Diversity and evolutionary relationship of nucleotide binding site-encoding disease-resistance gene analogues in sweet potato (Ipomoea batatas Lam.).

Most plant disease-resistance genes (R-genes) isolated so far encode proteins with a nucleotide binding site (NBS) domain and belong to a superfamily. NBS domains related to R-genes show a highly conserved backbone of an amino acid motif, which makes it possible to isolate resistance gene analogues (RGAs) by degenerate primers. Degenerate primers based on the conserved motif (P-loop and GLPL) of the NBS domain from R -genes were used to isolate RGAs from the genomic DNA of sweet potato cultivar Qingnong no.2. Five distinct clusters of RGAs (22 sequences) with the characteristic NBS representing a highly diverse sample were identified in sweet potato genomic DNA. Sequence identity among the 22 RGA nucleotide sequences ranged from 41.2% to 99.4%, while the deduced amino acid sequence identity from the 22 RGAs ranged from 20.6%to 100%. The analysis of sweet potato RGA sequences suggested mutation as the primary source of diversity. The phylogenetic analyses for RGA nucleotide sequences and deduced amino acids showed that RGAs from sweet potato were classified into two distinct groups--toll and interleukin receptor-1 (TIR)-NBS-LRR and non-TIR-NBS-LRR. The high degree of similarity between sweet potato RGAs and NBS sequences derived from R-genes cloned from tomato, tobacco, flax and potato suggest an ancestral relationship. Further studies showed that the ratio of non-synonymous to synonymous substitution within families was low. These data obtained from sweet potato suggest that the evolution of NBS-encoding sequences in sweet potato occur by the gradual accumulation of mutations leading to purifying selection and slow rates of divergence within distinct R-gene families.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

Ontogeny, glycosylation and modulation by dialysis, sodium and nucleotides of the rat brain delta opioid receptor studied with anti-idiotypic antibodies to anti-leucine enkephalin.

Ontogeny of the rat brain delta opioid receptor in 1-60 days old animals has been studied with anti-idiotypic antibodies to anti-leucine enkephalin. It is found that delta opioid receptors are present in rats from birth and attain adult levels by 28 days and these receptors are glycosylated and inhibited by Na+, GTP, ATP and CTP at all ages. Adult membrane-bound and solubilized delta opioid receptors are inhibited to similar extents by Na+ (100 mM), GTP, ATP and CTP (50 microM). Dialysis of the adult membrane-bound receptor led to 81% loss in binding which was restored by 100 mM Na+, 50 microM GTP, ATP and CTP to 77, 72, 87 and 94% respectively and by 100 mM NH4+, Mg2+, Ca2+ and Mn2+ to 63, 43, 57 and 73% respectively. Dialysis of the solubilized receptor resulted in 23% loss in binding with Na+ (100 mM), GTP and ATP (50 microM) inhibiting receptor binding to 46, 62 and 54% respectively, while CTP (50 microM) restored binding to 88%. These studies indicate that the delta opioid receptor can be probed with anti-idiotypic antibodies to anti-leucine enkephalin, that functional, glycosylated receptors are present at birth in rats and that the adult membrane-bound and solubilized receptors are modulated differently by dialysis.

Differences in the levels of aminoacylation and contents of modified nucleotides between total tRNAs from N2- and NH4(+)-grown Azospirillum lipoferum cells.

Total tRNAs isolated from N2- and NH4(+)-grown Azospirillum lipoferum cells were compared with respect to amino acid acceptance, isoacceptor tRNA species levels and extent of nucleotide modifications. Amino-acylation of these two tRNA preparations with ten different amino acids indicated differences in the relative acceptor activities. Comparison of aminoacyl-tRNA patterns by RPC-5 column chromatography revealed no qualitative differences in the elution profiles. However, quantitative differences in the relative amounts of some isoacceptors were observed. These results indicate that alterations of relative amounts of functional tRNA species occur to match cellular requirements of the bacterial cells using N2 or NH4+ as nitrogen source. In addition, the content of modified nucleotides in total tRNAs of N2- and NH4(+)-grown cells was determined. In the NH4(+)-grown cells, content of most of the modified nucleotides decreased significantly. Based upon these results, the relationship of chargeability of tRNAs to base modifications is discussed.