Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.     

Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes

We describe the identification of a mutant in the Arabidopsis accession Columbia (Col-0) that exhibits enhanced downy mildew (edm1) susceptibility to several Peronospora parasitica isolates, including the RPP7-diagnostic isolate Hiks1. The mutation was mapped to chromosome IV and characterized physically as a 35-kb deletion spanning seven genes. One of these genes complemented the mutant to full wild-type resistance against all of the Peronospora isolates tested. This gene (AtSGT1b) encodes a predicted protein of 39.8 kD and is an Arabidopsis ortholog of yeast SGT1, which was described originally as a key regulatory protein in centromere function and ubiquitin-mediated proteolysis. AtSGT1b contains three tetratricopeptide repeats at the N terminus followed by a bipartite chord-containing SGT domain and an SGT-specific domain at the C terminus. We discuss the role of AtSGT1b in disease resistance and its possible involvement in ubiquitin-mediated proteolysis in plants.     

A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development.

Brassinosteroids are widely distributed plant compounds that modulate cell elongation and division, but little is known about the mechanism of action of these plant growth regulators. To investigate brassinosteroids as signals influencing plant growth and development, we identified a brassinosteroid-insensitive mutant in Arabidopsis thaliana (L.) Henyh. ecotype Columbia. The mutant, termed bri1, did not respond to brassinosteroids in hypocotyl elongation and primary root inhibition assays, but it did retain sensitivity to auxins, cytokinins, ethylene, abscisic acid, and gibberellins. The bri1 mutant showed multiple deficiencies in developmental pathways that could not be rescued by brassinosteroid treatment including a severely dwarfed stature; dark green, thickened leaves; males sterility; reduced apical dominance; and de-etiolation of dark-grown seedlings. Genetic analysis suggests that the Bri1 phenotype is caused by a recessive mutation in a single gene with pleiotropic effects that maps 1.6 centimorgans from the cleaved, amplified, polymorphic sequence marker DHS1 on the bottom of chromosome IV. The multiple and dramatic effects of mutation of the BRI1 locus on development suggests that the BRI1 gene may play a critical role in brassinosteroid perception or signal transduction.     

Site-directed mutagenesis of exon 5 of p53: purification,analysis, and validation of amplicons for DHPLC

Reversed-phase ion-pair chromatography is used in its three modes of operation--(1) non-denaturing, (2) partially denaturing, (3) fully denaturing - to isolate and characterize five site-directed mutant amplicons of exon 5 of the p53 gene (GenBank X54156). The mutant fragments were prepared using a simplified mutagenesis procedure without cloning and isolated in high purity under non-denaturing conditions. Then, under partially denaturing conditions, heteroduplices formed by hybridization of each of the five mutants with wild-type were all detected by denaturing reversed-phase high-performance chromatography (DHPLC). The fidelity of the PCR step was also assessed. Finally, the homogeneity of each mutant was confirmed by minisequencing under fully denaturing conditions. The versatility and facility of the method for DNA manipulations was exploited to demonstrate a strategy for validating amplicons for mutation detection by DHPLC.     

Mutation detection in the human HSP7OB' gene by denaturing high-performance liquid chromatography

Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B' gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature-modulated heteroduplex analysis (TMHA). The underlying principles of TMHA for mutation detection using DHPLC technology are discussed. The procedures involved in amplicon design for mutation analysis by DHPLC are detailed. The melting behavior of the complete coding sequence of the target gene is characterized using WAVEMAKER software. Four overlapping amplicons, which span the complete coding region of the HSP70B' gene, amenable to mutation detection by DHPLC were identified based on the software-predicted melting profile of the target sequence. TMHA was performed on PCR products of individual amplicons of the HSP70B' gene on the WAVE Nucleic Acid Fragment Analysis System. The criteria for mutation calling by comparing wild-type and mutant chromatographic patterns are discussed.     

Fine-scale molecular genetic (RFLP) and physical mapping of a 8.9 cM region on the top arm of Arabidopsis chromosome 5 encompassing the male sterility gene, ms1

Fine-scale molecular mapping has been conducted using 183 recombinants between the markers lutescens ( lu; 17.6 cM) and transparent testa glabra ( ttg; 35.5 cM) on the top arm of Arabidopsis thaliana chromosome 5. This region contains a number of genes involved in floral development including Ms1 , a gene required for the post-meiotic development of pollen. In homozygous ms1 mutant plants, pollen development is aborted soon after microspore release, regardless of environmental conditions. The ms1 mutation is located at 29.8 ± 0.8 cM on chromosome 5. Markers have been identified which co-segregate with ms1 and should lie within 39 kb of the gene. The fine-scale map of the lu-ms1-ttg region that has been generated is significantly different from the published integrated map and provides substantially more accurate and higher marker density than the current recombinant inbred map for this region. Using clones derived from four yeast artificial chromosome libraries, a contig has been established between the RFLP markers 4111 and 4556, which encompasses the ms1 gene. This covers a genetic distance of 8.9 cM which corresponds to a physical distance of approximately 1.44 Mb, representing about 1.5–2.0% of the Arabidopsis genome. In this region, 1 cM represents a physical distance of approximately 160 kb.     

