Mutations in the spin gene are characterized by an extraordinarily strong rejection behavior of female flies in response to male courtship. They are also accompanied by decreases in the viability, adult life span, and oviposition rate of the flies. In spin mutants, some oocytes and adult neural cells undergo degeneration, which is preceded by reductions in programmed cell death of nurse cells in ovaries and of neurons in the pupal nervous system, respectively. The central nervous system (CNS) of spin mutant flies accumulates autofluorescent lipopigments with characteristics similar to those of lipofuscin. The spin locus generates at least five different transcripts, with only two of these being able to rescue the spin behavioral phenotype; each encodes a protein with multiple membrane-spanning domains that are expressed in both the surface glial cells in the CNS and the follicle cells in the ovaries. Orthologs of the spin gene have also been identified in a number of species from nematodes to humans. Analysis of the spin mutant will give us new insights into neurodegenerative diseases and aging.

Glycogen synthase kinase 3 (GSK3) plays important roles in Wnt and insulin signaling, cell fate determination, and Alzheimer-like tau phosphorylation. We discovered an isoform of tau protein kinase I (TPKI) / GSK3b with a 13 amino acid insert in the catalytic domain owing to alternative splicing. The alternative transcripts were found in the brains of the mouse, rat and human, with highly conserved sequences. The variant protein, named TPKI2 / GSK3b2, was abundant in the brain. Immunohistochemistry indicated differential distribution of the conventional and the new TPKI / GSK3b isoforms within young neurons. TPKI2 / GSK3b2 showed decreased kinase activities towards two phosphorylation sites on tau compared with the conventional isoform. Immunohistochemistry indicated that TPKI2 / GSK3b2 occurs predominantly in the neuronal soma, while TPKI1 / GSK3b1 is found both in the soma and processes. These results indicate that the new splice isoform has a different function. Because the amino acid insert occurs in the domain implicated in interaction with a protein phosphatase in a homologous kinase cdk-2, the alternative splicing can regulate multiprotein complex formation and function involving TPKI / GSK3b. Keywords: glycogen synthase kinase 3, protein kinase subdomain, splice variant, tau phosphorylation, tau protein kinase.

Most strains of Pseudomonas aeruginosa produce various types of bacteriocins (pyocins), namely, R-, F-, and S-type pyocins. The production of all types of pyocins was shown to be regulated by positive (prtN) and negative (prtR) regulatory genes. The prtN gene activates the expression of various pyocin genes, probably by the interaction of its product with the DNA sequences conserved in the 5' noncoding regions of the pyocin genes. The prtR gene represses the expression of the prtN gene, and its product, predicted from the nucleotide sequence, has a structure characteristic of phage repressors and seems to be inactivated by the RecA protein activated by DNA damage. A model for the regulation of the pyocin genes is proposed.

Pyocins S1 and S2 are S-type bacteriocins of Pseudomonas aeruginosa with different receptor recognition specificities. The genetic determinants of these pyocins have been cloned from the chromosomes of P. aeruginosa NIH-H and PAO, respectively. Each determinant constitutes an operon encoding two proteins of molecular weights 65,600 and 10,000 (pyocin S1) or 74,000 and 10,000 (pyocin S2) with a characteristic sequence (P box), a possible regulatory element involved in the induction of pyocin production, in the 5' upstream region. These pyocins have almost identical primary sequences; only the amino-terminal portions of the large proteins are substantially different. The sequence homology suggests that pyocins S1 and S2, like pyocin AP41, originated from a common ancestor of the E2 group colicins. Purified pyocins S1 and S2 make up a complex of the two proteins. Both pyocins cause breakdown of chromosomal DNA as well as complete inhibition of lipid synthesis in sensitive cells. The large protein, but not the pyocin complex, shows in vitro DNase activity. This activity is inhibited by the small protein of either pyocin. Putative domain structures of these pyocins and their killing mechanism are discussed.

The region adjacent to the 3' end of the recA gene is indispensable for normal cell division in a rec-2 strain of Pseudomonas aeruginosa when the recA gene is highly expressed. A putative protein encoded by this region may play a regulatory role(s) in recA function.

