Abstract
The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG.CAG)(n) in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG.CAG), (CGG.CCG), (GAA.TTC), (GTC.GAC), and (GTG.CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG.CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG.CAG regions. Furthermore, inversion products of the type [(CTG)(13)(CAG)(67)].[(CTG)(67)(CAG)(13)] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG.CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.
Highlights
Genetic instabilities of triplet repeat sequences (TRS)1 ((CTG1⁄7CAG), (CGG1⁄7CCG), or (GAA1⁄7TTC)) are a hallmark of certain hereditary neurological diseases [1, 2]
Since the other intragenic markers are located between the markers (CGG1⁄7CCG) and FMRb, this results in patches of normal and fragile X sequence in the FMR1 gene of the daughter and was explained on the basis of gene conversion
Interplasmid Recombination—To evaluate the potential role of homologous recombination in the expansion of TRS, a twoplasmid system was established in E. coli. (The term recombination is used in a general sense and includes gene conversion, unequal crossing-over, and sister chromatid exchange.) For our study, one family of plasmids (Fig. 1) was a derivative of pUC19 that contains the unidirectional ColE1 origin of replication and harbors the ampicillin resistance gene [20]
Summary
Plasmids—The plasmids used in these experiments are derivatives of the unidirectionally replicating pUC19 and pACYC184. PRW4100 and pRW4110 were constructed by digesting pRW3481 and pRW3463, respectively, with PvuII and recloning the fragments containing the triplet repeats into the PvuII sites of pACYC184. For the experiments involving cotransformation, cells were prepared for electrotransformation, and the transformation mixture contained various combinations of the pUC19 and pACYC184 derivatives. Cotransformants were selected on LB agar plates containing ampicillin (amp) and tetracycline (tet) since pUC19 and pACYC184, respectively, harbor these drug resistance genes. The cells were plated on LB agar that contained ampicillin (75 g/ml) and tetracycline (12 g/ml) and grown for 4 –16 h at 37 °C. E. coli AB1157 and JC10289 were washed and diluted to a concentration of 5 ϫ 107 cells/ml and cotransformed with combinations of plasmids as indicated: for example, pUC19 ϩ pRW3239; pRW3080 ϩ pACYC184; or pRW3080 ϩ pRW3239
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