RSF1, an Arabidopsis locus implicated in phytochrome A signaling

In Arabidopsis, phytochrome A (phyA) is the major photoreceptor both for high irradiance responses to far-red light and broad spectrum very low fluence responses, but little is known of its signaling pathway(s). rsf1 was isolated as a recessive mutant with reduced sensitivity to far-red inhibition of hypocotyl elongation. At the seedling stage rsf1 mutants are affected, to various degrees, in all described phyA-mediated responses. However, in adult rsf1 plants, the photoperiodic flowering response is normal. The rsf1 mutant has wild-type levels of phyA suggesting that RSF1 is required for phyA signaling rather than phyA stability or biosynthesis. RSF1 thus appears to be a major phyA signaling component in seedlings, but not in adult, Arabidopsis plants.     

Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.

Denaturing high performance liquid chromatography (DHPLC) has been described recently as a method for screening DNA samples for single nucleotide polymorphisms and inherited mutations. Thirty-eight DNAs, 22 of which were heterozygous for previously characterized rearranged transforming gene (RET) or cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations or polymorphisms, were examined using DHPLC analysis to assess the accuracy of this scanning method. Ninety-one per cent (20/22) of the PCR amplicons from specimens with heterozygous RET or CFTR sequence showed elution profiles distinct from corresponding homozygous normal patterns; whether the profiles for two amplicons containing heterozygous RET sequence were distinct from homozygous cases was equivocal. To investigate the usefulness of this method for detecting mutations in tumor DNAs, each of the phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) exons were examined for mutations in 63 malignant gliomas. Seventeen PTEN PCR products from this series of brain tumors showed elution profiles indicating sample heterozygosity and in each instance conventional sequencing confirmed the presence of a mutation. PTEN amplicons containing exons 1, 3 and 5 were sequenced for each of the 63 tumor DNAs to determine whether any mutations may have escaped DHPLC detection, and this analysis identified one such alteration in addition to the eight mutations that DHPLC had revealed. In total, DHPLC identified 37 of 40 (92.5%) PCR products containing defined sequence variation and no alterations were indicated among 196 amplicons containing homozygous normal sequence.     

Genetic and Mapping Analysis of Arabidopsis thaliana Male Sterile Mutant filament no elongation

To identify new genes important for anther development, we screened for male sterile mutants among a population of Arabidopsis ecotype Columbia (Col) mutagenized by T DNA insertion (provided by ARBC). A male sterile mutant line with normal vegetative and flora development but no seed yield was isolated from Salk_118481 line. T DNA insertion site identification showed that there were no T DNA sequences in the genome of the mutants. Genetic analysis indicated that the mutant was controlled by a single recessive nuclear gene named filament no elongation because the filament of the mutant remains very short at the 13-14 stage of anther development. The fne gene was mapped to a region of 97kb between the molecular makers MBD2 and MMG4 on chromosome 5 using map based cloning technique. No genes involved filament elongation were reported in this region, so we believe that FNE gene could be a new gene controlling filament elongation in Arabidopsis.     

High-accuracy DNA sequence variation screening by DHPLC

Genetic maps based on biallelic single-nucleotide polymorphisms amenable to microarray-based genotyping have significantly accelerated the mapping of mono- and multigenic traits in model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana. This advance needs to be matched by highly accurate, inexpensive and robust methodology for fine-structure mapping of the candidate region(s) and the eventual identification of the causative mutation(s). To establish the usefulness of denaturing high-performance liquid chromatography (DHPLC) for those purposes, we have amplified 476 fragments from two A. thaliana ecotypes with an average length of 563 bp covering various candidate regions on chromosomes 1, 2 and 4. Parallel analysis by DHPLC and dye terminator sequencing showed that DHPLC detected 165 out of 166 polymorphic fragments with only four false positives, amounting to a sensitivity, specificity and accuracy of 99.4%, 98.7% and 99%, respectively. It proved beneficial to analyze the fragments not only at the highest but also at the lower temperatures recommended by the algorithm freely available at http:?insertion.stanford.edu/melt.html.     

