Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.

An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.     

Divergence estimates and early evolutionary history of Figitidae (Hymenoptera: Cynipoidea)

We examine the phylogenetic relationships of Figitidae and discuss host use within this group in light of our own and previously published divergence time data. Our results suggest Figitidae, as currently defined, is not monophyletic. Furthermore, Mikeiinae and Pycnostigminae are sister‐groups, nested adjacent to Thrasorinae, Plectocynipinae and Euceroptrinae. The recovery of Pycnostigminae as sister‐group to Mikeiinae suggests two major patterns of evolution: (i) early Figitidae lineages demonstrate a Gondawanan origin (Plectocynipinae: Neotropical; Mikeiinae and Thrasorinae: Australia; Pycnostigminae: Africa); and (ii) based on host records for Mikeiinae, Thrasorinae and Plectocynipinae, Pycnostigminae are predicted to be parasitic on gall‐inducing Hymenoptera. The phylogenetic position of Parnips (Parnipinae) was unstable, and various analyses were conducted to determine the impact of this uncertainty on both the recovery of other clades and inferred divergence times; when Parnips was excluded from the total evidence analysis, Cynipidae was found to be sister‐group to [Euceroptrinae + (Plectocynipinae (Thrasorinae + (Mikeiinae + Pycnostigminae)))], with low support. Divergence dating analyses using BEAST indicate the stem‐group node of Figitidae to be c. 126 Ma; the dipteran parasitoids (Eucoilinae and Figitinae), were estimated to have a median age of 80 and 88 Ma, respectively; the neuropteran parasitoids (Anacharitinae), were estimated to have a median age of 97 Ma; sternorrhynchan hyperparasitoids (Charipinae), were estimated to have a median age of 110 Ma; the Hymenoptera‐parasitic subfamilies (Euceroptinae, Plectocynipinae, Trasorinae, Mikeiinae, Pycnostigminae, and Parnipinae), ranged in median ages from 48 to 108 Ma. Rapid radiation of Eucoilinae subclades appears chronologically synchronized with the origin of their hosts, Schizophora (Diptera). Overall, the exclusion of Parnips from the BEAST analysis did not result in significant changes to divergence estimates. Finally, though sparsely represented in the analysis, our data suggest Cynipidae have a median age of 54 Ma, which is somewhat older than the age of Quercus spp (30–50 Ma), their most common host.     

Nucleolar changes in liver before the onset of deoxyribonucleic acid replication.

A mixture of chemicals was previously devised (3, 3', 5'-triiodo-L-thyronine, amino acids, a butyryl derivative of cyclic adenosine 3':5'-monophosphate, theophylline, and heparin) that induces nuclear DNA replication in the liver of the unoperated rat (Short, J., Tsukada, K., Rudert, W.A. 7 Lieberman, I. (1975) J. Biol. Chem. 250, 3602-3606). The stimulation of DNA synthesis with the complete solution is greater than the sum of the responses to the thyroid hormone alone and to a mixture of the remaining components of the inductive solution alone. The effects of the complete mixture and of parts of it on three parameters involving the hepatocyte nucleolus have now been examined in intact animals. The complete solution increases the level of RNA polymerase I (measured with isolated nuclei), the rate of ribosome synthesis, and the total volume of nucleoli per nucleus. Nucleolar hypertrophy is unique among the three changes in showing a requirement, just as for DNA synthesis, for all or almost all of the components of the complete mixture, including the thyroid hormone, for a maximal effect. Enlargement of nucleoli is detectable as early as 2 hours after the start of treatment with the complete mixture and a large proportion of the total hepatocyte population is involved. Hypertrophy is accompanied by an increase in nucleolar RNA content. N2-Monobutyryl cyclic guanosine 3':5'-monophosphate is not able to substitute for the cyclic adenine nucleotide.