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PubMed2003

Genetic anthropology of the colorectal cancer-susceptibility allele APC I1307K: evidence of genetic drift within the Ashkenazim.

The adenomatous polyposis coli (APC) I1307K allele is found in 6% of the Ashkenazi Jewish population and in 1%-2% of Sephardi Jews; it confers a relative risk of 1.5-2.0 for colorectal cancer (CRC) on all carriers. Within the Ashkenazim, the existence of numerous high-prevalence mutations, including I1307K, has sparked controversy over whether genetic drift or selection is the underlying cause. For the present population-based case-control study of CRC in Israel, we tested whether selection has operated at I1307K. We also estimated the age of the I1307K allele, to understand its origin in the context of the Jewish diasporas and subsequent founder events. We genotyped 83 matched pairs, in which one or both members of the pair carried I1307K, at three microsatellites and two SNPs. Haplotypes were statistically constructed using PHASE software. Single-marker age estimates for I1307K were calculated using the approach described by Risch et al. A common progenitor haplotype spanned across APC I1307K from the centromeric marker D5S135 to the telomeric marker D5S346 and was observed in individuals of Ashkenazi, Sephardi, and Arab descent. The ancestor of modern I1307K alleles existed 87.9-118 generations ago ( approximately 2,200-2,950 years ago). This age estimate indicates that I1307K existed at about the time of the beginning of the Jewish diaspora, explaining its presence in non-Ashkenazi populations. Our data do not indicate that selection operated at I1307K (D5S346, P=.114; D5S135, P=.373), providing compelling evidence that the high frequency of disease-susceptibility alleles in the Ashkenazim is due to genetic drift, not selection. This research underscores the importance of the migratory patterns of ancestral populations in the ethnic and geographic distribution of APC I1307K.

Adenomatous Polyposis Coli↗
PubMed2005

Association analysis of estrogen receptor alpha gene polymorphisms with cross-sectional geometry of the femoral neck in Caucasian nuclear families.

Bone geometry is a key factor in bone strength, which is the ultimate intrinsic determinant of fracture risk. Though the heritability of bone geometry is high, little effort has been spent on searching for the underlying genes. In this study, employing a sample of 1,873 subjects from 405 Caucasian nuclear families, we studied seven single nucleotide polymorphisms (SNPs) and their haplotypes of the ER-alpha gene for association with six hip geometric variables, namely, cross-sectional area (CSA), cortical thickness (CT), endocortical diameter (ED), subperiosteal width (W), sectional modulus (Z) and buckling ratio (BR). The major method used was the quantitative transmission disequilibrium test (QTDT). Our major findings were summarized below. The within-family association between SNP4 (rs1801132) in exon 4 with endocortical diameter and subperiosteal width was detected in single locus analyses (P=0.008 and 0.021, respectively) and verified in haplotype analyses (P=0.034 and 0.058, respectively). The total association of SNP4 with these two diameters was also observed in both single locus and haplotype analyses (P=0.005 and 0.031 for ED, plus P=0.003 and 0.070 for W). In addition, the total association between SNP5 (rs932477) in intron 4 with cortical thickness and buckling ratio was detected (single locus analyses: P=0.035 and 0.041, respectively). Haplotype analyses further supported the above association (P=0.010 and 0.004, respectively). Similar patterns of associations with the studied SNPs and their haplotypes were present in subsamples stratified by sex, too. However, after permutation tests, the empirical significance level was set as P<0.011, which renders most associations insignificant. Therefore, we concluded that polymorphisms in the ER-alpha gene were nominally associated with femoral neck (FN) geometry variables estimated from DXA. Such genetic effects on hip geometry were not sex specific.

Adult↗
PubMed2006

Plasmodium falciparum: worldwide sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-2 (MSP-2).

We examined patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum, a major dimorphic malaria vaccine candidate antigen, by analyzing 448 msp-2 alleles from all continents. We describe several nucleotide replacements, insertion and deletion events, frameshift mutations, and proliferations of repeat units that generate the extraordinary diversity found in msp-2 alleles. We discuss the role of positive selection exerted by naturally acquired type- and variant-specific immunity in maintaining the observed levels of polymorphism and suggest that this is the most likely explanation for the significant excess of nonsynonymous nucleotide replacements found in dimorphic msp-2 domains. Hybrid sequences created by meiotic recombination between alleles of different dimorphic types were observed in few (3.1%) isolates, mostly from Africa. We found no evidence for an extremely ancient origin of allelic dimorphism at the msp-2 locus, predating P. falciparum speciation, in contrast with recent findings for other surface malarial antigens.

