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Biomedical subjects

N Willetts

Publications and source records attributed to N Willetts.

At least 19 recordsLinked to original sources
PubMed1994

Microorganisms in the development of subunit vaccines against parasites.

Because of the increasing problems of resistance to chemicals and chemical residues, preventative vaccination has increasing appeal as a way to control parasite infestations in humans and in animals. Such vaccines are now feasible through the application of genetic engineering technology to allow production of parasite protective antigens in microorganisms in commercially viable quantities at an acceptable cost. This concept is illustrated by describing research toward subunit vaccines against human malaria (P. falciparum) and against the tropical cattle tick (B. microplus). Although the concept is straightforward, difficulties include the identification of a protective antigen, refolding of the initial microbial product to achieve the native conformation, and its formulation to produce a vaccine eliciting an adequate and appropriate immune response.

Animals↗
PubMed1987

Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region.

The entire region required for mobilization of the non-conjugative plasmid RSF1010 has been cloned into a mobilization-deficient pBR322 derivative. The segment of DNA cloned was approximately 1.8 kb and included the origin of conjugal DNA transfer (oriT). The DNA sequence of the mobilization region has been determined, and revealed the presence of several overlapping reading frames. The isolation and mapping of both Tn1725 and BamH1-linker insertions and comparison with the DNA sequence data has allowed the identification of three genes required for mobilization. Two of these genes are overlapping and encode proteins of 16 kDa and greater than 65 kDa (although the truncated protein is functional, the gene extends outside the region cloned). The third gene is transcribed in the opposite direction. Promoters capable of transcribing these genes were located by S1 mapping in the inter-cistronic region between these divergently transcribed genes. The oriT site is located in this region, and the transcriptional patterns observed for mob+ and mob- plasmids implied that the promoters may be regulated by two of the mobilization proteins binding to the oriT site.

Base Sequence↗
PubMed1984

The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks.

The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT , has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT-rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand-specific nicks acquired by a lambda oriT genome upon propagation in Flac-carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .

Base Sequence↗
PubMed1983

Regulation of the F conjugation genes studied by hybridization and tra-lacZ fusion.

Hybridization experiments and tra-lacZ fusions were used to obtain further insight into the complex series of control systems that affect F conjugation. We confirmed that the regular IncF FinOP control system represses transcription of traJ, and found that the traJ product is required for transcription of traM as well as of the traY-Z operon. The chromosomal sfrA gene product may be required to prevent premature termination of traJ transcription, while the sfrB gene product prevents premature termination at two sites within the traY-Z operon. The FinQ inhibition system determined by several IncI plasmids caused termination at three different sites in the operon, and that of JR66a at one further site. JR66a and R485 strongly inhibit F transfer, but have weak, or no (respectively) effects on transcription: they may inhibit function of one or more transfer gene products.

Conjugation, Genetic↗
PubMed1982

Cloning, mutation, and location of the F origin of conjugal transfer.

pED806 , a pBR322 derivative carrying the origin of transfer ( oriT ) of F, was rapidly lost from cells carrying an F tra+ plasmid. Instability was increased in a RecA- host, and depended in particular upon the Ftra YZ genes that produce the nick at oriT at which transfer is initiated. Instability was also correlated with the orientation of the oriT fragment in the vector plasmid. Mutants of pED806 selected as being stable in the presence of Flac proved to carry cis-dominant oriT mutations. The oriT site was subcloned from pED806 on a HaeII fragment including a HaeII-Bg/II segment of F DNA approximately 385 base pair (bp) long into the 2.25 kilobase (kb) vector plasmid pED825 , giving pED822 . pED822 was fully proficient for oriT function, and recircularised in recipient cells by a recA- and tra-independent oriT -specific ligation/recombination event. ' Phasmids ' constructed by cloning pED806 or an oriT - mutant into a lambda vector were used to confirm that the nick site in lambda oriT phages grown in the presence of Flac tra+ is indeed at oriT . The nick site in a further lambda oriT phage (ED lambda 102) was then located 140 +/- 20 bp from the Bg/II site forming one terminus of the F fragment cloned in pED806 and pED822 .

