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1.
The cytoplasmic leucyl-tRNA synthetases were purified from a wild-type Neurospora crassa and from a temperature-sensitive leucine-auxotroph (leu-5) mutant. A detailed steady-state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out. These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The Kappm value for leucine was lowered by high ATP concentrations and correspondingly the Kappm,ATP was lowered by high leucine concentrations. The Kappm,Leu was lowered by high pH, a pK value of 6.7 (at 30 degrees C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 microM leucine, 0.3 microM tRNALeu, and pH 7 the apparent Km values were Kappm,ATP = 1.3 mM, Kappm,Leu = 49 microM and Kappm,tRNA = 0.15 microM. No essentially altered cytoplasmic leucyl-tRNA synthetase was produced by the temperature-sensitive mutant strain when kept at 37 degrees C. In none of these experiments could we find any difference between the wild-type enzyme and the enzyme from the mutant strain (whether grown at permissive temperature, 28 degrees C, or grown at permissive temperature for 24 h followed by growth at 37 degrees C). We therefore think that the small difference in the Km value for leucine of the wild-type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl-tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild-type and mutant strains. 相似文献
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The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from
nucleotide sequence variation across a 765-bp region in the cytochrome
oxidase I and II genes of the mitochondrial genome. Most parsimonious
relationships of 25 haplotypes from 16 Greya species and two outgroup
genera (Tetragma and Prodoxus) showed substantial congruence with the
species relationships indicated by morphological variation. Differences
between mitochondrial and morphological trees were found primarily in the
positions of two species, G. variabilis and G. pectinifera, and in the
branching order of the three major species groups in the genus. Conflicts
between the data sets were examined by comparing levels of homoplasy in
characters supporting alternative hypotheses. The phylogeny of Greya
species suggests that host-plant association at the family level and larval
feeding mode are conservative characters. Transition/transversion ratios
estimated by reconstruction of nucleotide substitutions on the phylogeny
had a range of 2.0-9.3, when different subsets of the phylogeny were used.
The decline of this ratio with the increase in maximum sequence divergence
among taxa indicates that transitions are masked by transversions along
deeper internodes or long branches of the phylogeny. Among transitions,
substitutions of A-->G and T-->C outnumbered their reciprocal
substitutions by 2-6 times, presumably because of the approximately 4:1
(77%) A+T-bias in nucleotide base composition. Of all transversions,
73%-80% were A<-->T substitutions, 85% of which occurred at third
positions of codons; these estimates did not decrease with an increase in
maximum sequence divergence of taxa included in the analysis. The high
frequency of A<-->T substitutions is either a reflection or an
explanation of the 92% A+T bias at third codon positions.
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