难转化物种中的基因组操作：来自大麦白粉病菌（一种大麦专性真菌病原体）的酵母人工染色体（YAC）文库的构建与表征

Genome manipulation in recalcitrant species: construction and characterization of a yeast artificial chromosome (YAC) library from Erysiphe graminis f. sp. hordei, an obligate fungal pathogen of barley.

作者信息

Borbye L, Giese H

机构信息

Environmental Science and Technology Department, Risø National Laboratory, Roskilde, Denmark.

出版信息

Gene. 1994 Jun 24;144(1):107-11. doi: 10.1016/0378-1119(94)90212-7.

DOI:10.1016/0378-1119(94)90212-7

PMID:7913075

Abstract

Extraction of DNA from organisms where spores are the only source of pure material is a major problem. Methods are described which allow the isolation of high-M(r) DNA, from small quantities of Erysiphe graminis f. sp. hordei (Egh) conidia, suitable for cloning in yeast artificial chromosomes (YACs). A YAC library of 1500 clones was constructed in the vectors, pYAC4 and pYACRC. The average size of YAC inserts is 220 kb and range from 70 to 500 kb, providing ten haploid genome equivalents. Multicopy RFLP markers and an Egh-specific repetitive SINE element were used to characterize the library. The SINE element is effective in fingerprint analysis and contig assembly. Four out of five representative clones containing more than one YAC were mitotically unstable.

摘要

从仅以孢子作为纯物质唯一来源的生物体中提取DNA是一个主要问题。本文描述了一些方法，这些方法能够从少量禾本科布氏白粉菌（Egh）分生孢子中分离出高分子量（M(r)）DNA，适用于克隆到酵母人工染色体（YAC）中。利用载体pYAC4和pYACRC构建了一个包含1500个克隆的YAC文库。YAC插入片段的平均大小为220 kb，范围在70至500 kb之间，相当于十个单倍体基因组。使用多拷贝RFLP标记和一个Egh特异性重复SINE元件对文库进行表征。该SINE元件在指纹分析和重叠群组装中有效。五个含有多个YAC的代表性克隆中有四个在有丝分裂时不稳定。