公开(公告)号：US06210945B1

公开(公告)日：2001-04-03

申请号：US09587066

申请日：2000-06-02

申请人： Keith D. Lunnen ,  Richard D. Morgan ,  Timothy Meixsell ,  Geoffrey G. Wilson

发明人： Keith D. Lunnen ,  Richard D. Morgan ,  Timothy Meixsell ,  Geoffrey G. Wilson

IPC分类号： C12N922

CPC分类号： C12N9/22

摘要： RsaI, a restriction enzyme from the bacterium Rhodopseudomonas sphaeroides, recognizes the DNA sequence 5′-GTAC-3′. Because RsaI is commercially valuable, we sought to overproduce it by cloning the genes for RsaI and its accompanying, modification, enzyme. The ‘methylase-selection’ method, the customary procedure for cloning restriction and modification genes, was applied to RsaI. The method yielded clones containing the methylase gene (rsaIM), but none containing both the methylase gene and the restriction gene (rsaIR). Inverse-PCR was then used to recover sections of the DNA downstream of rsaIM. These sections were sequenced, and the sequences were joined in silico to reveal the gene organization of the RsaI R-M system. By comparing the coding potential of the DNA with the N-terminal amino acid sequence of the purified RsaI restriction enzyme, we discovered that the RsaI R and M genes, rather than being adjacent-the situation that pertains in most R-M systems-are separated by an intervening gene of unknown function. Based on this information, the rsaIR gene was cloned by PCR instead of methylase-selection. These new clones proved to be highly unstable, however, even in the presence of the rsaIM gene. Various attempts were made to stabilize the gene, but most met with failure. Stability was finally achieved by introducing a second methylase gene, mjaVM, to augment the protection provided by rsaIM, and by tightly controlling the expression of rsaIR using a special two-promoter, anti-sense transcription, expression vector.

摘要翻译： 来自细菌Rhodopseudomonas sphaeroides的限制酶RsaI识别DNA序列5'-GTAC-3'。 由于RsaI具有商业价值，我们试图通过克隆RsaI及其伴随的修饰酶的基因来过度生产。 将“甲基化酶选择”方法，克隆限制和修饰基因的常规方法应用于RsaI，该方法产生含有甲基化酶基因（rsaIM）的克隆，但不含有甲基化酶基因和限制性基因（rsaIR）。 然后使用反向PCR来回收rsaIM下游DNA的切片，对这些切片进行测序，并将序列与硅胶连接以揭示RsaI RM系统的基因组织，通过将DNA的编码潜力与N 纯化的RsaI限制酶的末端氨基酸序列，我们发现RsaI R和M基因而不是与大多数RM系统相关的情况相邻 - 由未知功能的介入基因分离，基于该信息 ，通过PCR克隆rsaIR基因而不是甲基化酶选择，这些新克隆被证明是高度不稳定的，然而，即使在rsaIM基因的存在下，也进行了各种尝试 lize基因，但大多数遇到失败。 通过引入第二个甲基化酶基因mjaVM来增加rsaIM提供的保护，并通过使用特异的双启动子，反义转录，表达载体来严格控制rsaIR的表达，最终达到稳定性。

公开(公告)号：US20110076720A1

公开(公告)日：2011-03-31

申请号：US12906515

申请日：2010-10-18

申请人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

发明人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

IPC分类号： C12P21/06 ,  C12N9/22

CPC分类号： C12N9/22

摘要： A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or gutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

摘要翻译： 描述了一种切口内切核酸酶，其具有与SEQ ID NO：6具有至少70％同一性的氨基酸序列，并且包含分别对应于SEQ ID NO：6中的位置507和位置546的精氨酸或谷氨酸中的至少一种的突变 。

公开(公告)号：US07820424B2

公开(公告)日：2010-10-26

申请号：US11631438

申请日：2005-07-22

申请人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

发明人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

IPC分类号： C12N9/16 ,  C07K14/00 ,  C12P21/00 ,  C12N15/00 ,  C12N1/21 ,  C12N5/10 ,  C12Q1/44 ,  C07H21/00

CPC分类号： C12N9/22

摘要： A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or glutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

摘要翻译： 描述了一种切口内切核酸酶，其具有与SEQ ID NO：6具有至少70％同一性的氨基酸序列，并且包含分别对应于SEQ ID NO：6中的位置507和位置546的精氨酸或谷氨酸中的至少一种的突变 。

公开(公告)号：US20080213860A1

公开(公告)日：2008-09-04

申请号：US11631438

申请日：2005-07-22

申请人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

发明人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

IPC分类号： C12N9/14

CPC分类号： C12N9/22

摘要： A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or gutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

摘要翻译： 描述了一种切口内切核酸酶，其具有与SEQ ID NO：6具有至少70％同一性的氨基酸序列，并且包含分别对应于SEQ ID NO：6中的位置507和位置546的精氨酸或谷氨酸中的至少一种的突变 。

公开(公告)号：US08163529B2

公开(公告)日：2012-04-24

申请号：US12906515

申请日：2010-10-18

申请人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

发明人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

IPC分类号： C07K14/00 ,  C07H21/00 ,  C12N9/16 ,  C12N15/00 ,  C12N5/10 ,  C12N1/21 ,  C12P21/00 ,  C12Q1/44

CPC分类号： C12N9/22

摘要： A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or glutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

摘要翻译： 描述了一种切口内切核酸酶，其具有与SEQ ID NO：6具有至少70％同一性的氨基酸序列，并且包含分别对应于SEQ ID NO：6中的位置507和位置546的精氨酸或谷氨酸中的至少一种的突变 。