Induction of phenotypically altered mammary epithelial cells by neutrons and gamma rays.

These studies have examined alterations in the in vivo growth properties of mammary epithelial cells isolated at 1, 4, and 16 weeks after in vivo irradiation with -137Cs gamma rays or fission-spectrum neutrons. Altered in vitro growth potential was characterized by the proliferation of epithelial foci (EF) from irradiated animals under conditions in which mammary cells from nonexposed animals senesced. These EF were further characterized based on their ability to be subcultured. Both gamma and neutron irradiation resulted in the appearance of cells capable of forming EF. Further, with increased time in situ between irradiation and cell isolation, the frequency of EF which were capable of being subcultured indefinitely (EFs) increased. Reducing the gamma-ray dose rate resulted in fewer EFs while reducing the neutron dose rate resulted in increased frequencies of EFs. These data confirm earlier observations following gamma irradiation and show these cellular changes are also observed following neutron irradiation. In addition, these data indicate that changes in dose rate primarily influence the emergence of immortalized cell populations.     

Consistencies of individual DNA base-amino acid interactions in structures and sequences.

Amino acid-amino acid interaction energies have been derived from crystal structure data for a number of years. Here is reported the first derivation of normalized relative interaction from binding data for each of the four bases interacting with a specific amino acid, utilizing data from combinatorial multiplex DNA binding of zinc finger domains [Desjarlais, J. R. and Berg, J. M. (1994) Proc. Natl. Acad. Sci. USA, 91, 11099-11103]. The five strongest interactions are observed for lysine-guanine, lysine-thymine, arginine-guanine, aspartic acid-cytosine and asparagine-adenine. These rankings for interactions with the four bases appear to be related to base-amino acid partial charges. Also, similar normalized relative interaction energies are derived by using DNA binding data for Cro and lambda repressors and the R2R3 c-Myb protein domain [Takeda, Y., Sarai, A. and Rivera, V. M. (1989) Proc. Natl. Acad. Sci. USA, 86, 439-443; Sarai, A. and Takeda, Y. (1989) Proc. Natl. Acad. Sci. USA, 86, 6513-6517; Ogata, K. et al. (1995) submitted]. These energies correlate well with the combinatorial multiplex energies, and the strongest cases are similar between the two sets. They also correlate well with similar relative interaction energies derived directly from frequencies of bases in the bacteriophage lambda operator sequences. These results suggest that such potentials are general and that extensive combinatorial binding studies can be used to derive potential energies for DNA-protein interactions.     

Evolution of eutherian cytochrome c oxidase subunit II: heterogeneous rates of protein evolution and altered interaction with cytochrome c

Cytochrome c oxidase subunit II (COII), encoded by the mitochondrial genome, exhibits one of the most heterogeneous rates of amino acid replacement among placental mammals. Moreover, it has been demonstrated that cytochrome c oxidase has undergone a structural change in higher primates which has altered its physical interaction with cytochrome c. We collected a large data set of COII sequences from several orders of mammals with emphasis on primates, rodents, and artiodactyls. Using phylogenetic hypotheses based on data independent of the COII gene, we demonstrated that an increased number of amino acid replacements are concentrated among higher primates. Incorporating approximate divergence dates derived from the fossil record, we find that most of the change occurred independently along the New World monkey lineage and in a rapid burst before apes and Old World monkeys diverged. There is some evidence that Old World monkeys have undergone a faster rate of nonsynonymous substitution than have apes. Rates of substitution at four-fold degenerate sites in primates are relatively homogeneous, indicating that the rate heterogeneity is restricted to nondegenerate sites. Excluding the rate acceleration mentioned above, primates, rodents, and artiodactyls have remarkably similar nonsynonymous replacement rates. A different pattern is observed for transversions at four-fold degenerate sites, for which rodents exhibit a higher rate of replacement than do primates and artiodactyls. Finally, we hypothesize specific amino acid replacements which may account for much of the structural difference in cytochrome c oxidase between higher primates and other mammals.      

Solvation thermodynamics of biopolymers. I. Separation of the volume and surface interactions with estimates for proteins

The present paper is a systematic first approach to the problem of solvation thermodynamics of biomolecules. Most previous approaches have been only crude estimates of solvent contributions, and have simply assessed solvation free energy as proportional to surface areas. Here we estimate the various contributions and divide them into (a) hard-core interactions dependent upon the entire volume of solute and (b) the remainder of interactions manifested through surfaces, such as van der Waals, charge-charge, or hydrogen bonds. We have estimated the work to create a cavity with scaled-particle theory (SPT), the van der Waals interactions on the surface, and hydrogen bonds between the surface and the solvent. The conclusion here is that this latter term is the largest component of the solvation free energy of proteins. From estimates on nine diverse proteins, it is clear that the larger the protein, the more dominant is the hydrogen-bond term. In the next paper, we indicate that correlations between hydrogen-bonding groups on the surfaces could increase the magnitude of the hydrogen-bond contribution.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Paired sequence difference in ribosomal RNAs: evolutionary and phylogenetic implications

Ribosomal RNAs have secondary structures that are maintained by internal Watson-Crick pairing. Through analysis of chordate, arthropod, and plant 5S ribosomal RNA sequences, we show that Darwinian selection operates on these nucleotide sequences to maintain functionally important secondary structure. Insect phylogenies based on nucleotide positions involved in pairing and the production of secondary structure are incongruent with those constructed on the basis of positions that are not. Furthermore, phylogeny reconstruction using these nonpairing bases is concordant with other, morphological data.      

Sequence context of oligomer tracts in eukaryotic DNA: biological and conformational implications

Recent studies of homooligomer tracts suggest different characteristics from random sequence DNA (dA).(dT) and (dG).(dC) tracts are frequent in upstream regions and in some cases have been shown to be essential for regulation. Here we examine homooligomer occurrences in non-coding and coding eukaryotic sequences, focusing on the context in which the homooligomers occur. This analysis of sequences in the junction areas yields distinct and consistent characteristics. In particular, the nucleotide interrupting a run is most frequently complementary to the run. The base next to it is most frequently identical to the one constituting the run. For A or T runs the least frequent nearest and next to nearest neighbors are G or C. For G or C tracts the least frequent are A or T. Complementary oligomers behave similarly. These and additional trends are strongest for run lengths greater than or equal to 3. The computations are carried out on the whole eukaryotic database of greater than 4 x 10(6) nucleotides, separately for coding and non-coding regions. These same trends are evident for both groups, but are somewhat stronger for the non-coding regions. The context in which the homooligomers occur may yield some clues to DNA conformation and its biological implications.