Molecular markers for ozone stress isolated by suppression subtractive hybridization: specificity of gene expression and identification of a novel stress-regulated gene

Suppression subtractive hybridization was used to identify genes regulated by ozone (100 nmol mol ^{? 1}) in Pisum sativum. One novel gene (named PsUod1) was found. In addition, mRNA levels for four genes (encoding lipid transfer protein, pre‐hevein‐like protein, leucine‐rich repeat protein, and disease‐resistance response protein 230), which previously were shown to be regulated by biotic stress, increased. Finally, mRNA species for two genes (encoding extensin and pathogenesis‐related protein 4A), previously shown to be regulated by ozone in other species, were found to increase in abundance. The ozone‐specificity of the expression of these genes was studied by using UV‐B radiation. PsUod1 and the genes encoding extensin, leucine‐rich repeat protein, and disease‐resistance response protein 230, were differentially regulated when comparing ozone and UV‐B. Moreover, the mRNA levels for extensin, leucine‐rich repeat protein and disease‐resistance response protein 230 all increased under NaCl and aluminium stress and after wounding, whereas the message abundance for PsUod1 was unchanged under these stresses. Thus, in general, ozone caused changes similar to wounding, salt stress and aluminium stress, whereas UV‐B radiation regulated gene expression differently.     

Regulation of uterine 5 alpha-reductase type 1 in mice.

A null mutation in the murine gene encoding steroid 5 alpha-reductase type 1 (5 alpha R1) leads to failure of normal parturition at term. This observation, together with the finding that mRNA levels of uterine 5 alpha R1 increase significantly at term in normal pregnant animals, indicates that 5 alpha R1 plays an important role in murine parturition. The current studies were conducted to elucidate the regulation of 5 alpha R1 in uterine tissues of nonpregnant and pregnant animals. Nonpregnant, ovariectomized ICR mice were treated with vehicle (control), 17 beta-estradiol (E(2)), progesterone (P(4) ), or E(2)+P(4) for 3 days. Thereafter, uterine tissues were obtained for histology, quantification of 5 alpha R1 specific activity, and Northern blot analysis of 5 alpha R1 mRNA expression. The 5 alpha R1 enzyme activity was significantly increased in animals treated with E(2)+P(4). However, activity was much less in uterine tissues from E(2)+P(4)-treated animals than in uterine tissues from pregnant animals near term. To evaluate further the regulation of 5 alpha R1 during gestation, mice underwent unilateral tubal ligation before timed matings. The 5 alpha R1 activity increased eightfold in uterine tissues from the fetal horn from Gestational Days 12 to 18. This temporal pattern in 5 alpha R1 activity paralleled marked increases in uterine diameter. Taken together, these studies indicate that expression of 5 alpha R1 is regulated by E(2)+P(4) in uterine tissues. Whereas E(2) alone is insufficient to induce enzyme activity, E(2) may be required to increase P(4) receptors and, thereby, mediate the effects of P(4) on 5 alpha R1 gene expression. Further increases in enzyme activity during late gestation are mediated by fetal occupancy, possibly through stretch-induced increases in endometrial growth. Thus, like other genes involved in parturition, expression of 5 alpha R1 is regulated by both hormonal and fetal-derived signaling pathways.     

NMR structure of the pseudo-receiver domain of CikA

The circadian input kinase (CikA) is a major element of the pathway that provides environmental information to the circadian clock of the cyanobacterium Synechococcus elongatus. CikA is a polypeptide of 754 residues and has three recognizable domains: GAF, histidine protein kinase, and receiver-like. This latter domain of CikA lacks the conserved phospho-accepting aspartyl residue of bona fide receiver domains and is thus a pseudo-receiver (PsR). Recently, it was shown that the PsR domain (1) attenuates the autokinase activity of CikA, (2) is necessary to localize CikA to the cell pole, and (3) is necessary for the destabilization of CikA in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The solution structure of the PsR domain of CikA, CikAPsR, is presented here. A model of the interaction between the PsR domain and HPK portion of CikA provides a potential explanation for how the PsR domain attenuates the autokinase activity of CikA. Finally, a likely quinone-binding surface on CikAPsR is shown here.     

Regulation of the pAD1 sex pheromone response in Enterococcus faecalis: effects of host strain and traA, traB, and C region mutants on expression of an E region pheromone-inducible lacZ fusion.

Pheromone-induced conjugal transfer of the hemolysin-bacteriocin plasmid pAD1 of Enterococcus faecalis is regulated by a cluster of determinants designated traA, traB, and regions C and E. The E region is believed to include a positive regulator that controls many structural genes related to conjugation. The pheromone-inducible Tn917-lac fusion NR5, located in the E region, is regulated by the products of traA, traB, and the C region. To more closely examine the effects of these genes on the induction of E region products, inserts in each of these genes were combined with the NR5 fusion in a novel approach involving triparental matings with a pAD1 miniplasmid and recombinational mutagenesis. Results indicate that (i) the traA gene product is a key repressor of the pheromone response; (ii) the traB gene product, in cooperation with a gene within or regulated by the E region, controls pheromone shutdown; (iii) a primary function of the C region gene product is in pheromone sensing, with secondary functions in pheromone shutdown and negative regulation; and (iv) the host in which the plasmid resides has a dramatic effect on the regulation of the NR5 fusion in traB and C region mutants. Numerous parallels were observed between the regulation of the NR5 fusion and the regulation of the aggregation and transfer response. These parallels aided in further defining the functions of particular regulatory determinants as well as further establishing the link between the regulation of the E region and the regulation of the aggregation and transfer response.