t chnical tips Cosmid rescue strategy This co, mid rescue approach to the isolation of eukaryotic genes after gene transfer into animal cells is claimed to have the advantage that it relies solely on genetic selection to isolate recombinants, avoids the requirement for the existence of molecular probes to identify the clone of interest,
TIGand also allows the isolation of large stretches of DNA. Mouse Lewis lung tumor DNA was ligated to a cesmid containing a geneticin/kanamycin resistance gene and transferred into NIH3T3 cells. Recipient cells were first selected for geneticin resistance and subsequently for their ability to grow as a tumor when injected into nude mice.
Sequence analysis package put. The programs are modular Several features not available with other packages are claimed to be included in this set of programs, which provides tools for detailed primary analysis of nucleotide and amino acid sequences. Screen graphics are used extensively to enhance several types of out-
and easily adapted for individual needs. The programs, which can be integrated with other packages to combine abilities, are menu driven, autoinitializing, and self-prompting; they fit on one DSDD diskette (help and tutorial are on a second diskette) to eliminate diskette shuffling. This sequence analysis pack-
By repeating this transfection procedure with DNA from resultant tumors, geneticinresistant NIH3T3 cells were obtained which are tumorigenic and contain ~ I-5 copies of the transferred cosmid. The functional oncogene was cloned by preparing cosmid libraries of third-round tumor DNAs, using a cos,mid which does not contain a kanamvcin-resistance gene.
age was designed primarily for use with sequences generated using the Maxam-Gilbert sequencing method, but it is also useful for analysis of Sanger sequencing fragments and published sequences. Emphasis has been placed on the user's ability to rapidly obtain information pertinent to further sequencing (e.g. restriction mapping) or identification of the
I s o l a t i o n o f poly (A)RNA o n oligo(dT)-cellulose c o l u m n s brium of the column to give a flow rate of less than 2 ml/min for a column containing 1 g of Chromatography on oligo(dT)- report shows that an incom- oligo(dT)-cellulose. This setcellulose has become the plete binding of poly~A)RNA to ting is maintained during the m~hod of choice for the olig~dT)-celluloseis caused by application of the sample to the separation of poly(A) RNA flow rates that can be readily column. These conditions give from non-polyadenylatedRNA. achieved when the column is a good yield of poly(A) RNA. Several parameters have been run by gravity elution. To avoid This procedure has the advanevaluated but flow rate has this, the level of the exit tubing tages that constant readjustreceived little attention. This is adjusted during the equili- ments of the flow rate are not
Owing to the original linkage'of the oncogene with the cosmid containing the kanamycinresistance gene. a series of kanamycin-resistant cosmids were isolated. G. Brady, A. Funk, J. Mattem, G. Schfitz and R. Brown(1985) Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences. EMBO J. 4, 2583- 2588
fragment (e.g. translation, frame scanning, and homology searches), as well as user protection from data loss, and ease of operation.
Robert M. Stephens (1985). A sequencer's sequence analysis package for the IBM PC. Gene Anal T~ch 2. a7-75
necessary and the improved retention avoids the necessity of passing the RNA samples over multiple columns. S. Nadin-l)avis and V. A. Mezl (1985) Effect of flowrate on the isolation d polyadenyla~d RNA o~ oli~dT)-cellulose columns,f B/ochem. Biophys. Metha~ 11, 185--189