Base Sequence↗
PubMed1981

pED100, a conjugative F plasmid derivative without insertion sequences.

The largest HindIII fragment of F includes the entire replication and transfer regions, and its circularisation with ligase gave the conjugative plasmid pED100. This plasmid, which contains none of the F insertion sequences, was essentially unable to mobilise the E. coli chromosome or to give integrative suppression of a dnaA strain.

Chromosome Mapping↗
PubMed1981

sfrA and sfrB products of Escherichia coli K-12 are transcriptional control factors.

The mechanisms whereby mutations in Escherichia coli K-12 genes sfrA and sfrB reduce expression of the transfer functions of sex factor F have been examined by assaying the levels of tra messenger ribonucleic acid and of tra proteins. The sfrA product was necessary for efficient transcription of the control gene traJ and, directly or indirectly, for transcription of the traY leads to Z operon. In the absence of sfrA, reduced levels of the traJ and traT proteins were observed in the outer membrane. The sfrB product was needed to prevent premature transcription at one or more rho-dependent termination sites. sfrB mutations also reduced synthesis of full-length lipopolysaccharide molecules, of several chromosomally determined outer membrane proteins, and of functional flagella. Thus, the sfrB product may act as an antiterminator in transcription of several operons encording cell envelope components.

Bacterial Proteins↗
PubMed1980

Transfer-deficient cointegrates of Flac and lambda prophage.

Twelve transfer-deficient mutants of the plasmid Flac were obtained by insertion of prophage lambda into secondary attachment sites within the transfer region. Insertions into eight different tra genes were identified. These mutations were strongly polar on expression of tra genes previously mapped "downstream", and thus confirmed that the genes traA through traD form a single operon. However, some continued expression of traI suggested that this was transcribed in part from a promoter located between traD and traI, and in part from the transfer operon promoter. One insertion early in the transfer operon produced a plasmid-specific tra mutation not complemented by R100-1 or R1-19: this insertion was into a new gene (traY), located before traA as the first member of the transfer operon. Partial tra deletion mutants were obtained as 42 degrees C--survivors from several of the Flac tra::ED lambda 4 plasmids, and their properties are described.

Bacteriophage lambda↗
PubMed1980

Interactions between the F conjugal transfer system and CloDF13::Tna plasmids.

It was confirmed that all the F transfer genes required for the formation of stable mating pairs, including traN and traG, are essential for transfer of the two small multicopy plasmids ColE1 and cloDF13, whereas the traM, traI and traZ genes that are required for F conjugal DNA metabolism, are not. Differences between ColE1 and CloDF13 were that the F traD gene was needed for transfer of ColE1 but not of CloDF13, and that R100-1 efficiently transferred CloDF13 but not ColE1. A copy number mutant of CloDF13 inhibited F transfer and reduced plaque formation by the F-specific RNA phage f2, but not by Q beta or by the single-strand DNA phage f1. This phenotype suggests that the inhibition system (FinC) acts on traD, mutations in which give a similar phenotype. Hybridisation experiments with lambda tra phage DNA showed that transcription of traD was not reduced, and FinC probably inhibits function of the traD product rather than its synthesis. FinC-insensitive Flac mutants were isolated and characterised. One was shown to have an uppromoter mutation resulting in increased transcription of traJ and hence of the traY leads to Z operon including traD: the raised level of the traD product presumably then counteracted FinC inhibition.

Bacteriophage lambda↗
PubMed1980

Characterisation of a lambda transducing phage carrying the F conjugation gene traG.

A lambda transducing phage carrying the traGSTD genes of the E. coli K12 factor F was isolated by an in vivo technique, and characterized in tra complementation tests, by determining its restriction endonuclease fragment sizes, and by measuring heteroduplex molecules. The size and location on the F physical map of the tra transducing segment was thereby determined. Comparison of the proteins synthesized in UV-irradiated cells by this phage and by a derivative carrying the amber traG79 mutation, allowed the traG product to be identified as a protein of molecular weight 100,000. In the same experiments, the sizes of the traT and traD products made by the phage were also measured, being 25,000 and 85,000 daltons respectively.

Bacteriophage lambda↗