拟南芥黄心突变体yh基因的图位克隆及分析

在研究光合作用相关基因的过程中,获得了一个叶片为黄心(yellow heart,yh)的突变株,与野生型拟南芥(Col 0)相比,其新生叶片发黄,突变表型由隐性单基因控制。采用图位克隆及其精细定位技术,将yh突变基因定位在1号染色体的INS1_55_342与INS1_56_34区间,物理距离约为676 kb。通过测序得知yh在At1g64790第44个内含子剪接处有4个碱基的缺失,导致内含子剪切位点的变化。RT PCR分析显示,该基因表达降低,是At1g64790基因的一个新等位突变。研究表明,yh突变体与叶绿体的发育相关,可为进一步探究植物叶绿体和叶片发育机制提供新的遗传材料。     

拟南芥雄性不育突变体fne的遗传与定位分析

在T-DNA插入突变体Salk_118481株系的群体中,筛选到一株雄性不育突变体,用T-DNA序列上的一对引物进行PCR鉴定表明其基因组中没有T DNA插入。通过背景纯化与遗传分析发现该雄性不育突变体是由单个隐性基因控制的,引起不育的主要原因是在花药发育的第13~14期,花丝不能伸长以完成授粉,故该突变体命名为fne （filament no elongation）。利用图位克隆的方法对FNE基因进行了定位,结果表明FNE基因位于第五条染色体上分子标记MBD2和MMG4之间的97kb区间内。目前该区间内尚未见到控制花丝伸长基因的报道,因此,FNE基因是一个控制花丝伸长的新基因。     

Chromosome walking with YAC clones in Arabidopsis: isolation of 1700 kb of contiguous DNA on chromosome 5, including a 300 kb region containing the flowering-time gene CO

The co mutation of Arabidopsis thaliana causes a late-flowering phenotype that is insensitive to day-ength. The mutation was mapped previously to the upper arm of chromosome 5, approximately 1.6 cM from the chalcone synthase gene (CHS). We were provided with five yeast artificial chromosome (YAC) libraries and used these to perform a chromosome walk from CHS to the CO gene. In this paper we report the isolation of 1700 kb of contiguous Arabidopsis DNA, which represents approximately 1%–2% of the genome, inserted in YACs. This required the detailed analysis of 67 YACs, from which 87 end probes were isolated and examined in hybridisation experiments. This analysis showed that approximately 40% of the YACs presented problems in chromosome walking experiments because they contained repetitive sequence at one of their termini, were chimaeric or because part of the plant DNA was deleted. DNA fragments isolated from YACs were used as restriction fragment length polymorphism (RFLP) markers to localize CO to a 300 kb region within the cloned DNA. We compare the physical distance between CHS and CO with the genetic distance and find that in this region 1 cM is equivalent to approximately 200 kb.     

Chromosome walking with YAC clones in Arabidopsis: isolation of 1700 kb of contiguous DNA on chromosome 5, including a 300 kb region containing the flowering-time gene CO

The co mutation of Arabidopsis thaliana causes a late-flowering phenotype that is insensitive to day-ength. The mutation was mapped previously to the upper arm of chromosome 5, approximately 1.6 cM from the chalcone synthase gene (CHS). We were provided with five yeast artificial chromosome (YAC) libraries and used these to perform a chromosome walk from CHS to the CO gene. In this paper we report the isolation of 1700 kb of contiguous Arabidopsis DNA, which represents approximately 1%–2% of the genome, inserted in YACs. This required the detailed analysis of 67 YACs, from which 87 end probes were isolated and examined in hybridisation experiments. This analysis showed that approximately 40% of the YACs presented problems in chromosome walking experiments because they contained repetitive sequence at one of their termini, were chimaeric or because part of the plant DNA was deleted. DNA fragments isolated from YACs were used as restriction fragment length polymorphism (RFLP) markers to localize CO to a 300 kb region within the cloned DNA. We compare the physical distance between CHS and CO with the genetic distance and find that in this region 1 cM is equivalent to approximately 200 kb.     

