Biomedical subjects
Publications and source records attributed to R F Fowler.
New technologies for multiple chemical synthesis of oligonucleotides and stepwise hybridization on a solid-phase support enable the rapid and cost-effective preparation of long duplex DNA regions. Will these new technologies usher in a new era in protein engineering?
Sequence analyses show that deletions of 10 and 12 bp occur at homologous sites in a domain that is rich in alternating purines and pyrimidines (Pu/Py) in B42 and EXT, two cloned variants of a complex satellite DNA. A 3-bp deletion occurs 27 bp upstream from the site of the specific deletions in B42 and RU, a third cloned satellite variant that has not suffered the 10-bp deletion. Under torsional stress, the Pu/Py-rich domain adopts a Z-conformation as shown by (i) inhibition of cutting at a BssHII site that accounts for 2/5 of a 15-bp tract of pure Pu/Py in the domain; (ii) binding of polyclonal and monoclonal anti-Z-DNA antibodies to the domain; and (iii) antibody stabilization and subsequent relaxation of the Z-region.
A domain exhibiting major sequence divergences among three cloned repeat units of a complex satellite DNA of the Bermuda land crab contains a repetitive polypyrimidine.polypurine segment consisting of a long C.G tract embedded between runs of CCT.AGG and CGCAC.GTGCG and their variations. The domain adopts at least two types of altered conformations that are markedly affected by pH and negative superhelical density; only one is sensitive to ionic strength. Supercoil-dependent distortions in helical structure are most pronounced at points of interruption in compositional bias in this domain and a similar although less extensive, divergent domain nearby. Since the domain is the site of major sequence divergences among individual satellite repeat units, the altered conformations may be involved in site-specific recombination between repeat units, either those arranged in tandem or those scattered throughout the genome.
Major differences among the sequences of the repeat units of a very complex satellite DNA are located in domains which are sensitive to S1 nuclease under torsional stress, indicating that the domains assume unusual secondary or tertiary structures. Repeat units of the satellite, which accounts for 3% of the DNA of a land crab, have been inserted into pBR322 and the primary sequences of three cloned variants determined. The variants selected for sequencing include 1) RU (2089 base pairs (bp) ), representative of the average size of repeat units of cellular satellite; 2) TRU (1674 bp), truncated at an extra EcoRI site; and 3) EXT (2639 bp), extended by a 5-fold amplification of a 142-bp segment, one copy of which is present in RU and TRU (Bonnewell, V., Fowler, R.F., and Skinner, D.M. (1983) Science 221, 862-865). It appears that every copy of the satellite may be different and that the variants do not arise from cloning accidents. Extensive domains, as long as approximately 560 bp, are greater than 95% homologous among RU, TRU, and EXT; these conserved domains are composed of DNA whose base composition and sequences do not have remarkable features. By contrast, the sequences that comprise the divergent domains are unusually rich in 1) tracts of (dG X dC) 13-23 and arrangements of similar but not identical repetitive oligonucleotides or 2) alternating purines and pyrimidines (pu/py).
One major very highly repeated (VHR) DNA (approximately 7 X 10(6) copies/genome; repeat unit = 156 base pairs (bp)), a family of three minor VHR DNAs (approximately 2.8 X 10(6) copies/genome; repeat units = 71-74 bp), and a number of trace components account for almost 30% of the genome of a hermit crab. The repeat units of the three minor variants are defined by identical 14-bp G + C-rich inverted repeats that might form cruciforms. Two copies of the repeat unit (CCTA) of one of two patent satellites of this crab (Skinner, D. M., and Beattie, W. G. (1974) Biochemistry 13, 3922-3929; Skinner, D. M., Beattie, W. G., Blattner, F. R., Stark, B. P., and Dahlberg, J. E. (1974) Biochemistry 13, 3930-3937) occur at the center of one in seven of the G + C-rich inverted repeats; copies of the other patent satellite (Chambers, C. A., Schell, M. P., and Skinner, D. M. (1978) Cell 13, 97-110) are found in main component DNA. The sequences of both the major and minor VHR DNAs are characterized by short tracts of An and/or Tn (n = 4-7) residues whose presence would permit the formation of perfectly matched stems separated by loops of 8-16 bp. The An and/or Tn tracts are interspersed with segments of G + C-rich DNA and are arranged differently in the major and minor VHR DNAs. Although the repeat units of the major and the three minor VHR DNAs are arranged in tandem, the composition and sequence of their bases are such that they do not form distinct bands in CsCl gradients; they are cryptic satellites.
Repeat units of a complex G + C-rich satellite of the Bermuda land crab have been cloned by insertion into either the PstI or EcoRI site of pBR322 or the EcoRI site of pUC9. While most of the recombinants contained inserts of approx. 2.1 kb, the average size of repeat units seen in cellular satellite digests, several inserts were markedly different in size. Two domains that account for major sequence differences among the satellite variants and that may be 'hotspots' for sequence modification have been subcloned to permit characterization of their secondary and tertiary structures independent of the influence of the other unusual sequences present. One of these domains is striking in its content of simple repeats; one strand is highly biased in pyrimidines which may permit the formation of unusual secondary and/or tertiary conformations. The other subcloned domain is rich in Pu/Py; preliminary data indicate a transition from B----Z DNA in this region.