Alleles↗
PubMed1994

The zebrafish egr1 gene encodes a highly conserved, zinc-finger transcriptional regulator.

The Egr family of transcriptional regulators comprises a group of genes which encode members of the Cys2-His2 class of zinc-finger proteins. We have isolated a zebrafish egr1 homologue by screening a zebrafish genomic library with a mouse Egr1 zinc finger probe. Southern blotting indicated the existence of a single zebrafish egr1 gene and, as in higher vertebrates, the presence of related members of a larger gene family. Sequence analysis of the zebrafish egr1 coding region revealed a high level of homology to the mouse, rat, and human Egr1 genes with the notable exception of a polymorphic, triplet nucleotide repeat sequence in the region coding for the amino terminus of the Egr1 protein. The predicted DNA-binding, zinc-finger domain protein sequence was strictly conserved. The 5' region of the zebrafish egr1 gene contained a variety of transcription factor binding sites, also present in the mouse gene, for serum response factor, CREB and c-Ets. The zebrafish egr1 transcript was approximately 3.4 kb in size and was expressed in adult zebrafish brain and muscle RNA, a pattern of expression similar to that observed in mice. The potential for zebrafish egr1 to function as a transcriptional regulator was tested by constructing an expression vector containing zebrafish egr1 coding sequences under the control of a cytomegalovirus promoter. This construct was found to activate transcription of a reporter plasmid bearing multiple Egr1 binding sites when transiently cotransfected into mouse 3T3 cells. Our results indicate that the structure, regulation, and function of the Egr1 gene have been highly conserved during vertebrate evolution and suggest an important role for this gene in growth and development.

Amino Acid Sequence↗
PubMed2004

Molecular study of the 3 beta-hydroxysteroid dehydrogenase gene type II in patients with hypospadias.

To determine whether some patients with idiopathic hypospadias have HSD3B2 mutations, we genotyped this locus in 90 patients with hypospadias (age, 6.0 +/- 0.4 yr) and 101 healthy fertile male controls. We measured basal plasma renin activity and performed an ACTH test for determination of 17-OH-pregnenolone, 17-OH-progesterone, cortisol, dehydroepiandrosterone sulfate, and androstenedione and an human chorionic gonadotropin test for determination of androstenedione, testosterone, and dihydrotestosterone. We did not observe a clear steroidogenic pattern suggestive of 3 beta-HSD deficiency in any patient. DNA was extracted from peripheral lymphocytes; and exons 1, 2, 3, and 4 were amplified by PCR and analyzed by denaturing gradient gel electrophoresis. An abnormal electrophoretic migration pattern of exon 4 was observed in five patients. Two patients had missense heterozygous mutations (S213T and S284R). In another three patients, we observed heterozygous nucleotide variants in exon 4 that did not produce a change in amino acids (A238, T259, T320). In vitro enzymatic activity was diminished by 40% and 32% in the S213T and S284R heterozygous mutations, respectively. One control exhibited a heterozygous mutation in exon 3 (V78I), which did not alter in vitro enzyme activity. In addition, we observed possible polymorphisms in intron 1 in four patients and one control. We conclude that subtle molecular abnormalities in the HSD3B2 gene may be observed in some patients with apparent idiopathic hypospadias but that this finding is uncommon.

3-Hydroxysteroid Dehydrogenases↗
PubMed2001

Novel bacterial lineages at the (sub)division level as detected by signature nucleotide-targeted recovery of 16S rRNA genes from bulk soil and rice roots of flooded rice microcosms.

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.

Anaerobiosis↗
PubMed2006

Tracking Trichophyton tonsurans through a large urban child care center: defining infection prevalence and transmission patterns by molecular strain typing.