Arabidopsis downy mildew resistance gene RPP27 encodes a receptor-like protein similar to CLAVATA2 and tomato Cf-9

The Arabidopsis Ler-RPP27 gene confers AtSgt1b-independent resistance to downy mildew (Peronospora parasitica) isolate Hiks1. The RPP27 locus was mapped to a four-bacterial artificial chromosome interval on chromosome 1 from genetic analysis of a cross between the enhanced susceptibility mutant Col-edm1 (Col-sgt1) and Landsberg erecta (Ler-0). A Cf-like candidate gene in this interval was PCR amplified from Ler-0 and transformed into mutant Col-rpp7.1 plants. Homozygous transgenic lines conferred resistance to Hiks1 and at least four Ler-0 avirulent/Columbia-0 (Col-0) virulent isolates of downy mildew pathogen. A full-length RPP27 cDNA was isolated, and analysis of the deduced amino acid sequences showed that the gene encodes a receptor-like protein (RLP) with a distinct domain structure, composed of a signal peptide followed by extracellular Leu-rich repeats, a membrane spanning region, and a short cytoplasmic carboxyl domain. RPP27 is the first RLP-encoding gene to be implicated in disease resistance in Arabidopsis, enabling the deployment of Arabidopsis techniques to investigate the mechanisms of RLP function. Homology searches of the Arabidopsis genome, using the RPP27, Cf-9, and Cf-2 protein sequences as a starting point, identify 59 RLPs, including the already known CLAVATA2 and TOO MANY MOUTHS genes. A combination of sequence and phylogenetic analysis of these predicted RLPs reveals conserved structural features of the family.     

Structure of a frequently rearranged rRNA-encoding chromosome in Giardia lamblia.

It has been shown previously that the rRNA encoding chromosomes in Giardia lamblia undergo frequent rearrangements with an estimated rate of approximately 1% per cell per division (Le Blancq et al., 1992, Nucleic Acids Res., 17, 4539-4545). Following these observations, we searched for highly recombinogenic regions in one of the frequently rearranged rRNA encoding chromosomes, that is chromosome 1, a small, 1.1 Mb chromosome. Chromosome 1 undergoes frequent rearrangements that result in size variation of 5-20%. We analyzed the structure of chromosome 1 in clonal lineages from the WB strain. The two ends of chromosome 1 comprise telomere repeat [TAGGG] arrays joined to a truncated rRNA gene and a sequence referred to as '4e', respectively. Comparison of the structure of four polymorphic versions of chromosome 1, resulting from independent rearrangement events in four cloned lines, located a single polymorphic region to the variable rDNA-telomere domain. Chromosome 1 is organized into two domains: a core region spanning approximately 850 kb that does not exhibit size heterogeneity among different chromosome 1 and a variable region that spans 185-450 kb and includes the telomeric rRNA genes, referred to as the variable rDNA-telomere domain. The core region contains a conserved region, spanning approximately 550 kb adjacent to the telomeric 4e sequence, which is only present in the 4e containing chromosomes and a 300 kb region of repetitive sequences that are also components of other chromosomes as well. Changes in the number of rDNA repeats accounted for some, but not all, of the size variation. Since there are four chromosomes that share the core region of chromosome 1, we suggest that the genome is tetraploid for this chromosome.     

A frameshift mutation in exon 28 of the OPA1 gene explains the high prevalence of dominant optic atrophy in the Danish population: evidence for a founder effect

Dominant optic atrophy (DOA) is a hereditary optic neuropathy characterised by decreased visual acuity, colour vision deficits, centro-coecal scotoma and optic nerve pallor. The gene OPA1, encoding a dynamin-related GTPase, has recently been identified within the genetic linkage interval for the major locus for DOA on chromosome 3q28 and shown to harbour genetic aberrations segregating with disease in DOA families. The prevalence of the disorder in Denmark is reported to be the highest of any geographical location, suggestive of a founder effect. In order to establish the genetic basis of disease in a sample of 33 apparently unrelated Danish families, we screened DNA from affected members for OPA1 gene mutations by heteroduplex analysis and direct sequencing. A novel identical mutation in exon 28 (2826delT) was associated with DOA in 14 pedigrees and led to a frameshift and abnormal OPA1 protein -COOH terminus. Haplotype analysis of a region of approximately 1 Mb flanking the OPA1 gene using eight polymorphic markers revealed a common haplotype shared by all 14 patients; this haplotype was markedly over-represented compared with ethnically matched controls. Statistical analysis confirmed significant linkage disequilibrium with DOA over approximately 600 kb encompassing the disease mutation. We have therefore demonstrated that the relatively high frequency of DOA in Denmark is attributable to a founder mutation responsible for approximately 42% of the examined families and suggest that presymptomatic screening for the (2826delT) mutation may facilitate diagnosis and genetic counselling in a significant proportion of DOA patients of Danish ancestry.