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DNA polymorphisms at Amy locus of Drosophila melanogaster in Korean populations

Title
DNA polymorphisms at Amy locus of Drosophila melanogaster in Korean populations
Authors
박경희
Issue Date
1998
Department/Major
대학원 생물과학과
Keywords
DNA Polymorphismsamy locusdrosophila melanogasterkorean populations
Publisher
Graduate School of Ewha Womans University
Degree
Master
Abstract
The purpose of this study is to describe the restriction site variation of 20kb containing the Amy region in 27 isogenic second chromosome strains of Drosophila melanogaster, twelve from Cho^unan and fifteen from Pusan populations in Korea using the method of the restriction mapping. And the relationship between AMY activity levels measured by the spectrophotometer and restriction site variation is observed in the Amy region. Most restriction variants are present in both populations: most of site variants are shown in Cho^unan population, while those of length variants are shown in Pusan population. Among thirty-four restriction sites examined, nine restriction sites were polymorphic(26.5%). All polymorphic restriction sites were observed between the Amy transcriptional units and most of them were found only once. Among the site variants, BamHI(-l.7) is present only in Pusan population, three kinds of EcoRI site variants(-2.5, +0.6 and +4.6) are present in only Cho^unan population. Two kinds of large insertion and three kinds of large deletion(>0.3kb) were found around the Amy transcriptional units among the 27 alleles examined, The estimated heterozygosity per nucleotide pair, θ, for all strains was 0.007, so it means that an average pair of randomly chosen alleles will differ at 140 nucleotide positions over 20kb Amy region examined. The proportion of polymorphic nucleotides, p, for all strains was 0.0254 and the estimates of overall heterozygosity was 0.997. The heterozygosity of site and length variation for Pusan population was lower than that of Cho^unan population, and that for Amy^(1) was higher than that of non-Amy^(1) heterozygotes. There were three pairwise comparisons, which show relatively high level of the linkage disequilibria in both populations: EcoRI(-1.0) versus BamHI(-5.0), Del(i) versus Ins(e) and Del(j) versus EcoRI(-1.0). The enzyme activity for Cho^unan population is higher than activity for Pusan population. And enzyme activity for heterozygotes is higher than that of Amy^(1), so the Amy haplotype bearing non-Amy(1) isozymes is generally associated with higher adult enzyme activity. In the respect of the correlation between AMY activity and the amount of AMY protein, the associated differences in enzyme activities are largely attributable to variation in the amount of protein expression. There were no significant associations between the restriction site variation and AMY activity in Korean population. But strains bearing insertion/deletion were exhibited relatively low enzyme activities, therefore, there may be some effect on enzyme activity by the length variations. It is considered that the difference of AMY activity is due to the variations outside the Amy structural genes. There was no restriction site variation and insertion or deletion in the 1kb upstream regions of the Amy structural genes in Korean populations of D. melanogaster. So duplicated structural genes are very highly conserved including the upstream 1kb region in Korean population same as other populations in United states and Japan, shown the manner of concerted evolution.;한국산 노랑초파리(Drosophila melanogaster)의 천안집단으로부터 12계통, 부산집단으로부터 15계통 등 모두 27계통을 대상으로 하여 α-amylase isozyme type과 효소 활성도, 체내의 α-amylase 효소량, Amy유전자의 duplication과 Amy 구조유전자를 포함한 20kb부위의 DNA상의 유전자 변이 양상을 살펴보았다. 즉, 흡광도 측정을 통한 효소활성도 측정 및 single radial immunodiffusion 방법을 통한 AMY 효소량(CRM) 측정을 실시하였고, Southern blotting analysis를 이용하여 Amy유전자 부위에 대한 restriction map을 작성하고 DNA수준에서의 다형현상을 분석하였다. Amy유전자의 duplication을 확인하기 위하여 proximal 유전자와 distal유전자에 대해 PCR을 실시하고 그 증폭산물을 제한효소로 처리하여 구조유전자 서열의 보존성 여부를 분석하였다. 1. 27계통 중 24계통에서 AMY^(1) type의 isozyme을 나타냈고, C17계통과 P1 계통은 AMY^(1,3) type을 나타내었으며 P4계통은 AMY^(1,4) type을 나타내었다. 효소활성도를 측정한 결과 최저 0.105unit/min,㎎, 최고 0.520 unit/min,㎎의 폭 넓은 범위의 효소활성도를 나타내는 것을 확인할 수 있었다. 또한 부산집단의 효소활성도가 천안집단에 비해 약간 낮았으며, AMY^(1) type을 나타내는 계통의 효소활성도는 non-AMY^(1) heterozygote의 효소활성도에 비해 낮은 것으로 나타났는데, 다른 지역의 집단에서와 비슷한 양상을 보였다. 2. AMY 효소량(CRM)을 측정한 결과 부산집단의 효소량이 천안집단의 효소량보다 낮은 것으로 분석되었고 전반적으로 효소활성도의 경우와 유사한 양상을 나타내었다. 3. Southern blotting analysis를 실시한 결과 34개의 제한효소 좌위 중 9개의 좌위들이 다형현상을 나타냈고 5종류의 삽입과 6종류의 결실이 관찰되었다. 이러한 다양한 DNA상의 변이는 Amy 유전자의 전사부위 바깥에서 관찰되었다. 4. 27계통에 대한 전체적인 heterozygosity는 0.997이고, non-AMY^(1) heterozygote의 heterozygosity는 0.889로서 AMY^(1)의 heterozygosity, 0.997에 비해 낮은 것으로 나타났다. 5. 27계통을 대상으로 한 다형현상 간의 linkage disequilibrium을 분석한 결과 EcoRI(-1.0)와 BamHI(-5.0), Del(i)와 Ins(e), 그리고 Del(j)과 EcoRI(-1.0)사이에서 강한 연관관계를 갖는 것으로 나타났다. 6. PCR을 실시한 결과 27계통 모두에서 proximal 유전자와 distal유전자에 대한 증폭산물이 형성되었고, BamHI과 SalI 제한효소로 처리하여 Amy유전자의 duplication의 특징적인 구조인 "BSB" triad과 동일한 길이의 절편들을 확인할 수 있었다. 즉 Amy구조유전자는 DNA상의 변이가 없이 매우 보전적인 반면 구조유전자의 주변에서는 매우 다양한 양상의 다형현상들이 나타나는 것을 확인할 수 있었다. 따라서 한국산 노랑 초파리의 α-amylase 유전자는 concerted evolution의 양상을 나타낸다고 할 수 있다.
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