Giasson L, Specht C A, Milgrim C, Novotny C P, Ullrich R C
Department of Botany, University of Vermont, Burlington 05405.
Mol Gen Genet. 1989 Jul;218(1):72-7. doi: 10.1007/BF00330567.
An A alpha mating-type allele (A alpha 4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A alpha 4 allele isolated from the walk was used as a probe to recover the A alpha 1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A alpha allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A alpha encodes a diffusible product. Restriction mapping shows that A alpha 1 and A alpha 4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A alpha 1 or A alpha 4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alpha alleles. A alpha 1 and A alpha 4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.
通过从紧密连锁的PAB1基因沿染色体步移，分离出一个Aα交配型等位基因（Aα4）。从步移中分离出的含有Aα4等位基因的黏粒克隆用作探针，从另一个黏粒文库中回收Aα1等位基因。通过转化裂褶菌细胞并筛选A调控发育的激活来鉴定编码交配型活性的黏粒。在交配试验和子代的遗传分析中证实了推定的交配型转化体。通过表明它们在转化交配型方面的有效性仅限于具有与克隆序列编码的Aα等位基因不同的Aα等位基因的受体菌株，证明了分离出的特定等位基因的身份。转化DNA具有反式活性，表明Aα编码一种可扩散的产物。限制性图谱显示Aα1和Aα4编码在基因组的同一物理区域内，但在一个包含广泛序列差异的子区域内。此外，Southern分析表明，每个单倍体基因组中只有一个Aα1或Aα4拷贝，并且它们彼此之间或与任何其他Aα等位基因都不交叉杂交。Aα1和Aα4分别被亚克隆为2.8 kb和1.2 kb的片段，在转化中保留了原始黏粒所显示的所有交配型活性。
