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The complete nucleotide sequence of the Bacillus subtilis dnaB gene and its flanking regions was determined. The dnaB gene is essential for both replication initiation and membrane attachment of the origin region of the chromosome and plasmid pUB110. It has been known that there are two different classes (dnaBlI and dnaBII) in the dnaB mutants; dnaBI is essential for both chromosome and pUB110 replication, whereas dnaBII is necessary only for chromosome replication. The nucleotide sequence revealed that dnaBI and dnaBII are two functional domains in the single dnaB gene. The mutation sites of two mutants, belonging to dnaBI and dnaBII, respectively, were also determined as substitutions of amino acids. The putative DnaB protein deduced from nucleotide sequence consists of 472 amino acids (55 kDa) with no cysteine residue. A 55-kDa polypeptide produced in an in vitro transcription-translation system was labeled with (1), which corresponds to the position 255/360 from the origin (2). A fine mapping analysis using genetic transformation revealed two subunits that have a small but significant distance (0.3% recombination frequency) between them. Thus, it was concluded that either there are two different dnaB genes next to each other or there are two domains in the gene dnaB; these two regions were previously designated as dnaBI and dnaBII (1).Function of the dnaB gene has been extensively studied in both classes of mutants, dnaBI [dna-134 (3), dna-1 (4), dna-27 (5)] and dnaBII [dnaBl9 (6, 7)]. Both classes are clearly initiation mutants; when the temperature is raised to 45-48°C, the new round of replication initiation at the chromosomal origin is completely inhibited in both classes of mutants. In contrast, elongation at the preexisting replication forks continues until they reach the terminus. At least one round of initiation was recovered, without new protein synthesis, by bringing the culture to the normal temperature (4).Preferential attachment of the replication origin and the terminus of the bacterial chromosome was first observed in B. subtilis (8) and subsequently has been observed both in B. subtilis and in Escherichia coli (for reviews, see refs. 9 and 10). At the nonpermissive temperatures, the replication origin area of the B. subtilis chromosome detaches from the membrane in both dna-1 (dnaBI) and dnaBJ9 (dnaBII) mutants (7). The detachment has been shown both in vivo and in vitro. Subsequently, it was shown (7) that both the replication and membrane attachment of pUB110, a highcopy-number plasmid (-50 copies per cell) originally isolated from Staphylococcus aureus (11), require the function of the dnaBI region. In contrast, neither the replication nor the membrane attachment of pUB110 requires the function of the dnaBII region (7). These results indicate a functional distinction between the two regions. Whether the initiation or elongation of pUB110 replication is controlled by dnaBI has not been determined.The above results show that the function of the dnaB gene is essential not only for t...

RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.

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DNA-membrane association critical for initiation of DNA replication in Bacillus subtilis can be classified into two types. Type I is salt resistant and dependent on the initiation gene, dnaB, and type II is salt sensitive and independent of the dnaB gene. We found and sequenced two adjacent areas of type II binding within 1% of oriC on the B. subtiis chromosome.

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