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A genetic screen for cell division cycle mutants of Caulobacter crescentus identified a temperature-sensitive DNA replication mutant. Genetic complementation experiments revealed a mutation within the dnaE gene, encoding the ␣-catalytic subunit of DNA polymerase III holoenzyme. Sequencing of the temperature-sensitive dnaE allele indicated a single base pair substitution resulting in a change from valine to glutamic acid within the C-terminal portion of the protein. This mutation lies in a region of the DnaE protein shown in Escherichia coli, to be important in interactions with other essential DNA replication proteins. Using DNA replication assays and fluorescence flow cytometry, we show that the observed block in DNA synthesis in the Caulobacter dnaE mutant strain occurs at the initiation stage of replication and that there is also a partial block of DNA elongation.The initiation of DNA replication is a critical step in the cell cycle and requires the cooperative activities of a large number of proteins. The molecular events leading to the initiation of DNA replication have been clearly defined in Escherichia coli (15,23). The process begins with the DnaA protein binding to conserved regions within the oriC region (DnaA boxes), leading to the formation of an open complex (4,14,17,21). Upon formation of this single-stranded region, the helicase, DnaB, is recruited to the origin locus, followed by the primase, DnaG (2, 4, 16). This enzyme forms short ribonucleotide primers upon which DNA polymerase III (Pol III) holoenzyme units can assemble to drive bidirectional chromosomal replication.The E. coli Pol III holoenzyme is composed of 10 subunits that can be organized into three functional groups (24,32,36,39,41,45). The Pol III core consists of the ␣-catalytic subunit, encoded by the dnaE gene (35, 59); the 3Ј-5Ј exonuclease subunit, ε (52); and a third subunit, , which is not conserved in bacteria and whose function is unknown (6,41,54,56). The core enzyme associates with the ring-shaped ␤ 2 sliding clamp that holds the enzyme to the DNA and increases its polymerizing efficiency (7,18,25,31,44,49,55,57). The subunit associates with the DnaB helicase and the Pol III ␣ subunit, which results in the connection of two core polymerases at the replication forks (30, 62) and hence facilitates simultaneous leading-and lagging-strand DNA synthesis (7,29). It is proposed that during the initiation event, a ␤ dimer assembles onto primed DNA template sites and that its subsequent association with the Pol III core enzyme tethers the polymerases to the DNA (31, 57).The initiation step of DNA replication has not been as well characterized in the gram-negative, alpha-purple bacterium Caulobacter crescentus, where DNA replication is tightly linked to cell division and differentiation. In this organism, asymmetric cell division gives rise to a swarmer cell, with a single flagellum at one pole and a nonmotile stalked cell (5, 19). The stalked cell eventually forms a predivisional cell, synthesizing a flagellum at the pole opposite ...

A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, the PATH antigen detection method, and light microscopy were compared for their capacity to detect and identify Plasmodium species. One hundred and thirty-six blood specimens obtained from patients suspected of having malaria were examined by each of the three methods. Forty-four specimens were positive for malaria using microscopy as the "gold standard". The sensitivity for nested PCR was 100%, and the specificity was 98%. For the detection of Plasmodium falciparum, the antigen detection method had a sensitivity of 100% and a specificity of 97%. Species identification obtained using PCR-RFLP was identical or superior to light microscopy in 42 cases (96%). Although the nested PCR-RFLP method was more sensitive and specific, the rapid turnaround time and high sensitivity of the antigen detection method makes it a useful adjunct to standard microscopy.

A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, the PATH antigen detection method, and light microscopy were compared for their capacity to detect and identify Plasmodium species. One hundred and thirty-six blood specimens obtained from patients suspected of having malaria were examined by each of the three methods. Forty-four specimens were positive for malaria using microscopy as the "gold standard". The sensitivity for nested PCR was 100%, and the specificity was 98%. For the detection of Plasmodium falciparum, the antigen detection method had a sensitivity of 100% and a specificity of 97%. Species identification obtained using PCR-RFLP was identical or superior to light microscopy in 42 cases (96%). Although the nested PCR-RFLP method was more sensitive and specific, the rapid turnaround time and high sensitivity of the antigen detection method makes it a useful adjunct to standard microscopy.