OBJECTIVES: Trichophyton tonsurans is the single most common cause of pediatric dermatophytoses in North America and is observed with increasing frequency in other countries. This investigation was designed to gain insight into the natural course of T. tonsurans infection. PATIENTS AND METHODS: This 2-year prospective, longitudinal study evaluated all preschool-aged children attending a single child care center. Scalp cultures were collected monthly from each child in attendance, and the presence of disease symptoms recorded at each visit. Dermatophyte genotype was assigned based on the combination of stable sequence variations (2 length variants, 8 single-nucleotide polymorphisms, a 10-base pair insertion, a 14-base pair deletion) present in 2 gene loci. RESULTS: A total of 3541 scalp cultures were collected from 446 children during 24 months. Twenty-two percent to 51% of scalp cultures per month were positive, contributing 1390 fungal cultures of which 1048 were typeable. Among children with multiple typeable isolates, 51% exclusively carried the same strain, 37% demonstrated a single predominant strain with secondary strains transiently acquired, and 12% harbored a different strain of T. tonsurans with each typeable culture. The probability that the same strain persisted in subsequent months was 0.898 and unlikely to have arisen by chance. Rates of symptomatic disease were significantly different between exclusive, predominant, and transient carriers of T. tonsurans. CONCLUSIONS: In contrast to dermatophyte infections in older individuals, where symptomatic disease seems to be a consequence of pathogen acquisition and carriers can be traced to an index case, in this preschool-aged population infection was endemic, and symptomatic disease seemed to represent activation of a single strain that persisted on the scalp.

Child↗
PubMed1994

Taxonomic classification of 29 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis: a comparison of five different phenotypic and genotypic typing schemes.

Twenty-nine European and North American Borrelia burgdorferi strains isolated from patients with Lyme borreliosis, were investigated by restriction fragment length polymorphism (RFLP) of two phylogenetically highly conserved chromosomal genes encoding flagellin (fla) and the p60 common antigen (CA), as well as of the plasmid-borne outer surface protein A (ospA) gene. RFLP of the ospA, fla and CA gene revealed five, two and four distinct subspecies-specific patterns, respectively. RFLP classification of the B. burgdorferi strains was compared with four different classification schemes proposed by others: (i) molecular mass profile of OspA and OspB (Adam et al. [1]); (ii) OspA serotyping (Wilske et al. [34]); (iii) genomic fingerprinting on the central region of the B. burgdorferi fla gene (Picken [24]) and (iv) 16S rRNA signature nucleotide analysis (Marconi and Garon [19]). Results obtained with the different methods correlated highly. All strains classified as B. burgdorferi sensu stricto and B. afzelii could be unequivocally identified as one distinct group by all five typing methods. B. garinii isolates, however, were more heterogeneous and according to RFLP of the CA and ospA gene fell into either two or three subgroups. The agreement of the different approaches supports the recent concept that B. burgdorferi sensu lato strains should be delineated to three genomic groups and that B. burgdorferi sensu lato is clonal. All 12 US strains were B. burgdorferi sensu stricto, whereas the 17 European isolates belonged to any of three genospecies. Among European B. burgdorferi isolates there was an association between B. burgforferi genospecies and the clinical manifestation of Lyme borreliosis. B. afzelii strains were found to predominate in 11 skin isolates (75%), whereas all 6 cerebrospinal fluid isolates from patients with neuroborreliosis were B. garinii. These findings support the concept of a strain-dependent organotropism of B. burgdorferi.

Animals↗
PubMed2006

Efficient selection of tagging single-nucleotide polymorphisms in multiple populations.

Common genetic polymorphism may explain a portion of the heritable risk for common diseases, so considerable effort has been devoted to finding and typing common single-nucleotide polymorphisms (SNPs) in the human genome. Many SNPs show correlated genotypes, or linkage disequilibrium (LD), suggesting that only a subset of all SNPs (known as tagging SNPs, or tagSNPs) need to be genotyped for disease association studies. Based on the genetic differences that exist among human populations, most tagSNP sets are defined in a single population and applied only in populations that are closely related. To improve the efficiency of multi-population analyses, we have developed an algorithm called MultiPop-TagSelect that finds a near-minimal union of population-specific tagSNP sets across an arbitrary number of populations. We present this approach as an extension of LD-select, a tagSNP selection method that uses a greedy algorithm to group SNPs into bins based on their pairwise association patterns, although the MultiPop-TagSelect algorithm could be used with any SNP tagging approach that allows choices between nearly equivalent SNPs. We evaluate the algorithm by considering tagSNP selection in candidate-gene resequencing data and lower density whole-chromosome data. Our analysis reveals that an exhaustive search is often intractable, while the developed algorithm can quickly and reliably find near-optimal solutions even for difficult tagSNP selection problems. Using populations of African, Asian, and European ancestry, we also show that an optimal multi-population set of tagSNPs can be substantially smaller (up to 44%) than a typical set obtained through independent or sequential selection.

