Jeremy Dale - Academia.edu (original) (raw)

Papers by Jeremy Dale

Research paper thumbnail of Molecular epidemiology of tuberculosis in Malaysia

Journal of Clinical Microbiology, May 1, 1999

Molecular typing with IS6110 was applied to Mycobacterium tuberculosis isolates from all parts of... more Molecular typing with IS6110 was applied to Mycobacterium tuberculosis isolates from all parts of Malaysia. The degree of clustering increased with patient age, suggesting that reactivation may contribute to clustering. Identical banding patterns were also obtained for isolates from widely separate regions. Therefore, the use of clustering as a measure of recent transmission must be treated with caution. Strains related to the Beijing family were common in Peninsular Malaysia but were less common in Sabah and Sarawak, while a distinct group of strains comprised nearly 40% of isolates from East Malaysia but such strains were rare in Peninsular Malaysia. Single-copy strains, common in South and Southeastern Asia, constituted nearly 20% of isolates from the peninsula but were virtually absent in East Malaysia. The marked geographical difference in the prevailing strains indicates not only a restricted dissemination of M. tuberculosis but also a considerable degree of stability in the banding patterns.

Research paper thumbnail of Intracellular expression and regulation of mycobacterial promoters

Research paper thumbnail of Multivalent BCG vaccines

Research paper thumbnail of The purification and properties of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli and Proteus mirabilis. Biochem J

Biochemical Journal

ABSTRACT

Research paper thumbnail of The dimeric nature of an R-factor mediated beta-lactamase

Biochemical and Biophysical Research Communications

Research paper thumbnail of R-Factor-Mediated β-Lactamases That Hydrolyze Oxacillin: Evidence for Two Distinct Groups

Journal of Bacteriology

ABSTRACT

Research paper thumbnail of Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains

The Journal of applied bacteriology

probes derived from the insertion sequence IS986, which have previously been shown to differentia... more probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results Correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.

Research paper thumbnail of Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium

Molecular Microbiology

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain L... more A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

Research paper thumbnail of Insertion element IS986 from Mycobacterium tuberculosis: A useful tool for diagnosis and epidemiology of tuberculosis

Journal of Clinical Microbiology

IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it... more IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis.

Research paper thumbnail of Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect Immun

Infection and Immunity

Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like eleme... more Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only...

Research paper thumbnail of Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: A blind comparison study among seven laboratories

Journal of Clinical Microbiology

PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculo... more PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculosis. However, virtually no data are available on the reliability and reproducibility of the method. In order to assess the validity of PCR for the detection of mycobacteria in clinical samples, seven laboratories participated in a blinded study of 200 sputum, saliva, and water samples containing either known numbers of Mycobacterium bovis BCG cells or no added organisms. Each laboratory used its own protocol for pretreatment, DNA extraction, and detection of the amplification product. Insertion sequence IS6110 was the target for DNA amplification. Several participating laboratories reported high levels of false-positive PCR results, with rates ranging from 3 to 20% and with one extreme value of 77%. The levels of sensitivity also ranged widely among the different participants. A positive PCR result was reported for 2 to 90% of the samples with 10(3) mycobacteria. Although most participan...

Research paper thumbnail of Context-sensitive transposition of IS6110 in mycobacteria

Microbiology, 1999

The rational use of IS6110 fingerprinting for studies of the molecular epidemiology and evolution... more The rational use of IS6110 fingerprinting for studies of the molecular epidemiology and evolution of Mycobacterium tuberculosis requires understanding of the dynamics of transposition. In laboratory model systems, it has been shown that transposition is context-sensitive, i.e. it is influenced by the nature of the site in which the insertion sequence is presented. Stimulation of transposition by activation of an adjacent promoter supports the hypothesis that transposition occurs more readily from transcriptionally active locations. In addition, it has been shown that transposition can be enhanced by the expression of the transposase in trans. These findings imply that the frequency of transposition will vary substantially between different strains of M. tuberculosis, and furthermore that a hitherto stable strain may develop more rapid variation due to transposition into an active site. The use of IS6110 fingerprinting for the analysis of longer-range relationships between M. tuberculosis isolates therefore needs to be interpreted with care.

