instability of eukaryotic DNA in constructs propagated in prokaryotic hosts
is a frequently observed phenomenon. With the exception of a very high A T-content and the presence of multiple repetitions, no general rule at the
basis of this phenomenon is actually known. The intergenic spacer located
between the pi and alpha (D) chicken alpha-type globin genes is frequently
deleted from recombinant phages and plasmids. Here we have cloned this DNA
fragment using a specially designed bacterial strain (SURE competent cells,
Stratogene). Comparative analysis of DNA of recombinant clones bearing del
etions and clones containing the intact genomic DNA fragment has revealed t
wo important DNA sequence motifs that contribute to the unclonability of eu
karyotic DNA in prokaryotic cells. First, the similarity to bacterial trans
posons (i.e. the presence of repeats flanking a several kilobase DNA fragme
nt) may cause the loss of the fragment during propagation of the recombinan
t DNA in E. coli. Second, a high content of rotationally correlated kinkabl
e elements (TG*CA steps) may result in non-clonability of the DNA sequence,
interestingly, the latter type of "unclonable" DNA sequence motifs identif
ied in the globin gene domain is unstable (frequently rearranged) also in t
he eukaryotic chromosome resulting in a local polymorphism. In the chicken
domain of alpha globin genes this unstable DNA sequence seems to be partial
ly protected by interaction with nuclear matrix proteins. (C) 2001 Academic
Press.