User:Richard.F.Fowler

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Bold textThree Dimensional Higher Order Structures in Satellite DNABold text

Satellite DNA adopts higher-order three dimensional structures in eukaryotic organisms. This was demonstrated in the land crab Gecarcinus lateralis by Richard F. Fowler and coworkers in the lab of Dorothy M. Skinner at Oak Ridge National Laboratory (Fowler, Stringfellow, and Skinner, 1988; Fowler and Skinner, 1986; Stringfellow, Fowler, LaMarca, and Skinner, 1985; Skinner, Fowler, and Bonnewell, 1983; Fowler, Bonnewell, Spann, and Skinner, 1985). Satellite DNA structures are important for their functional role in chromatin structure and gene regulation.

Skinner's lab discovered that G. lateralis DNA contains 3% of a GC-rich sequence consisting of tandem repeats of a ~2100 base pair (bp) repeating unit (RU), the most complex repetitive sequence ever described (Skinner et al., 1982, Bonnewell et al., 1982). The RU is arranged in long tandem arrays with approximately 16,000 copies per genome. Several RU sequences were cloned and sequenced to reveal conserved regions of conventional DNA sequences interspersed with microsatellite repeats, in addition to long runs (20-25 bp) of G and C bases pairs with G on one strand and C on the other (Fowler, Bonnewell, Spann, and Skinner, 1985). The microsatellite repeats were also biased in strand composition in the microsatellite regions with pyrimidines (C,T) on one strand and purines (A,G) on the other (Stringfellow, Fowler, LaMarca, and Skinner, 1985). The most prevalent repeated sequences in the embedded microsatellite regions were CCT/AGG and CCCT/AGGG. The sequence CGCAC/GTGCG was repeated in one microsatellite region in all clones, and that sequence also appeared in a Z-DNA structure within RU (below).

Between the strand-biased microsatellite and GC stretches, all sequence variations retained one or two base pairs with an A residue interrupting the pyrimidines-rich strand and T interrupting the purine-rich strand. This sequence feature was highly distorted as shown by its response to nuclease enzymes (Fowler and Skinner, 1986). Regions consisting of microsatellites with bias in base composition adopted triple-helical structures under superhelical stress and other conditions. Triple-stranded structures imply that intermolecular interactions are modulated by the microsatellite domains and GC stretches.

Other regions of the RU sequence included variations of a symmetrical DNA sequence of alternating purines and pyrimidines shown to adopt a left-handed Z-DNA helical structure in equilibrium with a stem-loop structure under superhelical stress (Fowler, 1986; Fowler, Stringfellow, and Skinner, 1988). The palindromic sequence CGCACGTGCG/CGCACGTGCG, flanked by extended palindromic Z-DNA sequences over a 35 bp domain, adopted a Z-DNA structure with a symmetrical arrangement or alternatively a stem-loop structure centered on the CGCAC/GTGCG motif also seen in a microsatellite embedded within RU (above).

Conserved sequences showed virtually no differences among cloned RU sequences. Variations among cloned RU sequences were characterized by the number of microsatellite repeats, and also by the lengths of C and G stretches where triple stranded structures formed. Other regions of variability among cloned RU sequences were found adjacent to alternating purine and pyrimidine sequences with Z-DNA/stem-loop structures.

One RU sequence was shown to have multiple copies of an Alu repeat inserted into a region bordered by inverted repeats where most copies contained just one Alu sequence (Bonnewell, Fowler, and Skinner, 1983).

This work was one of the earliest indications that DNA analysis might be used to differentiate individual organisms as applied today in forensic identification. The results demonstrated that copy numbers of microsatellite repeats were sites of sequence differences. The authors hypothesized that the variations might be due to the fact that individual sequence variants could have been caused by mixing the DNA of multiple individual animals in order to obtain sufficient quantities of DNA. The research was carried out before PCR was available, and all DNA had to be extracted from natural materials. Attempts were made to perform the analysis on DNA from individual organisms, but sufficient amounts of DNA could not be recovered from individual animals using cesium chloride gradients.

References:

Fowler, R.F., L.A. Stringfellow, and D.M. Skinner (1988). A domain that assumes a Z-conformation includes a specific deletion in some cloned variants of a complex satellite. Gene 71: 165-176.

Fowler, R.F. (1986). Eukaryotic DNA Rich in Alternating Purines and Pyrimidines Adopts an Altered Conformation Similar to Z-DNA. The University of Tennessee, Knoxville, USA.

Fowler, R.F. and D.M. Skinner (1986). Eukaryotic DNA diverges at a long and complex pyrimidine-purine tract that can adopt altered conformations. J. Biol. Chem. 261: 8994-9001.

Stringfellow, L.A., R.F. Fowler, M.E. LaMarca, and D.M. Skinner (1985). Demonstration of remarkable sequence divergence in variants of a complex satellite by molecular cloning. Gene 38: 145-152.

Fowler, R.F., V. Bonnewell, M.S. Spann, and D.M. Skinner (1985). Sequences of three closely related variants of a complex satellite DNA diverge at specific domains. J. Biol. Chem. 260: 8964-8972.

Fowler, R.F. and D.M. Skinner (1985). Cryptic satellites rich in inverted repeats comprise 30% of the genome of a hermit crab. J. Biol. Chem. 260: 1296-1303.

Skinner, D.M., R.F. Fowler, and V. Bonnewell (1983). "Domains of simple sequences or alternating purines and pyrimidines are sites of sequence divergences in a complex satellite DNA" In: Mechanisms of DNA Replication and Recombination (N.R. Cozzarelli, ed.), A.R. Liss, New York. UCLA Symp. Molec. Cell Biol. 10: 849-861.

Bonnewell, V., R.F. Fowler, and D.M. Skinner (1983). An inverted repeat borders a fivefold amplification in satellite DNA. Science 221: 862-865.

Skinner, D.M., V. Bonnewell, and R.F. Fowler (1982). Sites of divergence in the sequence of a complex satellite and several cloned variants. Cold Spring Harbor Symp. Quant. Biol. 47: 1151-1157.