Research paper thumbnail of The use of alkaline phosphatase to prevent vector regeneration

Methods in molecular biology (Clifton, N.J.), 1985

Linearized plasmid vector molecules produced by restriction enzyme digestion can be reformed into... more Linearized plasmid vector molecules produced by restriction enzyme digestion can be reformed into circles by the action of DNA ligase. This is the most favorable reaction even when foreign DNA fragments are present. Thus, unless a direct selection technique is available, the reformed parental molecule will also give transformants and hence reduce the overall cloning efficiency. Usually most of the recircularized parental plasmids can readily be distinguished from recombinants by the absence of insertional inactivation; nevertheless, this may involve screening large numbers of transformants for each recombinant.

Research paper thumbnail of Preparation and assay of phage lambda

Methods in molecular biology (Clifton, N.J.), 1985

Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensiti... more Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensitive cells, known as the lytic and lysogenic cycles. In the lytic cycle, after the lambda DNA enters the cells, various phage functions are expressed that result in the production of a large number of mature phage particles and cell lysis. In the lysogenic mode, which normally occurs in only a small proportion of the infected cells, the phage forms a more or less stable relationship with the host bacterium; this stable state is known as lysogeny. In a lysogenic cell, phage DNA is normally incorporated into the chromosomal DNA via specific attachment sites on both the phage DNA and the host chromosome. Replication of lambda DNA then occurs only during replication of the host chromosome, and the phage genome is inherited by each daughter cell at cell division. The phage is maintained in this prophage state through the action of a repressor protein, coded for by the phage gene cl. This repres...

Research paper thumbnail of Preparation of Chromosomal DNA from E. coli

Methods in molecular biology (Clifton, N.J.), 1985

This chapter describes a simple and rapid way of extracting and purifying chromosomal DNA from E.... more This chapter describes a simple and rapid way of extracting and purifying chromosomal DNA from E. coli and many other species of bacteria. This procedure is essentially a simplified version of that described by Marmur in 1961 (1). The cells are lysed by treatment with a detergent and the mixture is deproteinized by phenol-chloroform extraction. Further purification can be achieved by treatment with ribonuclease and proteinase K. The resulting DNA, free of protein and RNA contamination, is sufficiently pure to be used for restriction digestion and cloning, e.g., in the preparation of gene libraries.

Research paper thumbnail of Plasmid DNA isolation (sheared lysate method)

Methods in molecular biology (Clifton, N.J.), 1985

The cleared lysate method (see Chapter 25 ) is not usually very effective for isolation of plasmi... more The cleared lysate method (see Chapter 25 ) is not usually very effective for isolation of plasmids larger than about 20 kb. Recovery of plasmid DNA is often poor, presumably because high molecular weight plasmids are removed by the clearing spin. An alternative procedure, therefore, is to load the complete cell lysate onto a cesium chloride-ethidium bromide gradient that will separate the plasmid DNA from the chromosomal material and also from other cell components.

Research paper thumbnail of Immunological detection of gene expression in recombinant clones

Methods in molecular biology (Clifton, N.J.), 1985

After ligation and transformation, a number of clones will (it is hoped) be obtained that can be ... more After ligation and transformation, a number of clones will (it is hoped) be obtained that can be identified as recombinants by the occurrence of insertional inactivation, by analysis of small-scale plasmid preparations, or more specifically by in situ hybridization. An additional strategy that is superficially attractive, but not without considerable problems, is to look directly for expression of the cloned gene(s). This may be applicable in primary cloning if no specific DNA probe is available, but is more generally used in subsequent studies of expression of specific foreign genes.

Research paper thumbnail of Plasmid screening using single colony lysates

Methods in molecular biology (Clifton, N.J.), 1985

There are a number of methods available for screening either potential recombinant clones or natu... more There are a number of methods available for screening either potential recombinant clones or natural isolates for the possession of plasmids. The method described in this chapter is based on that described by Sherratt (1) and is the simplest and most rapid of these. The name arises from the fact that the technique was devised to yield results using a single colony that can be taken directly from an appropriate agar plate. Although this procedure works best for high copy number plasmids, it can also be used to detect plasmids in environmental isolates, for example.

Research paper thumbnail of Identification of recombinant phages by plaque hybridization

Methods in molecular biology (Clifton, N.J.), 1985

Phage vectors are often used rather than plasmids, particularly for the production of gene "... more Phage vectors are often used rather than plasmids, particularly for the production of gene "banks" or "libraries." The plaques produced can be screened for the presence of specific DNA sequences by hybridization using a procedure similar to that used for colony hybridization (see Chapter 42 ). Plaque hybridization offers some advantages over colony hybridization, largely because the area of the filter to which the DNA is bound is smaller and more defined. A higher density of plaques can therefore be used, which in turn means that more plaques can be screened in a single hybridization-several thousand on an ordinary (8.5 cm) petri dish; using larger containers such as baking dishes or trays the number can run into hundreds of thousands in a single hybridization. In addition, since the location of the plaques is not disturbed by the process, nor are they smudged in the way that colonies can be, several replicates can be taken from the same plate. This means that th...

