repeats in one allele in HD patients exceeded 38 units (43 triplets on average). In two families an increase in the CAG repeat units number in the mutant allele upon its paternal transmission was recorded.