The recA gene of Pseudomonas aeruginosa has been isolated and its nucleotide sequence has been determined. The coding region of the recA gene has 1038 bp specifying 346 amino acids. The recA protein of P. aeruginosa showed a striking homology with that of Escherichia coli except for the carboxy-terminal region both at the nucleotide and amino acid level. The recA+-carrying plasmids restored the UV sensitivity and recombination ability of several rec mutants of P. aeruginosa. The precise location of the recA gene on the chromosome was deduced from the analysis of R' plasmids.

Functional domain structures of pyocins AP41, Si, and S2 were assigned by examining the functions of chimeric pyocins and deletion derivatives. Pyocins AP41, S1, and S2 are essentially composed of three domains, the receptor-binding domain, the translocation domain, and the DNase domain, in that order from the N terminus to the C terminus. The alignment of these domains is distinct from that in E2-group colicins with functions similar to those of these pyocins. Pyocins AP41 and S2 have a fourth domain between the receptor-binding and the translocation domains, which is dispensable for their killing functions.Pyocins Si, S2, and AP41 are the protease-sensitive bacteriocins most frequently found among Pseudomonas aeruginosa strains (19). They are distinguished by their different receptor specificities. Recently we have cloned and sequenced the genetic determinants for pyocins AP41, S1, and S2 (18,20). Each determinant for these three pyocins constitutes an operon encoding two proteins of different sizes, one responsible for killing (the killing protein) and the other conferring immunity to its own pyocin (the immunity protein). In the 5' upstream region of each operon, a characteristic sequence (a P box), a possible regulatory element for the induced pyocin production, is conserved (10,20). The molecular weights of the killing proteins are different for the pyocins (84,000, 65,600, and 74,000 for pyocins AP41, S1, and S2, respectively), whereas the immunity proteins are of similar sizes, around 10,000. As described previously (20), the amino acid sequences of the C-terminal halves of the killing proteins of pyocins AP41, Si, and S2 are highly conserved. Those of pyocins Si and S2 are identical except for one amino acid deletion (Si) or addition (S2) in this region. Furthermore, the distal ca. 130 amino acids show striking homology to the C-terminal sequences of E2-group colicins and possess DNase activity. The less highly conserved N-terminal halves have been suggested to be receptor-binding domains because these pyocins show different receptor specificities. In addition to causing breakdown of the chromosomal DNA, pyocins Si and S2, but not pyocin AP41, inhibit lipid synthesis in the susceptible bacteria, although their susceptible strains are not the same (16,18,20).In the study described here, we constructed various chimeric pyocins and domain-deleted pyocins on the basis of their sequence conservation and attempted to determine the functions of each domain by examining their functions in vivo and in vitro.JM109 (26), and MV1304 (25) were used as host strains. For preparation of pyocins and chimeric proteins, E. coli C600 carrying the appropriate plasmids was employed. P. aeruginosa PML1516d (SiS S2') (11) and its derivatives PML1567 (S1l S2r) (13) and PML1570 (Slr S2') (this study) were used as indicators for pyocins S1 and S2, and PA03092 (17) was used as an indicator for pyocin AP41. NIH3 (6) and its derivatives NIH3S1r, NIH3S2r, and NIH3AP41r (this study)were also used to determine the receptor specificity....

The genetic determinant for pyocin AP41, a bacteriocin produced by Pseudomonas aeruginosa, has been cloned. The determinant is located on the chromosome flanked by a pair of inverted repeats, forming a transposon-like structure (TnAP41). TnAP41 possesses some features characteristic of the Tn3 family of transposons. Based on a comparison with the structure of the corresponding region of the chromosome of a non-producer strain, we propose that P. aeruginosa has acquired pyocinogeny by the transposition of TnAP41 into the chromosome. The determinant comprises two ORFs encoding the protein subunits responsible for the killing action (the large component) and immunity (the small component). Amino acid sequences of the C-terminus of the large component (the deoxyribonuclease domain) and the immunity protein show remarkable homology to those of E2 group colicins, suggesting that these bacteriocins, which are produced by distantly related species, have originated from a common ancestor.