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We describe a general protocol for controlled gene amplification, which allows conditional expression of high levels of amber suppressor activity in monkey kidney cells, and we demonstrate its use in the genetic analysis of animal viruses by the generation and propagation of the first nonsense mutant of poliovirus. A human amber suppressor tRNASer gene linked to the SV40 origin of replication and a second DNA carrying a temperature-sensitive SV40 large T antigen gene were cotransfected into monkey cells. Cell lines having stably integrated the DNAs were isolated. Shifting the cells from the nonpermissive temperature to a lower permissive temperature caused the amplification of the suppressor tRNA gene, which resulted in suppression efficiencies at amber codons of 50%-70%, as measured by suppression of an amber codon in the E. coli chloramphenicol acetyltransferase gene. A mutant of poliovirus, in which a serine codon in the replicase gene was converted to an amber codon, was efficiently propagated on the suppressor-positive cell lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of approximately 2.5 x 10(-6), surprisingly low for a RNA genome. 相似文献
104.
The authors have investigated the electrical activity of corpus cavernous, first according to Wagner and Gerstenberg’ method, then, since one year, according to Stief method, in the basal state and following intracavernosal injection of vasoactive agents, or following various stimulation tests. They have found again the electrical activity described by these authors, but have been confronted with the difficulty in quantifying the data. Specially single potential analysis seems to them little reliable and reproducible for an objective interpretation. At this stage of their experience, they test two simpler criteria interpretation: the richness of the electrical activity, and the reactivity of the recording to various stimulations. Their preminary results suggest a correlation between those criteria and the state of the autonomic nervous system of the penis. 相似文献
105.
Fructose bisphosphatase of Escherichia coli: cloning of the structural gene (fbp) and preparation of a chromosomal deletion 总被引:12,自引:6,他引:6 下载免费PDF全文
The fbp locus at 96 min on the Escherichia coli chromosome governs fructose bisphosphatase (fructose-1,6-P2 1-phosphatase). We have cloned and subcloned fbp on vector pBR322 to obtain strains with high levels of the enzyme. In vivo mutagenesis of the clone was used to show that fbp is the structural gene. The gene was deleted on the plasmid in vitro, and the chromosomal wild-type locus was replaced with this deletion by a method involving stabilization of a heterozygous intermediate resulting from plasmid integration, followed by segregation of the wild-type gene. 相似文献
106.
Complete sequences of the rRNA genes of Drosophila melanogaster 总被引:19,自引:0,他引:19
In this, the first of three papers, we present the sequence of the
ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of
D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S
(123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat
of D. melanogaster is AT rich (65.9% overall), with the spacers being
particularly AT rich. Analysis of DNA simplicity reveals that, in contrast
to the intergenic spacer (IGS) and the external transcribed spacer (ETS),
most of the rRNA gene regions have been refractory to the action of
slippage-like events, with the exception of the 28S rRNA gene expansion
segments. It would seem that the 28S rRNA can accommodate the products of
slippage-like events without loss of activity. In the following two papers
we analyze the effects of sequence divergence on the evolution of (1) the
28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary
structures among eukaryotic species, respectively. Our detailed analyses
reveal, in addition to unequal crossing-over, (1) the involvement of
slippage and biased mutation in the evolution of the rDNA multigene family
and (2) the molecular coevolution of both expansion segments and the
nucleotides involved with compensatory changes required to maintain
secondary structures of RNA.
相似文献
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Physiological functions of mineral macronutrients 总被引:6,自引:0,他引:6
Frans JM Maathuis 《Current opinion in plant biology》2009,12(3):250-258
110.