MICROSOFT(R) EXCEL MACROS FOR ANALYZING PLASMID INSTABILITY AND THEIRAPPLICATION TO RECOMBINANT STRAINS OF SACCHAROMYCES-CEREVISIAE

We have developed a set of Microsoft (R) Excel 4 macros for the analys is of the kinetics of segregational plasmid instability. The custom SO LVER macro is used to solve non-linear equations and the error of the constants to derived is determined by the jackknife method. Jackknife analysis is especially useful when working with statistically complex data in situations where the error distribution on the required parame ters is undetermined, even though the distribution on the experimental data is known. The JACKKNIFE macro utilises three component parts to determine values for the rate of plasmid segregation (R) and the growt h rate difference between plasmid-containing and plasmid-free clones ( DELTAmu). Initial values of R and DELTAmu are determined using two lin ear regression methods describing two opposing situations (DELTAmu>R) and (DELTAmu less-than-or-equal-to R). These values are passed on to t he non-linear regression module which also carries out the jackknifing . Although jackknifed non-linear regression is time consuming, paramet ers obtained from jackknifed non-linear regression are robust. The mac ros work on both PC and Macintosh computers equipped with Microsoft Ex cel and can be easily modified to carry out jackknifed non-linear regr ession on other data sets and models. As an example, this method has b een used to determine the effect of the absence of endogenous 2 mum-pl asmid on the stability of a 2mum-based vector (pJB502) inserted into S accharomyces cerevisiae. Improvement in plasmid stability in 2mum-plas mid-free hosts was shown to be completely due to decreasedR even thoug h DELTAmu was the dominant factor in plasmid loss.