FRAGMENT DELETION AND BASE SUBSTITUTION OF STAPHYLOCOCCAL NUCLEASE GENE R
金黄色葡萄球菌核酸酶基因的片段缺失与定点突变

Staphylococcal nuclease R (SNase R) is an analogue of Staphylococcal nuc-ease A (SNase A), which has the same enzymatic activities as SNase A and six extra amino acid residues attached to the amino teminus of SNase A. In order to get an exact SNase A gene for expression, the 18 nucleotides coding for the six extra amino acid residues were deleted by uracil-containing single strand phagemid DNA template (U-ss pTZ19R) and one primer method. The mutagenesis frequency was about 90%. Furthermore, the SNase A was inserted into an expression vector pBV 221, at EcoRI/Sall restriction sites. The new expression plasmid was named pBVS-M2. The polyacrylamide gel electrophor-esis analysis results of the cell lysate from host cell harboring the pBVS-M2 showed that the SNase A gene was highly expressed in E. coli.Two different types of mutagenesis (the fragment deletion and hase substitution) were also performed simultaneously according to the same methods described above using two mutagerfetic primers. DNA sequencing analysis indicates that the mutagenesis frequency for two different types of mutagenesis with two different primers was 83.3%. This provides an useful approach to make more than one type of mutagenesis with high efficiency at the same time. Some experimental factors which affect mutagenesis frequency were also discussed.