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首页> 外文期刊>Molecular and Cellular Biology >Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.
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Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

机译：用含有克隆的谷氨酸脱氢酶（am）基因的重组质粒转化Crus Neurospora crassa：转化质粒自主复制的证据。

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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.
机译：我们已经表征了用质粒pJR2获得的crus Neurospora crassa转化子，该质粒由克隆在pUC8中的Neurospora谷氨酸脱氢酶（am）基因和一个am132宿主菌株组成，该菌株包含一个包含克隆片段的缺失。测试的33个转化子中的每一个都显示出极端的减数分裂不稳定：在am +转化子与am132之间的初始或连续回交中或在am +子代之间的杂交中，获得的am +后代少于1％或2％。此外，回交的am +后代在营养生长过程中产生了高比例的营养缺陷型（am）有丝分裂分离子。这些结果表明，在所测试的任何转化体中，am +特征均未稳定地整合到染色体DNA中。通过Southern杂交分析了来自六个转化体的核DNA。所有六个转化体均包含与pJR2同源的序列。有四个显示了预期的未修饰pJR2的限制性片段，但大多数显示了其他条带。对未消化的DNA的Southern印迹表明，质粒序列主要以高分子量形式（大于20 KB）存在。 Southern印迹显示，从回交到am132的营养缺陷型（am）后代丢失了与游离质粒相对应的限制条带，但保留了其他条带，显然以非功能方式整合到了染色体DNA中。一起考虑，这些结果最合理地解释如下：含有am +基因的重组质粒可以在克雷伯氏杆菌中自主复制，游离质粒以寡聚或修饰形式存在，或两者同时存在，并且质粒序列也整合在缺失的多个位点上主机，但功能不正常。无法完全排除另一种解释-质粒的串联重复序列已整合到染色体DNA中，但在减数分裂过程中被消除了。但是，使用克隆在噬菌体λ载体中的am基因，可以在多种其他条件下获得am基因的稳定整合（JA Kinsey和JA Rambosek，Mol。Cell。Biol。4：117-122， 1984），使用pJR2的衍生物，或使用pJR2转化移码突变体。

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