Algorithms↗
PubMed2005

Association of the 276G->T polymorphism of the adiponectin gene with cardiovascular disease risk factors in nondiabetic Koreans.

BACKGROUND: The adiponectin gene is known to modulate adiponectin concentrations and diabetes mellitus development. OBJECTIVE: We assessed whether adiponectin gene variants contribute to circulating adiponectin, insulin resistance (IR), or cardiovascular disease risk factors. DESIGN: Nondiabetic subjects [n = 902; x +/- SE age: 42.5 +/- 0.53 y; body mass index (BMI; in kg/m2): 24.7 +/- 0.11] were genotyped for 2 single-nucleotide polymorphisms (SNPs), 45T-->G and 276G-->T. RESULTS: After adjustment for age, sex, and BMI, subjects with the G allele for the SNP 276 had significantly higher concentrations of triacylglycerol and small dense LDL (sdLDL) and smaller LDL particle size than did T/T subjects. G/G subjects at SNP 276 had significantly lower plasma adiponectin and higher homeostasis model assessment (HOMA) of IR and urinary prostaglandin F2alpha than did T/T subjects. In the SNP 45-276 haplotype test, we also observed that subjects with the X/X haplotype had significantly higher plasma adiponectin after adjustment than did TG/TG or TG/X haplotype subjects. In the highest BMI group (BMI > or = 26), T/T subjects had lower HOMA-IR (P = 0.011) and higher plasma adiponectin (P = 0.026) at SNP 276 than did G/G or G/T subjects. These patterns were also seen for adiponectin in haplotype groups. However, no significant genotype effect for SNP 45T-->G was observed. CONCLUSIONS: The 276G-->T polymorphism of the adiponectin gene modulates circulating adiponectin and IR, particularly in obese states. G allele carriers also have higher oxidative stress, higher sdLDL concentrations, and smaller LDL particle size. Therefore, the presence of the G allele in the adiponectin gene at SNP 276 could be a significant contributor to higher cardiovascular disease risk in Koreans, independent of common environmental factors.

Adiponectin↗
PubMed1997

Discordant phylogeographic patterns between the Y chromosome and mitochondrial DNA in the house mouse: selection on the Y chromosome?

We have compared patterns of geographic variation and molecular divergence of mitochondrial DNA (mtDNA) and Y chromosome over the range of the different subspecies of Mus musculus. MtDNA was typed for 305 nucleotides in the control region, the Y chromosome for 834 base pairs (bp) in Zfy introns and 242 bp in Sry, a Zfy2 18-bp deletion, and two microsatellites. Apparent discrepancies exist between the distributions of the lineages of mtDNA and of the two major Y-chromosome lineages thus defined: some subspecies share the same mtDNA lineage but have different Y-chromosome lineages or vice versa. One microsatellite reveals a geographically clustered variation inside the distribution of each Y-chromosome lineage, showing that new Y-chromosome variants can rapidly spread locally. The two major Y-chromosome lineages have a divergence time only about one fourth of that between mtDNA lineages. Although this recent coalescence of the Y chromosomes between subspecies could partly be due to a lower ancestral polymorphism of the Y chromosome, it suggests that secondary introgression after the radiation of the subspecies might have occurred. There is evidence that the differentiation of the Y-chromosome lineages contributes to partial reproductive isolation between subspecies, and patterns of molecular evolution suggest that selection has played a role in the rapid spread across subspecies.

Animals↗
PubMed2001

Differentiation among three species of bovine Thelazia (Nematoda: Thelaziidae) by polymerase chain reaction-restriction fragment length polymorphism of the first internal transcribed spacer ITS-1 (rDNA).

Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini are nematodes transmitted by some species of Musca (Diptera: Muscidae) which cause ocular infestations in bovines. Differences in the rDNA of these species were determined by a PCR using different sets of relatively conserved oligonucleotide primers. PCR on the first internal transcribed spacer (ITS-1) revealed differences in size in Thelazia species (437 bp for T. gulosa, 370 bp for T. rhodesi and 506 bp for T. skrjabini) while the DNA control of Musca spp. was not amplified. The ITS-1 amplicons of the three species were sequenced and then analysed. The GC contents ranged from 26 to 36% and the level of differences in the nucleotide sequences of ITS-1 was lower between T. skrjabini and T. gulosa (39%) than the latter and T. rhodesi (49-56%). Restriction fragment length polymorphism (RFLP) of ITS-1 amplicons was also carried out and the restriction profiles compared. Clear genetic differences among the three Thelazia examined were demonstrated by using the enzymes HpaII, CpoI and SspI. This PCR-RFLP for the delineation of T. gulosa, T. rhodesi and T. skrjabini offers prospects as a molecular epidemiological tool to study parasite transmission patterns and prevalence.

Amino Acid Sequence↗
PubMed2000

New p53 mutations in hilar cholangiocarcinoma.

BACKGROUND: Inactivation of the tumour suppressor gene, p53, is the commonest genetic abnormality in human cancer. The study of the type of p53 mutation in a given tumour may provide prognostic information, clues to aetiology and become useful for therapeutics. MATERIALS AND METHODS: The molecular characterisation of p53 was performed by restriction analysis, denaturing gradient gel electrophoresis, and gene sequencing for exons 5-9. RESULTS: We report, p53 mutational analysis in exons 5-9 in 29 European patients with hilar cholangiocarcinoma who underwent attempted resection. Four patients (14%) showed somatic single nucleotide substitutions with amino acid changes (146, 163, 175, 158, and 175) with one showing mutations in two different positions involving a loss of two CfoI sites. All the mutations occurred in exon 5. Three patients had a germline polymorphism (10%) with a silent substitution in codon 213 (exon 6). CONCLUSIONS: The systematic screening for p53 mutations in European patients with hilar cholangiocarcinoma has shown that the type of mutation (except 175) is different and its incidence is much lower when compared to the pattern previously reported for intrahepatic cholangiocarcinoma in East Asian patients. A probable explanation is that the presence and type of p53 mutation is dependent on geographic and environmental factors which vary in different populations.

Adult↗
PubMed1996

Multi-locus selection and the structure of variation at the white gene of Drosophila melanogaster.

We surveyed sequence variation and divergence for the entire 5972-bp transcriptional unit of the white gene in 15 lines of Drosophila melanogaster and one line of D. simulans. We found a very high degree of haplotypic structuring for the polymorphisms in the 3' half of the gene, as opposed to the polymorphisms in the 5' half. To determine the evolutionary mechanisms responsible for this pattern we sequenced a 1612-bp segment of the white gene from an additional 33 lines of D. melanogaster from a European and a North American population. This 1612-bp segment encompasses an 834-bp region of the white gene in which the polymorphisms form high frequency haplotypes that cannot be explained by a neutral equilibrium model of molecular evolution. The small number of recombinants in the 834-bp region suggests epistatic selection as the cause of the haplotypic structuring, while an investigation of nucleotide diversity supports a directional selection hypothesis. A multi-locus selection model that combines features from both hypotheses and takes the recent history of D. melanogaster into account may be the best explanation for these data.

ATP-Binding Cassette Transporters↗
PubMed2006

The D category VI type 4 allele is prevalent in the Spanish population.

BACKGROUND: The D category VI (DVI) is one of the clinically most important partial D. Three different molecular structures causing the DVI phenotype have been described. STUDY DESIGN AND METHODS: To determine the molecular basis of the DVI phenotype in the Spanish population, 20 DVI samples, previously detected in serologic screening, were examined by polymerase chain reaction with RHD exon-specific primers. Unexpected findings were further pursued by cDNA nucleotide sequencing. RESULTS: A novel pattern of RHD exon amplification was detected, which did not correspond to any of the previously described molecular structures. The cDNA sequence led to the identification of the new hybrid RHD-Ce(3-5)-D allele. The origin of exon 2 is undeterminable, because the 5' breakpoint was located within a region of RHD and RHCE identical sequence, which encompasses this exon. Sequencing of intron 5 allowed the 3' breakpoint to be mapped between the sixth and seventh polymorphic sites. Serologically, the hybrid protein has a D epitope expression pattern identical to the previously described DVI phenotypes and an antigen density slightly lower than DVI type 3. The new DVI variant is linked to the DCe haplotype and expresses the low-incidence BARC antigen. CONCLUSION: A novel structure causing the DVI phenotype, here named DVI type 4, has been characterized. This novel structure is the most frequent cause of DVI in Spain.

