COMMUNICATION Non-clonability Correlates with Genomic Instability: A Case Study of a Unique DNA Region Sergey V. Razin 1,2 , Elena S. Ioudinkova 1,2 , Edward N. Trifonov 3 and Klaus Scherrer 1 * 1 Institut J. Monod/Universite  Paris 7, 2 Place Jussieu-tour 43, 75251 Paris CEDEX 05 France 2 Institute of Gene Biology Russian Academy of Sciences Vavilov Str. 34/5, 117334 Moscow, Russia 3 The Weizmann Institute of Science, Rehovot, Israel, and Genome Diversity CenterInstitute of Evolution University of Haifa, Haifa 31905, Israel 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 p and a D chicken alpha-type glo- bin 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 anal- ysis of DNA of recombinant clones bearing deletions and clones con- taining the intact genomic DNA fragment has revealed two important DNA sequence motifs that contribute to the unclonability of eukaryotic DNA in prokaryotic cells. First, the similarity to bacterial transposons (i.e. the presence of repeats ¯anking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombi- nant DNA in E. coli. Second, a high content of rotationally correlated kinkable elements (TG*CA steps) may result in non-clonability of the DNA sequence. Interestingly, the latter type of ``unclonable'' DNA sequence motifs identi®ed in the globin gene domain is unstable (fre- quently rearranged) also in the eukaryotic chromosome resulting in a local polymorphism. In the chicken domain of alpha globin genes this unstable DNA sequence seems to be partially protected by interaction with nuclear matrix proteins. # 2001 Academic Press Keywords: globin genes; ``unclonable'' DNA; kinkable dinucleotide steps; genomic instability; nuclear matrix *Corresponding author From the onset of molecular genetics it became clear that some eukaryotic sequences are unclonable in prokaryotic systems (Kang & Cox, 1996; Russo et al., 1996; Shirsat et al., 1992; Voet et al., 1997; Wyman et al., 1985). Although the problem is widely recognized, especially by specialists involved in large-scale genome sequencing projects, little is known about the nature of ``unclonable'' sequences, except for a few very speci®c cases (Bieth et al., 1997; Kang & Cox, 1996). There are well known cases of unclonability of eukaryotic DNA; these include the presence of multiple repeats (Kang & Cox, 1996; Muller et al., 1985), A  T-rich sequences and sequences capable of forming non-canonical secondary structures (i.e. z-DNA, palindromes, etc.). In some rare cases unclonability was explained by the fortuitous presence in eukary- otic DNA of prokaryotic-type regulatory elements (Bieth et al., 1997). Taken together, all these cases cannot, however, account for the relatively large portion of unclonable sequences in an average eukaryotic genome (Tang & Waterman, 1990). Analysis of unclonable DNA sequences determined using PCR-based approaches has not yet revealed any common characteristic feature. The results of the present study suggest that an important feature of unclonable DNA sequences is the presence of rotationally correlated kinkable dinucleotide steps. E-mail address of the corresponding author: Scherrer@ijm.jussieu.fr doi:10.1006/jmbi.2000.4372 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 307, 481±486 0022-2836/01/020481±6 $35.00/0 # 2001 Academic Press