Cloning, characterization, and properties of seven triplet repeat DNA sequences

Keiichi Ohshima, Seong Man Kang, Jacquelynn E. Larson, Robert D. Wells

Research output: Contribution to journalArticle

89 Citations (Scopus)

Abstract

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG·CAG, CGG·CCG, or AAG·CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA·TAC, GAT·ATC, GTT·AAC, CAC·GTG, AGG·CCT, TCG·CGA, and AAG·CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D. (1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA-TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of ~16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG·CTT and AGG·CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non- B structures. However, long tracts of TCG·CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG·CAG and CGG·CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D. (1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D. (1996) J. Biol. Chem. 271, 2875-2878) on CTG·CAG, CGG·CCG, and TTA·TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreich's ataxia based on intermolecular triplex formation is proposed.

Original languageEnglish
Pages (from-to)16773-16783
Number of pages11
JournalJournal of Biological Chemistry
Volume271
Issue number28
DOIs
Publication statusPublished - 1996 Jul 30
Externally publishedYes

Fingerprint

Trinucleotide Repeats
Cloning
DNA sequences
Organism Cloning
DNA
Neurodegenerative diseases
Friedreich Ataxia
Nucleic Acid Conformation
Neuromuscular Diseases
Agar Gel Electrophoresis
Electrophoresis, Gel, Two-Dimensional
DNA-Directed DNA Polymerase
Electrophoresis
Polymorphism
DNA Sequence Analysis
Oligonucleotides
Neurodegenerative Diseases
Sepharose
Escherichia coli
Conformations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning, characterization, and properties of seven triplet repeat DNA sequences. / Ohshima, Keiichi; Kang, Seong Man; Larson, Jacquelynn E.; Wells, Robert D.

In: Journal of Biological Chemistry, Vol. 271, No. 28, 30.07.1996, p. 16773-16783.

Research output: Contribution to journalArticle

Ohshima, Keiichi ; Kang, Seong Man ; Larson, Jacquelynn E. ; Wells, Robert D. / Cloning, characterization, and properties of seven triplet repeat DNA sequences. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 28. pp. 16773-16783.
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