Cloning of maize snap-back DNA

We are cloning and characterizing maize snapback DNA, that portion of the DNA reassociating at a Cot of 10-4 -10-5 M sec l-1. We expect to find several classes of snap-back sequences, including those associated with the inverted repeats of transposable elements.

Maize DNA was partially digested with HaeIII (300 ng DNA, 0.2 u HaeIII µg-1 for 30 min), which yielded DNA fragments with an average length of 20 kb. These fragments were denatured in 0.15 M NaOH at 65 C for 15 min, then renatured by adding an equal volume of 0.15 M HCl, 1 mM Tris-Cl pH 7.0 at 65 C, and quenched in ice at a Cot< 10-4 M sec l-1. Ice cold 2X S, nuclease buffer (as specified by BRL) was added along with 43 u S1 nuclease per ug DNA and the mix incubated for 4 hrs at 37 C. This treatment generated approximately 300 ng of S1 - resistant DNA <400 bp long. S1 nuclease should generate blunt-ended duplexes; however, for cloning it was necessary to fill in remaining single-stranded ends using T4 polymerase (4 u, 15 min, 37 C). This DNA was ligated into the SmaI site of the plasmid pUC18, and used to transform E. coli strain JM83. The transformation efficiency was 5.6 x 105 ampicillin-resistant, b-gal- clones per microgram snap-back DNA.

Ten clones, with insert sizes ranging from 42 to 200 bp, are currently being analyzed. The 200 bp insert from one of the clones, pCTE1409, has been used to probe a Southern blot of maize, teosinte, rice and petunia DNA. The banding pattern indicates that this clone represents sequences of variable copy number in different maize lines and present in at least one copy in all the genomes tested.

Kevin C. McElfresh and Judy Strommer
 
 


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