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1.
Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).
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P V Danenberg T Horikoshi M Volkenandt K Danenberg H J Lenz L C Shea A P Dicker A Simoneau P A Jones J R Bertino 《Nucleic acids research》1992,20(3):573-579
RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation. 相似文献
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The Drosophila melanogaster gene flightless-I, involved in gastrulation and
muscle degeneration, has Caenorhabditis elegans and human homologues. In
these highly conserved genes, two previously known gene families have been
brought together, families encoding the actin- binding proteins related to
gelsolin and the leucine-rich-repeat (LRR) group of proteins involved in
protein-protein interactions. Both these gene families exhibit
characteristics of molecular changes involving replication slippage and
exon shuffling. Phylogenetic analyses of 19 amino acid sequences of 6
related protein types indicate that actin- associated proteins related to
gelsolin are monophyletic to a common ancestor and include flightless
proteins. Conversely, comparison of 24 amino acid sequences of LRR proteins
including the flightless proteins indicates that flightless proteins are
members of a structurally related subgroup. Included in the flightless
cluster are human and mouse rsp-1 proteins involved in suppressing v-Ras
transformation of cells and the membrane-associated yeast (Saccharomyces
cerevisae) adenylate cyclase whose analogous LRRs are required for
interaction with Ras proteins. There is a strong possibility that ligands
for this group could be related and that flightless may have a similar role
in Ras signal transduction. It is hypothesized that an ancestral monomeric
gelsolin precursor protein has undergone at least four independent gene
reorganization events to account for the structural diversity of the extant
family of gelsolin-related proteins and that gene duplication and exon
shuffling events occurred prior to or at the beginning of multicellular
life, resulting in the evolution of some members of the family soon after
the appearance of actin-type proteins.
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3.
Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid
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Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge. 相似文献
4.
Raney nickel (Ni(H)) catalyzes a specific reductive cleavage of carbon-sulfur bonds and, therefore, can be used to determine whether compounds are covalently bound to proteins through a sulfide linkage. When the covalent thymidylate synthetase-[3H]5-fluoro-2'-deoxyuridylic acid-[14C]-5,10-CH2H4-folate complex (Langenbach et al. (1972a), Biochem, Biophys. Res. Commun. 48, 1565) was denatured and then shaken with Ni(H) at 25 degrees C, both isotopes were rapidly cleaved from the protein, with identical reaction halftimes of less than 10 min. The liberated radioactivity was filterable through nitro-cellulose filters and comigrated with small molecules on Sephadex G-25. Both labels migrated identically upon paper chromatography. A [3H]5-fluoro-2'-deoxyuridylic acid-[35S]thymidylate synthetase complex was formed with enzyme isolated from Lactobacillus casei grown in the presence of [35S]cysteine. This complex, upon Ni(H) treatment, released both tritium and sulfur-35 at identical rates. Control experiments on amino acids showed that only the sulfur-containing amino acids are degraded by Ni(H). Cysteine was rapidly converted to alanine and methionine to alpha-aminobutyric acid. 5-Carboxymethylcysteine and 5-uracilylcysteine, simple models for the tenary enzyme-5-fluoro-2'-deoxyuridylic acid-5,10-CH2H4-folate complex, were converted to alanine at the same rate that 5-fluoro-2'-deoxyuridylic acid (FdUrd-5'-P) was cleaved from the enzyme. Native ribonuclease, which has a tightly coiled structure, was not affected by the reagent, but carboxymethylated ribonuclease was desulfurized. Amino acid analysis of Ni(H)-treated thymidylate synthetase showed that cysteine was the only amino acid degraded. Gel electrophoresis of the proteins after exposure to Ni(H) showed no breakage of polypeptide chains. These results support a sulfide linkage between FdUrd-5'-P and thymidylate synthetase in the covalent complex. 相似文献
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Rates and patterns of evolution in partial sequences of five mitochondrial
genes (cytochrome b, ATPase 6, NADH dehydrogenase subunit 5, tRNA(Glu), and
the control region) were compared among taxa in the passerine bird genera
Fringilla and Carduelis. Rates of divergence do not vary significantly
among genes, even in comparisons with the control region. Rate variation
among lineages is significant only for the control region and NADH
dehydrogenase subunit 5, and patterns of variation are consistent with the
expectations of neutral theory. Base composition is biased in all genes but
is stationary among lineages, and there is evidence for directional
mutation pressure only in the control region. Despite these similarities,
patterns of substitution differ among genes, consistent with alternative
regimes of selective constraint. Rates of nonsynonymous substitution are
higher in NADH dehydrogenase subunit 5 than in other protein-coding genes,
and transitions exist in elevated proportions relative to transversions.
Transitions appear to accumulate linearly with time in tRNA(Glu), and
despite exhibiting the highest overall rate of divergence among species,
there are no transversional changes in this gene. Finally, for resolving
phylogenetic relationships among Fringilla taxa, the combined
protein-coding data are broadly similar to those of the control region in
terms of phylogenetic informativeness and statistical support.
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