Genes involved in the biogenesis of the flagellum in Caulobacter crescentus are expressed in a temporal order and are controUed by a trans-acting regulatory hierarchy. Strains with mutations in one of these genes, flaS, cannot transcribe flageUlar structural genes and divide abnormally. This gene was cloned, and it was found that its transcription is initiated early in the cell cycle. Subclones that restored motility toflaS mutants also restored normal cell division. Although transcription offlaS was not dependent on any other known gene in the flagellar hierarchy, it was autoregulated and subject to mild negative control by other genes at the same level of the hierarchy. An additional level of control was revealed when it was found that an interruption of DNA replication caused the inhibition offlaS transcription. TheflaS transcript initiation site was identified, and an apparently unique promoter sequence was found to be highly conserved among the genes at the same level of the hierarchy. The flagellar genes with this conserved 5' region all initiate transcription eariy in the cell cycle and are all sensitive to a disruption in DNA replication. Mutations in these genes also cause an aberrant cell division phenotype. Therefore, flagellar genes at or near the top of the hierarchy may be controlled, in part, by a unique transcription factor and may be responsive to the same DNA replication cues that mediate other cell cycle events, such as cell division.Each Caulobacter crescentus cell division produces morphologically distinct progeny that exhibit different programs of gene expression ( Fig. 1) (14, 23, 40). The progeny stalked cell immediately initiates DNA replication, whereas chromosome replication is delayed in the progeny swarmer cell until its transition to a stalked cell later in the cell cycle (12,34). This transition is marked by the loss of the polar flagellum and initiation of stalk formation at the site previously occupied by the flagellum. Shortly after DNA replication begins, the stalked cell embarks upon the ordered transcription of a group of flagellar genes that culminates in the biogenesis of a new flagellum at the cell pole opposite the stalk.The biogenesis and function of the C. crescentus flagellum require the coordinated expression of at least 48 genes (17). These genes are organized in a trans-acting regulatory hierarchy in which the temporal order of transcription of the structural genes reflects the order of flagellar component assembly (14,23,40). Differential activation of or'4 promoters contributes to the temporal sequence of class II and class III flagellar gene transcription (13,22,24,(37)(38)(39)43) and the localized positioning of flagellar gene products (21,33). An additional level of control was revealed when Newton and coworkers showed that the synthesis of the hook protein and the flagellins is dependent on DNA replication (45)(46)(47)(48)(49)52).

Each Caulobacter crescentus cell division generates distinct progeny cells: a stalked cell which is competent for DNA replication, and a motile swarmer cell which initiates DNA replication only after it sheds its flagellum and differentiates into a stalked cell later in the cell cycle. Following the initiation of DNA replication, the flagellar transcriptional hierarchy is activated, culminating in the assembly of a single flagellum at the cell pole opposite that bearing the stalk. The biogenesis of the flagellum is a landmark in the establishment of asymmetry in the predivisional cell (Fig. 1).A large number of the flagellar structural and regulatory genes have been grouped into classes (Fig. 1) that form a regulatory hierarchy (7,9,11,43,62). We have preliminary evidence that the class I genes respond to cell cycle cues (44a). The class II flagellar genes are transcribed early in the cell cycle and are required for the transcription of genes in classes III and IV. Two class II genes, rpoN and flbD, encode a sigma factor ( 54 ) (6), and a transcriptional activator, FlbD (45, 60), respectively, which are required directly for the transcription of class III and class IV genes. Other class II genes encode proteins required for flagellar assembly and function (24, 45a, 65). The order of flagellar gene expression approximates the order of assembly of the gene products into the nascent structure (11,13,17,43,62). In C. crescentus, Salmonella typhimurium, and Escherichia coli, if flagellar assembly is aborted, transcription of the remaining flagellar genes is blocked, suggesting that the two processes are coupled (19,23,24,31).We reported previously that a nonmotile mutant with a deletion in the flaS locus not only diminished or abolished the transcription of class III and class IV genes but also exhibited a cell division defect (13). To understand the role of this class II flaS locus in the assembly of the flagellum and in cell divi- At each division, a predivisional cell gives rise to two different progeny, a stalked cell and a motile swarmer cell. As the cell cycle proceeds, the swarmer cell sheds its flagellum and assembles a stalk at the previously flagellated pole. The new stalked cell begins to divide, assembling a new flagellum at the pole opposite the stalk. The flagellar genes are expressed in an order that reflects their positions in the regulatory hierarchy and the order of assembly of their protein products into the flagellum (7,42). Arrows indicate positive regulation, such that transcription of each class of genes requires the expression of the gene products of the preceding class. Both rpoN and flbD encode transcription proteins (6, 45), whereas other class II genes appear to encode proteins involved in the assembly, structure, and function of the flagellum (24, 65).

No abstract

Half-smooth tongue sole (Cynoglossus semilaevis: Pleuronectiformes) is a commercially important cultured marine flatfish in China and forms an important fishery resource, but the research of its genome is underdeveloped. In this study, we constructed a female C. semilaevis fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 49,920 clones with an average insert size of about 39 kb, amounting to 3.23 genome equivalents. Fosmid stability assays indicate that female C. semilaevis DNA was stable during propagation in the fosmid system. Library screening with eight microsatellite markers yielded between two and five positive clones, and none of those tested was absent from the library. End-sequencing of both 5' and 3' ends of 1,152 individual clones generated 2,247 sequences after trimming, with an average sequence length of 855 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 259 (11.53%) and 287 (12.77%) significant hits (E < e (-5)), respectively. Repetitive sequences analysis resulted in 5.23% of base pairs masked using both the Fugu and Danio databases, repetitive elements were composed of retroelements, DNA transposons, satellites, simple repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for large-scale genome sequencing, physical mapping, and positional cloning, and provide a better understanding of female C. semilaevis genome.