Amino acid replacements resulting from super-suppression of nonsense mutants of iso-1-cytochrome c from yeast

The eight class I, set 1 super-suppressor genes, SUP2, SUP3, SUP4, SUP5, SUP6, SUP7, SUP8 and SUP11 are not closely linked and map at distinct loci throughout the genome of yeast. Each of these suppressors causes the production of 5 to 10% of the normal amount of iso-1-cytochrome c when it is individually coupled to the ochre (UAA) mutant cy1-2. All eight iso-1-cytochromes c contain a residue of tyrosine at position 20 which corresponds to the site of the ochre codon. Several of these super-suppressors also were shown to act on cy1-9, but at a much lower efficiency. It was shown that iso-1-cytochrome c from one of the suppressed cy1-9 strains contains a tyrosine at position 2, which corresponds to the site of the ochre codon in this mutant. It is suggested that the gene product of the eight super-suppressors is tyrosine transfer RNA.     

Quantitation of mitochondrial injury in cultured rat heart endothelioid cells with nitroblue tetrazolium

Synopsis A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondriain situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.     

Geographical patterns of persistence in duck guilds

Geographical gradients of persistence in community structure have been suggested to be causally related to underlying gradients of species diversity, environmental variability and/or productivity. In order to test whether the persistence of breeding duck communities was dependent on any one of these three factors, thirty-three years of census data from the Canadian prairie and boreal forest regions was examined along geographical gradients of wetland habitat variability and productivity. For breeding ducks, locally derived patterns of persistence were generally independent of local habitat conditions. Persistence appeared to be related more to patterns of emigration and immigration in response to climatic conditions (i.e., drought) in the southern prairies than to local species richness, wetland habitat variability or productivity. It is suggested, therefore, that analyses of community persistence derived at small spatial scales may be of limited value if the structure of communities is not regulated by local conditions.     

Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana

Thirteen Arabidopsis thaliana mutants with deviating epicuticular wax layers (i.e., cer mutants) were isolated by screening 13 000 transformed lines produced by the seed transformation method. After crossing the 13 mutants to some of the previously known cer mutant lines, 12 of our mutants mapped to 6 of the 21 known complementation groups (cer1 through cer4 as well as cer6 and cer10), while the other mutant corresponded to a previously unknown locus, cer21. Mutant phenotypes of 6 of the 13 mutant lines were caused by T-DNA insertions within cer genes. We also analyzed the chemical composition of the epicuticular wax layers of the cer mutants isolated in this study relative to that of Arabidopsis wild-type plants. Our results suggest that the five genes we tagged regulate different steps in wax biosynthesis, i.e., the decarbonylation of fatty aldehydes to alkanes, the elongation of hexacosanoic acid to octacosanoic acid, the reduction of fatty aldehydes to primary alcohols and the production of free aldehydes, while an insertion in the fifth gene causes an alteration in the chain length distribution of the different classes of wax compounds.     

In vivo suppression of phytochrome aggregation by the GroE chaperonins in Escherichia coli

When expressed in Escherichia coli, a truncated form of phytochrome (oat PHYA AP3 residues 464-1129) self associates to form a series of products ranging in size from monomers to aggregates of greater than 20 subunits. When these same phytochrome sequences are coexpressed with the chaperonins GroEL and GroES, the truncated phytochrome migrates as a native-like dimer in size exclusion chromatography and no higher-order aggregates were detected. GroEL and GroES inhibition of phytochrome aggregation in E. coli presumably occurs via the suppression of folding pathways leading to incorrectly folded phytochrome.     

Specific enzymatic dephosphorylation of the retinoblastoma protein.

The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.