Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells.

Journal ArticleWe have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity

Polypetide chain termination. Purification of the release factors, R1 and R2, from Escherichia coli.

Journal ArticleWe have extensively purified the release factors RI and Rz from Escherichia coli. These proteins can each mediate polypeptide chain termination. The physiological substrate for this reaction is a completed polypeptide chain in a peptidyl- transfer RNA-messenger RNA-ribosome complex. The reaction consists of recognition of a chain-terminating signal in the mRNA and hydrolysis of the peptidyl-tRNA ester bond, releasing the polypeptide chain. For the purification we were guided by two kinds of assay, the hexapeptide release assay and the formylmethionme release assay, each named according to the molecule released in a model reaction analogous to physiological chain termination. The two factors have different codon specificities, RI acting in response to UAG or UAA, and Re in response to UGA or UAA. Both factors were purified from a l-kg batch of frozen E. coli MRE600 by a scheme which carried the material through four steps before reaching a branch point at the Bth step, when RI- and R2-rich fractions were produced. The two fractions were then treated similarly but separately through three more steps. The products were studied by polyacrylamide gel lectrophoresis using both routine and sodium dodecyl sulfate techniques. For the 6.4 mg of purified RI, we estimate 85% purity after 2,000-fold purification. For the 9.4 mg of purified Rz, we estimate 99% purity after 1,500-fold purification. Our results indicate that each release factor consists of a single polypeptide chain with a molecular weight of 44,000 for RI and 47,000 for Rz. We calculate that there are about 500 molecules of RI and 700 molecules of Rz per E. coli cell

Swaying is a mutant allele of the proto-oncogene Wnt-1

Journal ArticleMice homozygous for the recessive mutation swaying (SW) are characterized by ataxia and hypertonia, attributed to the malformation of anterior regions of the cerebellum. We show that SW is a deletion of a single base pair from the proto-oncogene Wnf-1. The deletion is predicted to cause premature termination of translation, eliminating the carboxy-terminal half of the Writ-1 protein. Histological examination shows that SW is phenotypically identical to a previously described writ-1 mutation introduced into mice by gene targeting

Epoxy/ graphene nanocomposites – processing and properties: a review

Graphene has recently attracted significant academic and industrial interest because of its excellent performance in mechanical, electrical and thermal applications. Graphene can significantly improve physical properties of epoxy at extremely small loading when incorporated appropriately. Herein, the structure, preparation and properties of epoxy/graphene nanocomposites are reviewed in general, along with detailed examples drawn from the key scientific literature. The modification of graphene and the utilization of these materials in the fabrication of nanocomposites with different processing methods have been explored. This review has been focused on the processing methods and mechanical, electrical, thermal, and fire retardant properties of the nanocomposites. The synergic effects of graphene and other fillers in epoxy matrix have been summarised as well

Location and function of retroviral and SV40 sequences that enhance biochemical transformation after microinjection of DNA.

Journal ArticleBiochemical transformation of thymidine-kinase-deficient (TK-) mouse L cells is enhanced 20 to 40 fold when microinjected plasmid DNA contains regions of the genomes of Rous sarcoma virus or simian virus 40 in addition to the complete herpes simplex virus tk gene, irrespective of the orientation and location of the enhancer. The enhancing sequence of RSV DNA has been localized to 143 bp that include 88 bp at the 5' end of the long terminal repeat; the enhancing segment of SV40 DNA lies 42-276 bp upstream from the initiation site for early transcription. The RSV enhancer does not provide a favored integration site within microinjected plasmids and does not affect the frequency of integration into host cell DNA. When microinjected cells were grown into cell lines in nonselective medium, few cells containing integrated HSV tk without RSV DNA could grow in selective medium. In contrast, over 80% of cells from lines containing the HSV tk gene linked to the RSV enhancer could grow in selective medium. Thus the RSV enhancer affects the expression rather than the integration of the tk gene

Characterization of three proteins involved in polypeptide chain termination.

Journal ArticleAt each stage of elongation, the growing polypeptide chain is bound to the ribosome-messenger RNA complex through the transfer RNA of the most recently incorporated amino acid residue. When the chain is complete, the last polypeptide-transfer RNA (tuna) ester linkage is cleaved, releasing the chain from the tuna and thus from the ribosomal complex. This hydrolysis occurs when the ribosome in the course of moving along the messenger RNA (mina) reaches a chain terminating signal

Isolation of a suppressible nonsense mutant in mammalian cells

Journal ArticleAn HGPRT- cell line derived from mouse L cells has been shown to have the following properties: it is CRM'; the defective HGPRT molecules are altered in the carboxyterminal peptide; the mutant cells regain HGPRT activity when ochre-suppressor tRNA is microinjected into them, but not when amber-suppressor or wild-type tRNAs are injected. We conclude from these properties that this mutant cell line contains an ochre nonsense mutation (UAA) in the structural gene for HGPRT