Grant D M, Lambowitz A M, Rambosek J A, Kinsey J A
Mol Cell Biol. 1984 Oct;4(10):2041-51. doi: 10.1128/mcb.4.10.2041-2051.1984.
We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am+ transformants and am132 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am132 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation--that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis--cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.
我们已经对用质粒pJR2获得的粗糙脉孢菌转化体进行了特性分析，该质粒由克隆于pUC8中的粗糙脉孢菌谷氨酸脱氢酶（am）基因和一个am132宿主菌株组成，该宿主菌株含有一个包含克隆片段的缺失。测试的33个转化体中的每一个都表现出极端的减数分裂不稳定性：在am +转化体与am132之间的初始或连续回交中，或在am +后代之间的杂交中，获得的am +后代少于1%或2%。此外，回交产生的am +后代在营养生长期间产生了高比例的营养缺陷型（am）有丝分裂分离体。这些结果表明，在所测试的任何转化体中，am +性状都没有稳定地整合到染色体DNA中。通过Southern杂交分析了六个转化体的核DNA。所有六个转化体都含有与pJR2同源的序列。四个显示出未修饰的pJR2预期的限制性片段，但大多数显示出额外的条带。未消化DNA的Southern印迹显示，质粒序列主要以高分子量形式存在（大于20千碱基）。Southern印迹显示，与am132回交产生的营养缺陷型（am）后代失去了与游离质粒相对应的限制性条带，但保留了额外的条带，显然以无功能的方式整合到染色体DNA中。综合考虑这些结果，最合理的解释如下：含有am +基因的重组质粒可以在粗糙脉孢菌中自主复制，游离质粒以寡聚体形式或修饰形式存在或两者皆有，并且质粒序列也在缺失宿主的多个位点整合，但方式无功能。另一种解释——质粒串联重复序列整合到染色体DNA中但在减数分裂期间被消除——不能被完全排除。然而，在多种其他条件下可以获得am基因的稳定整合，即使用克隆于噬菌体λ载体中的am基因（J. A. Kinsey和J. A. Rambosek，《分子细胞生物学》4：117 - 122，1984），使用pJR2的衍生物，或使用pJR2转化移码突变体。
