McReynolds L A, DeSimone S M, Williams S A
Proc Natl Acad Sci U S A. 1986 Feb;83(3):797-801. doi: 10.1073/pnas.83.3.797.
A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved by Alu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% homologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes.
当用限制性内切酶Hha I消化来自马来布鲁线虫和彭亨布鲁线虫的丝状寄生虫的DNA样本时，观察到一个320个碱基对的重复序列。来自马来布鲁线虫的重复序列的一个640个碱基对的二聚体被插入到质粒pBR322中。当使用点杂交时，发现马来布鲁线虫中该重复序列的拷贝数约为30,000个。320个碱基对的Hha I重复序列以直接串联阵列排列，约占基因组的12%。彭亨布鲁线虫有一个相关的重复序列，它与克隆的马来布鲁线虫Hha I重复序列交叉杂交。与克隆的重复序列进行点杂交表明，该序列存在于马来布鲁线虫和彭亨布鲁线虫中，但不存在于其他四种丝状寄生虫中。克隆的重复DNA序列是检测血液样本中布鲁线虫的极其敏感的探针。与克隆的重复序列杂交可以检测从感染马来布鲁线虫的动物的一份血液中分离出的单个寄生虫的DNA。重复序列中存在的限制性位点存在差异，可用于区分这两种布鲁线虫。马来布鲁线虫的重复DNA序列可被Alu I和Rsa I切割，但彭亨布鲁线虫的序列则不能。通过DNA序列分析对这两个物种的重复序列进行比较表明，单个重复序列的某些区域同源性超过95%，而其他短区域的同源性仅为60-65%。DNA序列的这些差异将有助于构建物种特异性杂交探针。
