Non-clonability correlates with genomic instability: a case study of a unique DNA region.
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 deletions and clones containing 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 flanking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombinant 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 identified in the globin gene domain is unstable (frequently 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.
Institut J., Monod/Université Paris 7, 2 Place Jussieu-tour 43, 75251 CECDEX Paris, France.
S V
SV
Razin
E S
ES
Ioudinkova
E N
EN
Trifonov
K
K
Scherrer
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002875:Chromosomes / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D021901:DNA, Intergenic / Q000235:genetics*; D005057:Eukaryotic Cells; D005914:Globins / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011110:Polymorphism, Genetic; D011387:Prokaryotic Cells; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid
D004251:DNA Transposable Elements; D021901:DNA, Intergenic; D009687:Nuclear Proteins; D005914:Globins