Lubega G W, Klein R D, Geary T G, Prichard R K
Institute of Parasitology, McGill University, Ste. Anne de Bellevue, Quebec, Canada.
Biochem Pharmacol. 1994 Apr 29;47(9):1705-15. doi: 10.1016/0006-2952(94)90551-7.
The role of beta-tubulin genes in benzimidazole (BZ) resistance was investigated using one susceptible (S) and two resistant (Rt and Rc) strains of Haemonchus contortus. The Rt strain was isolated from the field on the basis of thiabendazole resistance. The Rc strain was derived from the S strain by treatment with cambendazole. cDNAs, derived from the S strain, encoding two isoforms of beta-tubulin (beta 12-16 and beta 8-9), alpha-tubulin and phosphofructokinase (Pfk) were used as probes for Southern hybridization analysis of genomic DNA digested by restriction enzymes. Genomic DNA was isolated from a pool of worms or single worms. The restriction-enzyme fragment length polymorphism (RFLP) differences among these strains depended on the enzyme and the probe used. When digested with Stu I or Hpa I, and probed under stringent conditions with beta 8-9 or beta 12-16, fewer fragments were seen in the Rt and Rc strains than in the S strain. Different hybridizing fragments were found in different individuals. The frequency of individuals bearing certain fragments hybridizing to beta 12-16 or beta 8-9 in the susceptible population was reduced significantly in the resistant populations. Some differences in RFLP between these strains were observed when probed with alpha-tubulin or Pfk, but the changes were not consistent with fragments being lost from the resistant strains as observed for beta-tubulin probes. These changes in RFLP pattern correlate with changes in the binding profiles of BZs and isoelectric isoform patterns reported previously for these strains. The data confirm that reduced heterogeneity within the population is associated with BZ resistance. Our results show that both the beta 8-9 and the beta 12-16 subfamilies of beta-tubulin are affected to a similar extent by this reduction in heterogeneity in a resistant population.
利用捻转血矛线虫的一个敏感株（S）和两个抗性株（Rt和Rc）研究了β-微管蛋白基因在苯并咪唑（BZ）抗性中的作用。Rt株是根据对噻苯达唑的抗性从田间分离得到的。Rc株是通过用坎苯达唑处理S株而获得的。来源于S株的编码β-微管蛋白两种同工型（β12 - 16和β8 - 9）、α-微管蛋白和磷酸果糖激酶（Pfk）的cDNA用作探针，对经限制性酶消化的基因组DNA进行Southern杂交分析。基因组DNA从虫体群体或单个虫体中分离。这些菌株之间的限制性酶片段长度多态性（RFLP）差异取决于所用的酶和探针。用Stu I或Hpa I消化，并用β8 - 9或β12 - 16在严格条件下进行探针杂交时，Rt和Rc菌株中观察到的片段比S菌株少。在不同个体中发现了不同的杂交片段。在抗性群体中，敏感群体中与β12 - 16或β8 - 9杂交的某些片段的个体频率显著降低。用α-微管蛋白或Pfk作为探针时，观察到这些菌株之间在RFLP上有一些差异，但这些变化与β-微管蛋白探针所观察到的抗性菌株中片段丢失不一致。RFLP模式的这些变化与先前报道的这些菌株的BZ结合谱和等电同工型模式的变化相关。数据证实群体内异质性降低与BZ抗性相关。我们的结果表明，在抗性群体中，β-微管蛋白的β8 - 9和β12 - 16亚家族受这种异质性降低的影响程度相似。
