IDENTIFICATION AND CLONING OF FULL LENGTH MARINER TRANSPOSON FROM WILD SILKMOTHS AND UZI FLY

MAY 6-JULY 6 1999

CENTRE FOR DNA DIAGNOSTICS AND FINGERPRINTING

GUIDE:Dr.J.Nagaraju

SUMMER RESEARCH AS AN MSc STUDENT

The mariner family of transposable elements was being considered for use as a vector in silkmoth transgenesis as they are independent of host specific factors for transmission. The aim of my study was to identify and clone full length(~1.2kb with 30bp inverted terminal repeats)mariner elements from wild silkmoths. PCR amplifications were done for the 490bp conserved region of A.proylei, A.roylei, A.assama, Philosamia cynthia ricini (eri) and Uzi fly with the following primers

MAR124F TGGGTNCCNCAYGARYT (17mer, 128-fold degenerate)

MAR276R GGNGCNARRTCNGGNSWRTA (20mer, 4096-fold degenerate)

20-30 ng of genomic DNA was taken in a final volume of 20ml with 200mM dNTPs, 8mM of each primer, 50mM KCl, 10mMTris-Cl, 1.5mM MgCl2 and MBI Taq polymerase. Conditions were:

For the 1.2kb region of A.roylei 2.5mM MgCl2 and single primer MTRP TAGGTCCTTACCTATGAAATTGCCGTTT (28mer for inverted terminal repeats) was used and other conditions were maintained as such. For the 1.2kb region of Eri, B.mandarina and Uzi fly, touchdown PCR was done as they gave multiple bands with regular PCR

These were resolved on LMP agarose and the required 490bp and 1.2kb fragments were eluted.DNA samples were then ligated with pMos Blue vector and transformed into DH5a Rubidium chloride competent cells using heat shock method.White colonies were selected with IPTG/Xgal and the insert presence was verified by PCR amplification with M13 forward and reverse primers.The clones containing insert were cultured in LB ampicllin liquid broth and purifird DNA was used for sequencing.Sequencing was done in an ABI Prism 377 Automated DNA Sequencer with Big Dye Terminator sequence kit.

SOUTHERN BLOTTING Genomic DNA samples were resolved on 1% aarose gel and transferred to Hybond N+ membrane (Amersham) using capillary transfer and UV crosslinked.The probe (radiolabelled with aP32 using random primer labelling kit LCK2) used was 490bp PCR fragment of A.mylitta obtained using degenerate primers. PCR amplification and visualization with ethidium bromide indicated the possible presence of 1.2kb and 490bp region in all silkmoths and in Uzi fly. Multiple bands were observed in B.mandarina while a band slightly lower than 1.2kb was obtained for Philosamia cynthia ricini. Exorista bombycis(uzi fly) also gave multiple bands of various lengths. Southern blotting and Hybridization verified the presence of 1.2kb region in all silkmoths except in A.assama. A.roylei and A.proylei gave high intensity bands. Three PCR amplified products of A.roylei , cloned into pMos Blue vector were found to contain the required insert. Confirmation of insertion was done by restriction digestion with SalI and stark mobility differences between Pks+SalI and control. A 490 bp sequence of Uzi fly was sequenced directly with Degenerate F primer and a partial 100bp sequence was obtained.