Evidence from nuclear sequences that invariable sites should be considered when sequence divergence is calculated

It has long been known, from the distribution of multiple amino acid replacements, that not all amino acids of a sequence are replaceable. More recently, the phenomenon was observed at the nucleotide level in mitochondrial DNA even after allowing for different rates of transition and transversion substitutions. We have extended the search to globin gene sequences from various organisms, with the following results: (1) Nearly every data set showed evidence of invariable nucleotide positions. (2) In all data sets, substitution rates of transversions and transitions were never in the ratio of 2/1, and rarely was the ratio even constant. (3) Only rarely (e.g., the third codon position of beta hemoglobins) was it possible to fit the data set solely by making allowance for the number of invariable positions and for the relative rates of transversion and transition substitutions. (4) For one data set (the second codon position of beta hemoglobins) we were able to simulate the observed data by making the allowance in (3) and having the set of covariotides (concomitantly variable nucleotides) be small in number and be turned over in a stochastic manner with a probability that was appreciable. (5) The fit in the latter case suggests, if the assumptions are correct and at all common, that current procedures for estimating the total number of nucleotide substitutions in two genes since their divergence from their common ancestor could be low by as much as an order of magnitude. (6) The fact that only a small fraction of the nucleotide positions differ is no guarantee that one is not seriously underestimating the total amount of divergence (substitutions). (7) Most data sets are so heterogeneous in their number of transition and transversion differences that none of the current models of nucleotide substitution seem to fit them even after (a) segregation of coding from noncoding sequences and (b) splitting of the codon into three subsets by codon position. (8) These frequently occurring problems cannot be seen unless several reasonably divergent orthologous genes are examined together.      

Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes

Differentiation of Ob1771 preadipocytes to adipocytes wascharacterized by morphological changes and elevated expression of thespecific marker enzyme, glycerol-3-phosphate dehydrogenase. Adifferentiation response substantially more complete and rapid thanthat obtained with insulin and 3,5,3'-triiodothyronine was observed with established inhibitors of adenylyl cyclases:2',5'-dideoxyadenosine (2',5'-dd-Ado),9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or byN⁶-(2-phenylisopropyl)adenosineor 1,3-diethyl-8-phenylxanthine, agonist and antagonist, respectively,for adenosine A₁ receptors. Alsoineffective were IBMX or forskolin, agents that can raise intracellularcAMP levels. Potency of the differentiation response followed the order2',5'-dd-Ado (1-20 µM) > 9-CP-Ade (10-100µM) = 9-Ara-Ade (10-100 µM) >> inosine, consistent withtheir potencies to inhibit adenylyl cyclases. The data suggest thatinhibition of adenylyl cyclase via the P-site and the consequentreduction in cell cAMP levels facilitate the induction ofdifferentiation in Ob1771 cells. The findings raise the questionwhether the known endogenous P-site ligands participate in thedifferentiation response induced by hormones.