Base Sequence↗
PubMedDate not supplied

Determination of close genetic relatedness of the major Salmonella enteritidis phage types by pulsed-field gel electrophoresis and DNA sequence analysis of several Salmonella virulence genes.

Salmonella enteritidis (SE) is an important cause of egg-associated outbreaks in both Europe and the United States. Phage typing has become an important epidemiologic tool in identifying the source of outbreaks. Limitations of phage typing have become apparent with wholesale egg distributors that have multiple suppliers in an area where a particular phage type is endemic. Several different molecular typing methods were evaluated for their discriminatory power to identify genetic differences among different SE phage types isolated in Europe and the United States. Pulsed-field gel electrophoresis (PFGE) identified a single DNA pattern among the different SE phage types. Comparison of the nucleotide sequence for several Salmonella virulence genes failed to identify a single nucleotide change in the gene sequences from most SE isolates, regardless of phage type. On the basis of these results, the different SE phage types appear to be genetically related or clonal. However, with primers 1283 and Opa4, it was possible to differentiate not only SE isolates from different geographic locations but those within a specific geographic locale as well by random amplified polymorphic DNA polymerase chain reaction. Any chance for discerning genetic differences among isolates will need to rely on molecular techniques other than PFGE.

Animals↗
PubMedDate not supplied

Analysis of the HLA-DR gene locus by temperature gradient gel electrophoresis and its application for the rapid selection of unrelated bone marrow donors.

The human leucocyte antigen (HLA) class II compatibility of bone marrow donor and recipient is an essential prerequisite for the prevention of severe graft versus host disease and therefore for the successful outcome of bone marrow transplantation. In this study an efficient protocol was developed for the rapid analysis of the polymorphic HLA-DR gene locus, based on DNA-polymerase chain reaction (PCR) amplification of the variable exon II of the HLA class II DR genes and subsequent temperature gradient gel electrophoresis (TGGE). Computer-assisted melting map calculations were carried out to determine the melting behavior of the different HLA-DR fragments. Despite the high variability of the DR alleles on the nucleotide sequence level the calculations revealed a common melting domain structure of the different HLA-DR fragments, which was experimentally confirmed by perpendicular TGGE. On parallel TGGE, all samples were separated under the same electrophoretical conditions using a single PCR fragment without GC-clamp. TGGE was applied for the analysis of the DR alleles of numerous bone marrow receipt pairs and compared to the corresponding serological and DNA typing results. The TGGE patterns were found to be different for all samples with different HLA-DR typing results. Identical homoduplex and heteroduplex patterns occurred only in the case of complete genotypic HLA-DR identity as determined by direct sequencing of PCR products.

Alleles↗
PubMed2003

Discrimination of homoeologous gene expression in hexaploid wheat by SNP analysis of contigs grouped from a large number of expressed sequence tags.

Single-nucleotide polymorphisms (SNPs) are useful markers for gene diagnosis and mapping of genes on chromosomes. However, polyploidy, which is characteristic of the evolution of higher plants, complicates the analysis of SNPs in the duplicated genes. We have developed a new method for SNP analysis in hexaploid wheat. First, we classified a large number of expressed sequence tags (ESTs) from wheat in silico. Those grouped into contigs were anticipated to correspond to transcripts from homoeologous loci. We then selected relatively abundant ESTs, and assigned these contigs to each of the homoeologous chromosomes using a nullisomic/tetrasomic series of Chinese Spring wheat strains in combination with pyrosequencing. The ninety genes assigned were almost evenly distributed into seven homologous chromosomes. We then created a virtual display of the relative expression of these genes. Expression patterns of genes from the three genomes in hexaploid wheat were classified into two major groups: (1) genes almost equally expressed from all three genomes; and (2) genes expressed with a significant preference, which changed from tissue to tissue, from certain genomes. In 11 cases, one of the three genes in the allopolyploid was found to be silenced. No preference for gene-silencing in particular genomes or chromosomes was observed, suggesting that gene-silencing occurred after polyploidization, and at the gene level, not at the chromosome or genome level. Thus, the use of this SNP method to distinguish the expression profiles of three homoeologous genes may help to elucidate the molecular basis of heterosis in polyploid plants.

Base Sequence↗