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N Glanville

Publications and source records attributed to N Glanville.

15 recordsLinked to original sources
PubMed1985

Unstable expression and amplification of a transfected oncogene in confluent and subconfluent cells.

NIH 3T3 cells were transfected with a plasmid containing the transforming gene, v-src, from Rous sarcoma virus. One of the transformed cell lines isolated reverted to a flat, nontransformed morphology after cloning through soft agar. This cell line did not express the src gene and could no longer grow in soft agar. When these cells were held at confluence, spontaneous foci appeared which eventually covered the dish. The appearance of foci correlated with an increase in v-src gene expression, ability to grow in soft agar, and tumorigenicity in mice. When these transformed cells were trypsinized and held at subconfluence, both v-src expression and the transformed phenotype were progressively lost. Whereas rearrangement of the transfected gene was not detected, the gene copy number in the transformed cells was markedly increased (greater than 50-fold). Confluence-dependent gene amplification and deamplification have been retained after several cycles of growth alternately at high and low density, in cells recloned through soft agar, and after cells had been maintained continuously at high or low density. The results suggest that, in this cell line, reversible gene amplification plays a central role in expression of the transfected gene.

Animals↗
PubMed1981

Structure of mouse metallothionein-I gene and its mRNA.

Metallothioneins are small cysteine-rich proteins that bind heavy metals such as zinc, cadmium, copper and mercury. Recent interest in these proteins has focused on the part they play in zinc metabolism and heavy metal detoxification. Our interest in metallothionein genes stems largely from the observations that these proteins are inducible by both heavy metals and glucocorticoid hormones. To explore the regulation of these genes, we have isolated cDNA and genomic clones corresponding to mouse metallothionein-I (MT-I), and have used them to show that both inducers act at the transcriptional level in vivo and in a wide variety of cell lines. We have also shown that the MT-I gene is amplified during selection for cadmium resistance. To investigate the mechanisms of gene regulation, knowledge of the primary DNA sequence is necessary. Here we present the entire sequence of mouse MT-I gene along with approximately 300 bases of 5' flanking region that presumably includes promoter and regulatory sites. The 5' mRNA sequence, defined by S1 nuclease mapping, was combined with sequences of the coding and 3' untranslated regions obtained previously to allow a computer prediction of the most stable secondary structure of MT-I mRNA.

Animals↗
PubMed1981

The ovalbumin gene family: hormonal control of X and Y gene transcription and mRNA accumulation.

The ovalbumin gene family is composed of three genes, X, Y and ovalbumin, which are expressed in laying hen oviduct. We have analyzed the in vivo transcription products of X and Y genes and the effect of steroid hormones on their synthesis and accumulation. As in the case of ovalbumin, the complete gene transcripts and processing intermediates are present in the poly(A)+ RNA fraction. The mature RNAs are found in polysomes and are translated into proteins. The expression of X and Y genes is controlled by steroid hormones: X and Y RNAs are not detectable in oviducts from chicks withdrawn from estrogen stimulation, whereas in chicks stimulated with estrogen for 7 days, X RNA represents 0.3% and Y RNA 0.8% of ovalbumin mRNA. In laying hen, however, the levels of X and Y RNAs are about 2% of ovalbumin mRNA. After stimulation with other steroid hormones, alone or in combination, the level of X and Y RNA does not achieve that detected in laying hen. Progesterone has a much weaker effect on X RNA accumulation than on that of Y and ovalbumin mRNAs. Studies with isolated nuclei show that X and Y gene expression is regulated by hormones at the level of transcription. However, the differences observed between the transcription rates and the accumulation of X and Y mRNAs suggest that the expression of X and Y genes could also be controlled at the levels of RNA processing and/or mRNA stability.

Animals↗
PubMed1976

Simultaneous translation of structural and nonstructural proteins from Semliki-forest-virus RNA in two eukaryotic systems in vitro.

The Semliki Forest virus genome, 42-S RNA, and the virus-specific intracellular 26-S RNA were translated in two cell-free protein-synthesising systems, the wheat germ extract, and a partially purified system from mammalian tissues. The 26-S RNA directed the synthesis of structual proteins only, as revealed by tryptic peptide mapping. About 75--80% of the radioactivity in the products comigrated with capsid and about 4--8% with envelope protein peptides. All the capsid peptides and the full-sized capsid protein were found in the products in vitro, no complete envelope protein was formed and fewer than half of the envelope peptides were detected. This result is consistent with reports that there is only one initiation site for the translation of virus structural proteins, and that the capsid protein is N-terminal in the polyprotein followed by envelope proteins. The systems programmed with 42-S RNA yielded virtually the same structural peptides. However, the bulk of the radioactivity was in peptides which did not comigrate with the structural ones. These peptides were mostly associated with relatively small-sized products. This shows that Semliki Forest virus 42-S RNA has at least two initiation sites, one for the structural proteins and the other(s) for the nonstructural proteins.

