Faculty Bibliography

Endoproteinase Asp-N cleaves the 581-amino acid Escherichia coli primase (65,564 Da) into several major fragments. One of these, a 47-kDa fragment containing the complete N terminus and the first 422 amino acids of primase, is capable of primer RNA (pRNA) synthesis in the G4oric/single-stranded DNA binding protein/primase pRNA synthesis system. A cloned 398-amino acid N-terminal fragment of primase can also synthesize pRNA. The sizes of the pRNA synthesized by these N-terminal fragments, however, are smaller than those synthesized by intact primase, suggesting that the C-terminal region of primase plays a role in processivity or regulation of pRNA synthesis. Primase mutants with the last 10 and 40 C-terminal amino acids deleted synthesize pRNA as wild-type primase, indicating that any regulatory sequences must be internal to the C terminus of primase

The rpsU-dnaG-rpoD operon messenger RNA that encodes S21, primase, and sigma-70 is cleaved under normal physiological conditions. The dnaG coding portion of the mRNA is then rapidly degraded. An endonuclease activity has been isolated from wild type Escherichia coli cells that cleaves dnaG mRNA. This activity has been identified as RNase E, and the identity confirmed by the accumulation of the unprocessed operon polycistronic mRNA in RNase E mutants, rne-3071 and ams-1, when incubated at nonpermissive temperatures. Extracts prepared from RNase E mutant strains failed to cleave dnaG mRNA in vitro. The dnaG mRNA RNase E cleavage site (5'-AAGUGAUUUA-3') is only 50% homologous to the ribosomal RNA RNase E cleavage site. By using computer programs predicting secondary structure, the dnaG RNase E cleavage site appears to be in a single stranded RNA loop

The origin of phage G4 DNA complementary strand synthesis (G4oric) consists of three stem-loop structures (stem loops I, II, and III) that have been proposed as a recognition site for primase during primer RNA (pRNA) synthesis (Godson, G. N., Barrell, B. G., Staden, R., and Fiddes, J. C. (1978) Nat. New Biol. 276, 236-247; Fiddes, J. C., Barrell, B. G., and Godson, G. N. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1081-1085; Sims, J., Capon, D., and Dressler, D. (1979) J. Biol. Chem. 254, 12615-12628). It is generally considered that the double-stranded DNA stem-loop structure is not coated with Escherichia coli single-stranded DNA-binding protein (SSB), but is recognized by primase as naked DNA (Kornberg, A., and Baker, J. (1992) DNA Replication, 2nd Ed., p. 280, W. H. Freeman & Co., New York). Using small G4oric single-stranded DNA fragments of various sizes (302, 278, 149, and 100 nucleotides) consisting of the core 100-nucleotide stem-loop region plus differing lengths of 3'- and 5'-flanking sequence as substrates for gel retardation and DNase I and micrococcal nuclease digestion, we show that under conditions of pRNA synthesis, two SSB tetramers bind to the stem-loop structure. With increasing lengths of 5'- and 3'-flanking sequence, more SSB tetramers are added. Regardless of the number of SSB tetramers bound, however, the region of DNA containing the pRNA initiation site is always left accessible to nuclease digestion. In situ copper-phenanthroline footprinting of individual gel shift assembly intermediates shows that on the 302-nucleotide G4oric, the first two SSB tetramers assemble at random, but the addition of more SSB tetramers results in formation of a unique structure. In this structure, SSB tetramers protect both sides of stem loop III plus the intervening region between stem loops III and I, but leave most of stem loop I and the CTG pRNA initiation site accessible to copper-phenanthroline. Primase can only synthesize pRNA when the stem-loop structure is saturated with SSB and presumably in the unique configuration. The G4oric stem-loop structure therefore appears to dictate the phasing of SSB to leave a primase recognition site as free DNA

