α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding to Rat Striatal Membranes: Effects of Selective Brain Lesions

The binding of alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl-/Ca2+, saturation-curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 +/- 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several other excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, L-Glu, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N-Methyl-D-aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intrastriatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2-3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopaminergic pathway (using nigral injection of 6-hydroxy-dopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA-preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1

Summary The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.     

2-Aminopurine and 5-bromouracil induce alleviation of type I restriction in Escherichia coli: Mismatches function as inducing signals?

Summary The EcoK restriction of unmodified phage is 1000-fold alleviated in Escherichia coli grown in the presence of base analogs 2-aminopurine (2AP) and 5-bromouracil (5BU). 2AP treatment of bacteria affects specificially the type I restriction systems (EcoA, EcoB, EcoD and EcoK) and does not influence type II (EcoRI) and type III (EcoP1) restriction. 2AP-induced alleviation of restriction occurs in bacteria which are deficient in the SOS response (recA and lexA) and mismatch repair (mutH, mutL and mutS) and can be distinguished from the alleviation of restriction observed in dam ^- strains. We suggest that mismatches induced by 2AP and 5BU may function as an inducing signal for the alleviation of restriction observed in the presence of base analogs.     

The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.     

A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae

Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.     

Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in leaves of Citrus explants throughout the annual cycle and its regulation by ethylene

Leaf senescence and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) degradation in orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] explants have been investigated. Explants consisted of a segment of stem (ca 15 cm) and 5 mature leaves. In vitro RuBP carboxylase degradation was determined by culturing the explants in water for different periods of time (3 days usually) and quantifying the two RuBP carboxylase subunits in the extracts following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vitro RuBP carboxylase degradation was estimated by autodigestion of leaf extracts and SDS-PAGE. The extent of in vivo RuBP carboxylase degradation in explants cultured under 16 h light/8 h dark photoperiod varied throughout the year and showed a cyclic behaviour correlated with the growth cycle of Citrus. The highest proteolytic activity both in vivo and in vitro was found in explants made from April to August coinciding with the maximum vegetative growth period of the tree.
Leaf senescence and abscission could be retarded significantly at any time of the year by maintaining the explants continuously in the dark. Treatment of the explants in the dark with a continuous flow of ethylene enhanced both leaf abscission and rate of RuBP carboxylase degradation, proportionally to ethylene concentration (0.1-0.6 ppm). Ethylene-induced senescence of Citrus leaf explants in the dark appears to be a convenient model system to study the regulation of the proteolytic degradation of RuBP carboxylase.     

数种昆虫5S rRNA结构特点的比较

比较已知结构的昆虫5S rRNA的核苷酸顺序,发现同科、同目的昆虫比不同科、不同目的昆虫有较少的核苷酸差别.根据Kimura和Ohta(1972)提出的经验公式,绘制了数种昆虫的系统发育图.结果表明,从分子进化得到的结论和经典分类基本上是一致的.根据DeWachter等(1982)提出的二级结构模型,归纳分析这些昆虫5S rRNA,发现保守位点与半保守位点(同一位点仅出现二种核苷酸残基)之和几乎占整个5S rRNA分子的100%.