Cell Line↗
PubMed1976

Initiation of translation directed by 42S and 26S RNAs from Semliki Forest virus in vitro.

The proteins synthesized in vitro in response to 42S and 26S RNAs from Semliki Forest virus were labeled with formyl-[35S]methionine from initiator tRNA. One protein which comigrated with viral capsid protein was labeled under the direction of 26S RNA, and only one labeled peptide was detected after digestion with trypsin. Further digestion with pronase gave rise to the dipeptide fMet-AsN. Several labeled polypeptides were found in the 42S RNA directed product and these had molecular weights of up to 150,000. However, tryptic digestion of the product yielded only one formylmethionyl-labeled peptide, which had a different mobility from that directed by the 26S RNA. Further digestion with pronase gave a single dipeptide, fMet-Ala. This indicates that nonstructural proteins as large as 150,000 daltons are probably synthesized from one initiation site on the 42S RNA. Translation starting from the internal initiation site on the 42S RNA, which is equivalent to that on the 26S RNA, could not be detected under the conditions used. Internal initiation sites which are similarly inactive have also been detected in other viral RNAs (e.g., brome mosaic virus, tobacco mosaic virus, and polyoma 19S RNA) and this suggests that, although eukaryotic mRNAs can contain more than one initiation site for protein synthesis, only the site nearer the 5' terminus is active in vitro.

Cell-Free System↗
PubMed1976

Transitional control in Semliki forest virus infected cells.

The cistron of the 42 S RNA, which codes for other than structural proteins, is poorly translated in the middle of the growth cycle. The translational control operating in the infected cells is not expressed in cell-free protein synthesising systems programmed with the 42 S RNA, since mostly non-structural proteins are made in vitro. One of our temperature-sensitive mutants of SFV, ts-1, directs the synthesis of two non-structural proteins with molecular weights of 78,000 (ns-78) and 86,000 (ns-86) but only at the restrictive temperature. This indicates that the synthesis of the non-structural proteins is controlled by viral rather than cellular factor(s). Temperature shift experiments revealed that the control mechanism was only slowly affected by the change of the temperature.

Cell Line↗
PubMed1975

Tryptic peptide analysis on nonstructural and structural precursor proteins from Semliki Forest virus mutant-infected cells.

Analysis of [35S]methionine-labeled tryptic peptides of the large proteins induced by temperature-sensitive mutants of Semliki Forest virus was carried out. The 130,000-molecular-weight protein induced by ts-2 and ts-3 mutants contained the peptides of capsid protein and of both major envelope proteins E1 and E2. The ts-3-induced protein with molecular weight of 97,000 contained peptides of the capsid and envelope protein E2 but not those of E1. Two proteins with molecular weights of 78,000 and 86,000 from ts-1-infected cells did not contain the peptides of the virion structural proteins. They are evidently expressions of the nonstructural part of the 42S RNA genome of Semliki Forest virus.

Autoradiography↗
PubMed1975

Translation of Semliki forest virus 42S and 26S RNAs in a cell-free system derived from Escherichia coli.

The SFV 42S RNA and the intracellular 26S RNA have been translated in a prokaryotic cell-free system, the E. coli S30. About half of the [35S]methionine-labelled products directed by both RNAs had molecular weights larger than 20,000 on polyacrylamide gels. Both products contained tryptic peptides which comigrated with all the capsid and envelope protein-derived peptides. The most striking difference between the prokaryotic and eukaryotic systems lay in the translation of the 42 S RNA: The "42S RNA-specific nonstructural" peptides, which predominate in the eukaryotic systems, were apparently absent from the product translated by the prokaryotic system.

Cell-Free System↗
PubMed1975

Polysomes and initiation complexes in vitro induced by Semliki Forest virus 42S and 26S RNA.

Semliki Forest virus 42S and 26S RNA induce the formation of polysomes when translated in vitro in cell-free systems. After analysis on sucrose gradients the polysomal structures, containing prelabelled RNA and nascent peptide chains labelled with [35S]methionine, had sedimentation values from 100 to 200S in the case of 26S RNA and from 150 to over 250S with 42S RNA. After incubation in the presence of inhibitors of elongation 26S RNA was found in initiation complexes sedimenting at about 80S. Under identical conditions 42S RNA had a heterogenous sedimentation pattern, being attached to complexes sedimenting even faster than 250S. These structures, however, fulfilled the criteria of initiation complexes suggesting that there were more than one ribosome attached to each 42S RNA under conditions where 26S RNA bound only one.

Cell-Free System↗