We have identified a DNA site involved in chromosome partitioning in B. subtilis. This site was identified in vivo as the binding site for the chromosome partitioning protein Spo0J, a member of the ParB family of partitioning proteins. Spo0J is a site-specific DNA-binding protein that recognizes a 16 bp sequence found in spo0J. Allowing two mismatches, this sequence occurs ten times in the entire B. subtilis chromosome, all in the origin-proximal approximately 20%. Eight of the ten sequences are bound to Spo0J in vivo. The presence of a site on an otherwise unstable plasmid stabilized the plasmid in a Spo0J-dependent manner, demonstrating that this site, called parS, can function as a partitioning site. This site and Spo0J are conserved in a wide range of bacterial species.

We previously described two mammalian secreted proteins, prokineticin 1 and prokineticin 2, that potently contract gastrointestinal smooth muscle. Prokineticin 1 has also been shown to promote angiogenesis by stimulating proliferation, migration, and fenestration of endocrine organ-derived endothelial cells. Here we report the cloning and characterization of two closely related G protein-coupled receptors as receptors for prokineticins. Expression of prokineticin receptors in heterologous systems shows that these receptors bind to and are activated by nanomolar concentrations of recombinant prokineticins. Activation of prokineticin receptors leads to mobilization of calcium, stimulation of phosphoinositide turnover, and activation of p44/p42 MAPK signaling pathways that are consistent with the effects of prokineticins on smooth muscle contraction and angiogenesis. mRNA expression analysis reveals that prokineticin receptors are expressed in gastrointestinal organs, endocrine glands, and other tissues. Diseases involving altered gastrointestinal (GI)1 motility are among the most common human disorders (1, 2). Understanding the physiological functions and mechanisms of action of GI motility regulatory factors is a prerequisite for developing effective treatments for GI motility disorders (3, 4). We have previously discovered two mammalian proteins, prokineticin 1 and 2 (PK1 and PK2, respectively), that are potentially important regulators of GI motility (5). Prokineticins are ϳ10-kDa cysteine-rich secreted proteins expressed in the GI tract and several other tissues. Recently a second function for PK1 was reported. This protein, which LeCouter et al. (6) named endocrine gland vascular endothelial growth factor, stimulates cell proliferation, migration, and fenestrations in several endothelial cell lines derived from endocrine glands. In addition, expression of PK1 from recombinant adenovirus injected into the mouse ovary stimulates angiogenesis (6). Thus, prokineticins regulate diverse biological functions that include contraction of GI smooth muscle and angiogenesis. Based on pharmacological evidence, we have demonstrated that a receptor(s) for prokineticins belongs to the family of G protein-coupled receptors (GPCRs) (5). Here we report the cloning of two closely related G protein-coupled receptors as receptors for prokineticins. EXPERIMENTAL PROCEDURES Cloning of Prokineticin Receptor 1 (PKR1) and ProkineticinReceptor 2 (PKR2) cDNAs-Human PKR1 and PKR2 sequences were identified in human sequence genome searches as described previously (7). Fulllength cDNAs were cloned from Marathon RACE Ready cDNA (CLON-TECH) by PCR. The PKR1 gene was amplified from testis Marathon RACE Ready cDNA using PCR and the following oligonucleotide primers: 5Ј-ggtgacatcagccttgcagacattgccc and 5Ј-ATGTGCATCCAAGCACA-CTAGTCAGTGTCC. This was followed by nested PCR using the following oligonucleotide primers: 5Ј-CACCATGGAGACCACCATGGG-GTTCATG and 5Ј-ATGTGCATCCAAGCACACTAGTCAGTGTCC. PKR2 was amplified from pooled testis and fetal brain Marathon...

smc of Bacillus subtilis encodes a homolog of eukaryotic SMC proteins involved in chromosome condensation, pairing, and partitioning. A null mutation in B. subtilis smc caused a temperature-sensitive-lethal phenotype in rich medium. Under permissive conditions, the mutant had abnormal nucleoids, ∼10% of the cells were anucleate, and assembly of foci of the chromosome partitioning protein Spo0J was altered. In combination with a null mutation in spo0J, the smc mutation caused a synthetic phenotype; cell growth was slower and ∼25% of the cells were anucleate. Our results demonstrate that the B. subtilis Smc protein, like its eukaryotic counterpart, plays an important role in chromosome structure and partitioning.

In B. subtilis, the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) regulate the initiation of sporulation. Soj is a negative regulator of sporulation gene expression, and Spo0J antagonizes Soj function. Using fusions of Soj to green fluorescent protein, we found that Soj localized near the cell poles and upon entry into stationary phase oscillated from pole to pole. In the absence of Spo0J, Soj was associated predominantly with DNA. By in vivo cross-linking and immunoprecipitation, we found that Soj physically associates with developmentally regulated promoters, and this association increased in the absence of Spo0J. These results show that Soj switches localization and function depending on the chromosome partitioning protein Spo0J. We further show that mutations in the Soj ATPase domain disrupt localization and function and render Soj insensitive to regulation by Spo0J.

We have determined the subcellular localization of the chromosome partition protein Spo0J of Bacillus subtilis by immunof luorescence microscopy and visualizing f luorescence of a Spo0J-GFP fusion protein. Spo0J was associated with a region of the nucleoid proximal to the cell pole, both in growing cells dividing symmetrically and in sporulating cells dividing asymmetrically. Additional experiments indicated that Spo0J was bound to sites in the originproximal third of the chromosome. These results show that the replicating chromosomes are oriented in a specific manner during the division cycle, with the Spo0J binding region positioned toward the cell poles. Experiments characterizing cells at different stages of the cell cycle showed that chromosome orientation is established prior to the initiation of cell division. Our results indicate that there is a mechanism for orienting the chromosomes and that the chromosome partition protein Spo0J might be part of a bacterial mitotic-like apparatus.