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Biomedical subjects

G N Godson

Publications and source records attributed to G N Godson.

At least 19 recordsLinked to original sources
PubMed1991

An over-expression plasmid for Escherichia coli primase.

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.

Amino Acid Sequence↗
PubMed1990

Structural features of the priming signal recognized by primase: mutational analysis of the phage G4 origin of complementary DNA strand synthesis.

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.

Bacteriophages↗
PubMed1990

Hypersensitive mung bean nuclease cleavage sites in Plasmodium knowlesi DNA.

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.

Animals↗
PubMed1990

Isolation of alpha- and beta-tubulin genes of Plasmodium falciparum using a single oligonucleotide probe.

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.

Amino Acid Sequence↗
PubMed1990

Cloning and nucleotide sequence of a chromosomally encoded tetracycline resistance determinant, tetA(M), from a pathogenic, methicillin-resistant strain of Staphylococcus aureus.

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.

Amino Acid Sequence↗
PubMed1989

Mutational analysis of the primer RNA template region in the replication origin (oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase.

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.

Base Sequence↗
PubMed1989

Distinct functional contributions of three potential secondary structures in the phage G4 origin of complementary DNA strand synthesis.

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.

Base Sequence↗
PubMed1989

DNA----DNA, and DNA----RNA----protein: orchestration by a single complex operon.

In Escherichia coli, the workhorse of molecular biology, a single operon is involved in the replication, transcription and translation of genetic information. This operon is controlled in a complex manner involving multiple cis-acting regulatory sequences and trans-acting regulatory proteins. It interacts with global regulatory networks by mechanisms which are presently being dissected.

Amino Acid Sequence↗
PubMed1989

An Escherichia coli cis-acting antiterminator sequence: the dnaG nut site.

The Escherichia coli rpsU-dnaG-rpoD operon contains an internal transcription terminator T1 located in the intergenic region between the rpsU and dnaG genes (Smiley et al. 1982). By cloning T1 as a small 127 bp fragment into the terminator probe plasmid pDR720 between the trp operator promoter and the assayable galK gene, it was shown that T1 acts as a strong transcription terminator, comparable in strength to the 3' operon terminator T2. However, an operon sequence that occurs 5' to T1 within the coding region of the rpsU gene and which has homology with the lambda nut site, (Lupski et al. 1983) when placed 5' to T1 in the pDR720 plasmid construct, modifies transcription through T1 allowing expression of the galK gene. This sequence, called the dnaG nut site also modifies the termination activity of the external operon terminator T2. It is proposed that the dnaG nut site is a cis-acting element of an antitermination system in E. coli.

Base Sequence↗
PubMed1988

Role of the potential secondary structures in phage G4 origin of complementary DNA strand synthesis.

Phage G4 origin of complementary DNA strand synthesis (oric) consists of three stable stem-loop structures (I, II, and III). Mutant oric sequences with alterations in the structure of stem-loop II, stem-loop III, and the stem-loop II-III spacer region have been constructed and cloned into the filamentous phage vectors to assay their functional activity. Changes in the lowermost GC base pair in the stem of stem-loop III, in the 9-bp spacer region between the stems of stem-loops II and III, and in the loop of stem-loop II, impair or abolish in vivo oric function. The results suggest that recognition sequences for dnaG primase must be present in the loop of stem-loop II, and in the spacer region between the stems of stem-loops II and III.

Bacteriophages↗
PubMed1988

Role of the 5' upstream sequence and tandem promoters in regulation of the rpsU-dnaG-rpoD macromolecular synthesis operon.

Bal31 exonuclease deletion analysis and transposon Tn5 mutagenesis of the 5' regulatory region of the rpsU-dnaG-rpoD macromolecular synthesis operon fused to the chloramphenicol acetyltransferase gene (pGLR301) demonstrated that sequences 5' to the operon promoters were not involved in operon transcriptional regulation and that the three tandem promoters P1, P2, and P3 were functionally independent. P2 was the strongest promoter, and P3 was the weakest. P1, P2, and P3 acting in combination appeared to be stronger than the individual promoters.

