The pem locus is responsible for stable maintenance of plasmid R100 and consists of two genes, pemI and pemK. The pemK gene product is a growth inhibitor, while the pemI gene product is a suppressor of this inhibitory function. We found that the PemI amino acid sequence is homologous to two open reading frames from Escherichia coli called mazE and orf-83, which are located at 60 and 100 min on the chromosome, respectively. We cloned and sequenced these loci and found additional open reading frames, one downstream of each pemI homolog, both of which encode proteins homologous to PemK. The pem locus homolog at 60 min was named chpA and consists of two genes, chpAI and chpAK; the other, at 100 min, was named chpB and consists of two genes, chpBI and chpBK. The distal portion of chpBK was found to be adjacent to the ppa gene that encodes pyrophosphatase, whose map position had not been previously determined. We then demonstrated that the chpAK and chpBK genes encode growth inhibitors, while the chpAI and chpBI genes encode suppressors for the inhibitory function of the ChpAK and ChpBK proteins, respectively. These E. coli pem locus homologs may be involved in regulation of cell growth.

A circadian clock located in the suprachiasmatic nucleus (SCN) regulates the period of physiological and behavioural rhythms to approximately 24 h. Lithium can lengthen the period of circadian rhythms in most organisms although little is known about the underlying mechanism. In the present study, we examined Drosophila shaggy ortholog glycogen synthase kinase-3 (GSK-3) protein expression in the SCN after lithium treatment. When locomotor activity was assessed, we found an association between the effect of lithium and the period of circadian oscillation as well as the level of GSK-3 protein expression. The decreased expression of GSK-3 and increased expression of phosphorylated GSK-3 (pGSK-3) resulted in an antiphasic circadian rhythm between the two in the SCN of lithium-treated mice housed under both light-dark and constant dark conditions. The enzyme activity of GSK-3 in the SCN was low when the level of pGSK-3 protein was high, as examined by immunoblotting analysis. Thus, GSK-3 enzyme activity has a correlation with the expression of GSK-3 protein in the SCN. Although both GSK-3 and pGSK-3 proteins are also expressed in the arcuate nucleus, lithium did not affect their expression. Based on the association that we found between lengthened circadian period and GSK-3 protein and GSK-3 activity in the SCN, we suggest that GSK-3 plays a role in regulating the period of the mammalian circadian pacemaker.

No abstract

Fibroblast growth factor (FGF)-9 is a glycosylated neurotrophic polypeptide highly expressed in brain. The mechanism for its secretion from expressing cells is unclear, because its primary structure lacks a cleavable signal sequence. We, therefore, investigated the mechanism and structural requirements for secretion of FGF-9. As with other secreted proteins, in vitro translation of FGF-9 was inhibited by signal recognition particle, which binds to the signal sequence. When translated in vitro, full-length FGF-9 was translocated into microsomes, glycosylated, and protected from trypsin digestion. By using various FGF-9 deletion mutants, we found that two hydrophobic domains, located at the N terminus and at the center of the FGF-9 primary structure, were crucial for translocation. Examination of various point mutants revealed that local hydrophobicity of the central hydrophobic domain, but not the N terminus, was crucial for translocation. Analogous results were obtained with respect to FGF-9 secretion from transfectant cells. Upon deletion of the complete sequence preceding it, the previously uncleavable hydrophobic domain appeared to serve as a cleavable signal sequence. Our results suggest that nascent FGF-9 polypeptides translocate into endoplasmic reticulum without peptide cleavage via a co-translational pathway in which both the N terminus and the central hydrophobic domain are important; thereafter, FGF-9 is glycosylated and secreted.