No abstract

Abstract. Catabolite repression is defined as the inhibition of enzyme induction by glucose or related substances. In the bacterium E. coli, the effect of glucose appears to be due to a lowering of the cyclic AMP level. A DNAdirected cell-free system for #3-galactosidase synthesis has served as a model system for studying the mechanism of action of cyclic AMP. Previously, it was reported that in this system cyclic AMP is required for normal initiation of mRNA synthesis. A protein factor which acts in conjunction with the cyclic AMP has been partially purified. This protein factor has a high affinity for cyclic AMP. These and other results presented herein lead us to the conclusion that cyclic AMP and a protein factor called the catabolite gene activator protein are part of a positive control system for activating catabolite-sensitive genes.

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages.Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNAAGG in the cell to become sequestered in translating peptidyl_tRNAAGG_mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome to frameshift, hop, or terminate translation.Most amino acids are encoded by more than one codon, and frequencies of use of synonymous codons tend to reflect the relative abundance of the tRNAs that recognize them (7,29). Synonymous codons may be translated at different rates (16,17) MATERUILS AND METHODSCodon test plasmids. Plasmids pCT1, pCT2, and pCT3 ( Fig. 1) were assembled from elements of pET vectors (13,24) and of T7 DNA (6). Elements were assembled by using a natural XbaI site (T'CTAGA, where the primer indicates the position of cleavage) between the 410 promoter and the slO translation initiation region for the gene 10 major capsid protein of T7, a natural NdeI site (CA'TATG) at the slO initiation codon, and newly introduced XbaI or NheI (G'CTAGC) sites at the ends of elements. Plasmids were assembled in modular fashion by fusions of compatible ends of DNA fragments from plasmids carrying individual elements or combinations of elements. Most of the fusions were between fragments that had one end at the PstI site in the on May 7, 2018 by guest

Oriented fibres of extracted nuclcohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tctroxide solution at pH 6, containing l0 -2 M Ca ++, and embedding in Araldite enabled sections of the fibres to be cut in which the oricntation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqucous solution. Strongest staining of whole tissue was obtained with very short fixation times--5 minutes or so at 0°C. Particularly intensc staining was obtaincd when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissucs fixed for longer times (~ hour to 2 hours, at 0°C or 20°C), in briefly fixed material the DNA-containing regions appearcd relatively unstaincd by lcad hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.