To facilitate the overexpression of Escherichia coli primase, the dnaG gene has been reconstructed using polymerase chain reaction to remove the 5' transcription terminator and the 3' RNA processing site. This construct was cloned into the T7 polymerase-transcribed expression vector, pET-3d. Cells containing the resulting plasmid (pGNG1) express up to 30% of the cellular protein as primase. The pGNG1-encoded primase has normal activity in synthesizing primer RNA on a single-stranded DNA template in vitro. Plasmid pGNG1 can also be used to synthesize [35S]methionine-labelled primase in in vitro transcription-translation systems. In addition, the small amount of transcription in the absence of T7 polymerase is sufficient to complement temperature-sensitive and amber dnaG chromosomal mutations in vivo. Plasmid pGNG1 can therefore be used not only to overproduce wild-type primase, but to change and manipulate the primase structure in vivo and in vitro. These mutant proteins can be overproduced and used for structural and functional studies

This report describes the cloning and sequencing of a chromosomally encoded tetracycline resistance determinant from a clinical isolate of methicillin-resistant Staphylococcus aureus. On the basis of the sequence, the gene is in the tet(M) class, and it was shown that the S. aureus tetA(M) gene is induced at the level of transcription

45 mutations (insertion, deletion and base substitution) of the G4 Goric were tested for their functional activity in M13 and R199 in vivo. The critical mutants were also assayed for their ability to synthesize pRNA in vitro using SSB and primase. The results demonstrate that the secondary structure and spacing of stem-loops I and III are essential for Goric activity and that the 5'-CTG-3' sequence flanking stem-loop I is essential for initiation of pRNA synthesis

Nucleotide sequences of Plasmodium knowlesi DNA that are cleaved by mung bean nuclease (Mbn) at low enzyme concentration (0.2 units enzyme per micrograms DNA) are listed. They are tandemly repeated purine/pyrimidine (RpY) stretches of DNA with (ApT) dimers predominating. Most cut sites are within almost 100% RpY tracts. The enzyme cleaves at many points within the RpY stretch and usually hydrolyzes the 5'-ApT-3' linkage. These alternating RpY target sites are flanked by homopurine and homopyrimidine stretches. At least one Mbn target site lies next to an in vivo transcribed region

An oligonucleotide probe (315) specific for the alpha- and beta-tubulin genes of Plasmodium falciparum was synthesized utilizing codon usage of P. falciparum determined from published gene sequences. By screening genomic and cDNA libraries with the oligonucleotide probe, alpha- and beta-tubulin clones were isolated. Positive clones were identified by partial sequencing and comparing the deduced amino acid sequence with the chicken brain alpha- and beta-tubulin amino acid sequences. The beta-tubulin gene was completely sequenced at the genomic level and partially at cDNA level. The deduced polypeptide is 445 amino acids long, shares 88% homology with chicken brain beta-tubulin, and contains two introns of 362 and 163 bp long, respectively. alpha- and beta-tubulin genes of P. falciparum are unlinked and dispersed; more than one copy of each gene may be present. Northern blot analysis of total RNA of the blood-stage parasite indicates the presence of three transcripts of alpha-tubulin (3.3 kb, 2.6 kb, 1.9 kb) and three transcripts of beta-tubulin gene (3.6 kb, 2.9 kb, 2.0 kb). The significance of these transcripts is presently unknown

Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4oric, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA. In a previous publication [Sakai et al., Gene 71 (1988) 323-330], we suggested that base pairing between the loops of stem-loops I, and II, and/or II and III, might play a role in G4oric function. To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing. These mutations, however, did not seriously affect G4oric activity. This indicates that base pairing between the loops is not essential for G4oric functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4oric activity. To complete an analysis of the effects of altering the structure of the G4oric stem-loops, insertions were made into stem-loop III. In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4oric activity

The primase-dependent phage G4 origin of complementary DNA strand synthesis (G4oric) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA). Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4oric pRNA template region. Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4oric activity. However, functional G4oric activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I. Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4oric functional activity. These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4oric function