The ATP binding site on rho protein. Affinity labeling of Lys181 by pyridoxal 5'-diphospho-5'-adenosine

We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein. Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C). The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation. A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C). Amino acid sequence analysis of the single [3H]PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP. These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins. Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation.     

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.     

Studies of the interaction of RecA protein with DNA

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Does Fire Trigger Seed Germination in the Neotropical Savannas? Experimental Tests with Six Cerrado Species

The Cerrado (Brazilian savanna) is a biodiversity hotspot with a history of fire that goes back as far as 10 million years. Fire has influenced the evolution of several aspects of the vegetation, including reproduction and life cycles. This study tested how fire by‐products such as heat and smoke affect the germination of six species common to two Cerrado open physiognomies: wet grasslands and the campo sujo (grassland with scattered shrubs and dwarf trees). We subjected seeds collected in northern Brazil to heat shock and smoke treatments, both separately and combined, using different temperatures, exposure times, and smoke concentrations in aqueous solutions. High temperatures and smoke did not break seed dormancy nor stimulate germination of the Cerrado study species. However, seeds were not killed by high temperatures, indicating that they are fire‐tolerant. Our findings differed from those of other fire‐prone ecosystems (mostly of Mediterranean vegetation), where fire stimulates germination. Moreover, we provide important information regarding germination strategies of non‐woody Cerrado plants, showing the importance of considering the tolerance of seeds to high temperatures when evaluating fire‐related traits in fire‐prone ecosystems.