Bacteriophage lambda↗
PubMed1987

Possible new genes as revealed by molecular analysis of a 5-kb Escherichia coli chromosomal region 5' to the rpsU-dnaG-rpoD macromolecular-synthesis operon.

The complete nucleotide sequence of 5-kb DNA fragment immediately 5' to the rpsU-dnaG-rpoD macromolecular-synthesis operon in Escherichia coli has been determined. It encodes for six open reading frames. Transcriptional and translational analysis have shown that three of them (orfx, orfz1 and orfz2) are expressed in exponentially growing E. coli cells. The orfx, directly 5' to the rpsU gene but transcribed in the opposite direction, may be part of the rpsU-dnaG-rpoD macromolecular-synthesis operon.

Base Sequence↗
PubMed1986

Immunogenicity of the repetitive and nonrepetitive peptide regions of the divergent CS protein of Plasmodium knowlesi.

The circumsporozoite (CS) protein of the Nuri strain of the simian malarial parasite Plasmodium knowlesi was expressed as a fusion protein in E. coli. This fusion protein cross-reacted with the polyclonal monkey sera raised against irradiated sporozoites of another strain (H strain) of P. knowlesi. The antibody against the repeat units of the H strain CS protein was affinity purified from the polyclonal sera by using synthetic repeat peptides. The affinity-purified antibody did not cross-react with the Nuri CS fusion protein. The immunogenicity of different regions of the CS protein was additionally studied by using several synthetic peptides. All but the most COOH-terminal peptide showed cross-reactivity with the polyclonal sera. Because the repeat regions of the CS protein of the two strains are diverse, whereas the non-repetitive regions are immunogenic and conserved, the latter may be better suited for a potential vaccine.

Amino Acid Sequence↗
PubMed1986

Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase.

The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction.

Amino Acid Sequence↗
PubMed1986

A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect.

In the process of randomly mutagenizing a recombinant pBR322 clone with transposon Tn5, a high copy number plasmid mutant, pLO88, has been isolated. The copy number phenotype of pLO88 is observed only at elevated temperatures, greater than or equal to 37 degrees C, and is due to the precise position of a Tn5 insertion. Nucleotide sequence of the Tn5-pBR322 junction reveals that Tn5-88 has inserted into an open reading frame that codes for a 63 amino acid protein previously shown to negatively regulate pBR322 plasmid copy number. By deleting portions of the Tn5 it is shown that the copy number phenotype is due not only to the insertion of Tn5 in pBR322 but also to the requirement that some Tn5 sequences remain intact. It appears that an outwardly directed Tn5 promoter initiates the synthesis of a transcript (RNA X) that interferes with the normal repressor RNA (RNA I)-primer RNA (RNA II) interaction at elevated temperatures.

DNA Replication↗
PubMed1985

Isolation and construction of mutants of the G4 minus strand origin: analysis of their in vivo activity.

An active, rifampicin-resistant primase-dependent bacteriophage G4 origin of complementary DNA strand synthesis has been cloned as a 274 bp fragment into the filamentous phase M13 and its secondary structure altered by deletion and insertion. It has been found that the entire 136 bp G4 intergenic region containing the secondary structure loops I and III is necessary for rifampicin-resistant conversion of SS----RF DNA in vivo. The secondary structures, however, can be widely separated by insertion between them of both random DNA sequences, and sequences that form strong additional secondary structure configurations and the origins still retain activity. Primase therefore probably recognises two DNA domains on loops I and III, the physical separation of which is not important.

Base Sequence↗
PubMed1985

Comparison of left-end DNA sequences of bacteriophages Mu and D108.

The nucleotide sequences of the left ends of bacteriophage Mu DNA and that of its close relative D108 have been determined. The first 100 bp of phages Mu and D108 are substantially the same except for an octanucleotide change from bp 53 to 61 and other small interspersed base-pair changes from bp 61 to 200. The first five host nucleotides preceding the host-phage junction are generally, but not always, G + C-rich and these five nucleotides display no obvious consensus sequence. Both phages Mu and D108 share striking similarity in their end DNA sequences to the end sequences of the newly described Escherichia coli movable genetic element IS30.

Bacteriophage mu↗