The structure of M6, a recombinant plasmid containing Dictyostelium DNA homologous to actin messenger RNA

The recombinant plasmid M6 contains a DNA sequence from the cellular slime mold Dictyostelium discoideum which hybridizes to actin messenger RNA. The plasmid contains 6 kilobase pairs (kb) of Dictyostelium DNA inserted into a pMB9 vector. Ten cleavage sites for four different restriction enzymes have been mapped. Other work has shown that a central restriction fragment, 1.7 kb in length, contains sequences repeated about fifteen times in the genome, and that this fragment hybridizes to actin mRNA. Heteroduplexes between M6 and pDd actin 2, a chromosomal plasmid which contains two copies of the actin repeated sequence, were used to define the position of this repeat in M6. Two plasmids with inserts of cDNA made from actin mRNA were heteroduplexed to M6 to define the position and orientation of the message complementary region. This orientation was confirmed by inserting the fragment into phage λ and determining which of the separated λ strands was complementary to actin mRNA. An electron microscope technique has been developed for identifying poly(dA) sequences by hybridizing to them dBrU polymers attached to suitable markers. The mapping of the (dA) tracts that occur in the Dictyostelium insert of M6 is described here. The positions of the A:T tracts do not correlate in any simple way with the position of the actin gene sequence.