Expression of the p16 and p15 cyclin-dependent kinase inhibitors in lymphocyte activation and neuronal differentiation.

The cyclin-dependent kinase inhibitors p16INK4/MTS1 and p15INK4B/MTS2 have been mapped to a region in chromosome 9 (921) that is deleted frequently in acute lymphoblastic leukemias and malignant gliomas. To gain insight into the functions of these inhibitors in lymphocytes and neuronal cells, we studied the expression of p15 and p16 during lymphocyte mitogenesis and neuronal differentiation. Expression of p15 was extinguished during lymphocyte activation, concomitant with an increase in retinoblastoma kinase activity. The differentiation of the embryonic teratocarcinoma cell line NT2 into postmitotic neurons (hNT) was associated with enhanced expression of p15 and p16 proteins. These findings suggest that p15 and p16 play a role in maintaining cell quiescence in lymphocytes and neuronal cells, respectively. Deletions of these genes may thus promote unrestrained growth.

Behavior of Rhizobium meliloti in oxygen gradients.

Rhizobium meliloti cells responded to an abrupt change in oxygen concentration by changing the cell speed (chemokinesis), but they did not alter the frequency at which swimming cells stopped briefly (aerotaxis). Changes in cell speed upon stimulation with oxygen coincided with changes in membrane potential. The cells did not form an aerotactic band in a spatial gradient of oxygen as do the cells of other bacterial species. The fixL and fixJ genes which encode a heme-containing protein kinase that senses oxygen and a response regulator, respectively, were not involved in the behavior of R. meliloti in oxygen gradients.

Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter.

FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation genes during symbiosis with alfalfa. FixJ is a positive activator required for transcription of the regulatory genes nifA and fixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphorylation status of both itself and FixJ, in response to oxygen availability. In this study, we isolated four FixJ mutants that display increased activity at the nifA promoter (PnifA) in Escherichia coli. All four mutants possess amino acid changes in a domain of FixJ that is conserved in other response regulator proteins, and all exhibit increased activity at PnifA in R. meliloti that is dependent on the presence of FixL. One of the mutant proteins, while less efficient at accepting phosphate from a truncated derivative of FixL (FixL*), nevertheless has a phosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL*. The wt FixJ-phosphate was found to have a half-life of approximately 4 h, which makes it an unusually long-lived response regulator protein. The exceptional stability of wt FixJ-phosphate and the altered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.

Autophosphorylation and phosphatase activities of the oxygen-sensing protein FixL of Rhizobium meliloti are coordinately regulated by oxygen.

The FixL and FixJ proteins of Rhizobium meliloti control the expression of other nif and fix genes in response to oxygen levels. FixL is a hemoprotein kinase that senses oxygen availability and responds to the absence of oxygen by activation of its autophosphorylating activity followed by transfer of the phosphate to FixJ. FixJ in turn activates the nifA and fixK promoters. In vitro studies reported here with a soluble truncated version of FixL (FixL*) indicate that, while low oxygen tension specifically increases the autophosphorylating activity of FixL*, the ability of phospho-FixL* to act as a phosphate donor to FixJ is not affected by the presence or absence of oxygen. FixL* is also shown to possess a phosphatase activity that is repressed under anaerobic conditions only when the protein is in the phosphorylated form. A fixL mutant that induces a higher level of nifA promoter activity in the presence of fixJ in vivo displayed both an increased autophosphorylating activity and a decreased phosphatase activity in vitro. These data provide evidence for a role for both autophosphorylation and phosphatase activities of FixL in the mechanism by which oxygen tension within the alfalfa nodule induces expression of bacterial nitrogen fixation genes during symbiosis.

The oxygen sensor FixL of Rhizobium meliloti is a membrane protein containing four possible transmembrane segments.

Regulation of nitrogen fixation genes in Rhizobium meliloti is mediated by two proteins, FixL and FixJ, in response to oxygen availability. FixL is an oxygen-binding hemoprotein with kinase and phosphatase activities that is thought to sense oxygen levels directly and to transmit this signal to FixJ via phosphorylation-dephosphorylation reactions. FixJ controls the expression of other regulatory genes, including nifA, that regulate the transcription of genes required for symbiotic nitrogen fixation. We have been studying the structural and functional features of FixL that are required for oxygen sensing. We constructed mutant derivatives and confirmed that FixL consists of 505 amino acids instead of 464, as originally reported. Hydropathy plots of the full-length protein, together with TnphoA insertional analysis, lead us to propose that FixL is likely to be a polytopic integral membrane protein containing four membrane-spanning segments. We have also constructed an N-terminal deletion of the FixL protein whose in vivo activity indicates that the hydrophobic membrane-spanning regions are not absolutely required for oxygen sensing in vivo. We also report that FixL shares homology in its N terminus with other sensor proteins, including KinA from Bacillus subtilis and NtrB from Bradyrhizobium parasponia. The region of homology comprises a 70-amino-acid residue stretch that is also conserved in two oxygenases, P-450 and isopenicillin synthase.

Isolation of phosphorylation-deficient mutants of the Rhizobium meliloti two-component regulatory protein, FixJ.

Rhizobium meliloti FixL and FixJ are members of a symbiotically essential two-component system that regulates nitrogen-fixation genes in response to environmental oxygen concentrations. FixL is a membrane protein that is thought to relay information about oxygen availability to FixJ via a phosphotransfer mechanism. FixJ increases expression of the nifA and fixK genes by activating transcription of the nifA and fixK promoters (p-nifA and p-fixK, respectively). In this study, we examined the relationship between the in vivo activity of FixJ as a transcriptional regulator and its ability to be phosphorylated in vitro by the sensor FixL. FixJ mutants were isolated that showed decreased activity on p-nifA in Escherichia coli. Most of the FixJ mutant proteins also showed decreased activity on the fixK promoter. These mutants were analysed in R. meliloti for activity on p-nifA during vegetative growth, where similarities and differences were observed when compared with their phenotypes in E. coli. Three mutants showing significantly less activity in R. meliloti were examined for symbiotic activity in planta and were found to be ineffective. When these three mutant FixJ proteins were examined in vitro for their ability to be phosphorylated by FixL, two mutants were found to have a significantly decreased ability to accept phosphate from FixL. These findings are discussed in relation to signal transduction in the FixLJ system.

Regulation of expression of the casbene synthetase gene during elicitation of castor bean seedlings with pectic fragments.

A castor bean cDNA library has been constructed in the expression vector lambda gt11. Screening of 30,000 plaques with antibodies against casbene synthetase yielded six positive clones, from which three purified clones were obtained after sequential screening. The cDNA inserts in these clones ranged in size from 200 to 500 base pairs and were shown to share homologous sequences. One of the clones (pCS4) was examined in hybrid-selected and hybrid-arrested translation experiments and shown to contain a partial sequence of the casbene synthetase coding region. Northern analysis using the pCS4 clone as a probe revealed that the total hybridizable casbene synthetase mRNA amount increased to a maximum at 6 h after treatment of seedlings with pectic fragment elicitor and then decreased steadily with almost the same kinetics as observed previously for the changes in the translatable casbene synthetase mRNA activity under these same conditions. Run-on transcription experiments were performed with nuclei isolated from castor bean seedlings at various time periods after treatment with pectic fragment elicitors. Transcription of the casbene synthetase gene was first detected at 2 h after elicitation, rose to a maximum at 5 h, and fell rapidly thereafter. These results suggest that the accumulation of casbene synthetase, and hence casbene, is governed primarily by the rate of transcription of the casbene synthetase gene during elicitation by pectic fragments.