Research paper thumbnail of Use of an arrayed promoter-probe library for the identification of macrophage-regulated genes in Mycobacterium tuberculosis

Microbiology (Reading, England), 2002

The survival of Mycobacterium tuberculosis within the human host after infection, especially with... more The survival of Mycobacterium tuberculosis within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes. To identify such genes, a promoter-probe library was constructed in which fragments of M. tuberculosis H37Rv DNA were inserted upstream of a lacZ reporter gene, using an Escherichia coli-mycobacterial shuttle vector. Mycobacterium bovis Bacille Calmette-Guérin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions. From preliminary screening, 41 clones were selected that exhibited upregulation of lacZ expression when subjected to acidified sodium nitrite. Subsequent sequence analyses identified 26 of these clones as containing potential promoters. After measuring lacZ expression in BCG clones recovered from a THP-1 macrophage cell line, three gene...

Research paper thumbnail of Molecular epidemiology of tuberculosis in Malaysia

Journal of Clinical Microbiology, May 1, 1999

Molecular typing with IS6110 was applied to Mycobacterium tuberculosis isolates from all parts of... more Molecular typing with IS6110 was applied to Mycobacterium tuberculosis isolates from all parts of Malaysia. The degree of clustering increased with patient age, suggesting that reactivation may contribute to clustering. Identical banding patterns were also obtained for isolates from widely separate regions. Therefore, the use of clustering as a measure of recent transmission must be treated with caution. Strains related to the Beijing family were common in Peninsular Malaysia but were less common in Sabah and Sarawak, while a distinct group of strains comprised nearly 40% of isolates from East Malaysia but such strains were rare in Peninsular Malaysia. Single-copy strains, common in South and Southeastern Asia, constituted nearly 20% of isolates from the peninsula but were virtually absent in East Malaysia. The marked geographical difference in the prevailing strains indicates not only a restricted dissemination of M. tuberculosis but also a considerable degree of stability in the banding patterns.

Research paper thumbnail of Intracellular expression and regulation of mycobacterial promoters

Research paper thumbnail of Multivalent BCG vaccines

Research paper thumbnail of The purification and properties of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli and Proteus mirabilis. Biochem J

Biochemical Journal

ABSTRACT

Research paper thumbnail of The dimeric nature of an R-factor mediated beta-lactamase

Biochemical and Biophysical Research Communications

Research paper thumbnail of R-Factor-Mediated β-Lactamases That Hydrolyze Oxacillin: Evidence for Two Distinct Groups

Journal of Bacteriology

ABSTRACT

Research paper thumbnail of Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains

The Journal of applied bacteriology

probes derived from the insertion sequence IS986, which have previously been shown to differentia... more probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results Correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.

Research paper thumbnail of Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium

Molecular Microbiology

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain L... more A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

Research paper thumbnail of Insertion element IS986 from Mycobacterium tuberculosis: A useful tool for diagnosis and epidemiology of tuberculosis

Journal of Clinical Microbiology

IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it... more IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis.

Research paper thumbnail of Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect Immun

Infection and Immunity

Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like eleme... more Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only...

Research paper thumbnail of Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: A blind comparison study among seven laboratories

Journal of Clinical Microbiology

PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculo... more PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculosis. However, virtually no data are available on the reliability and reproducibility of the method. In order to assess the validity of PCR for the detection of mycobacteria in clinical samples, seven laboratories participated in a blinded study of 200 sputum, saliva, and water samples containing either known numbers of Mycobacterium bovis BCG cells or no added organisms. Each laboratory used its own protocol for pretreatment, DNA extraction, and detection of the amplification product. Insertion sequence IS6110 was the target for DNA amplification. Several participating laboratories reported high levels of false-positive PCR results, with rates ranging from 3 to 20% and with one extreme value of 77%. The levels of sensitivity also ranged widely among the different participants. A positive PCR result was reported for 2 to 90% of the samples with 10(3) mycobacteria. Although most participan...

Research paper thumbnail of Context-sensitive transposition of IS6110 in mycobacteria

Microbiology, 1999

The rational use of IS6110 fingerprinting for studies of the molecular epidemiology and evolution... more The rational use of IS6110 fingerprinting for studies of the molecular epidemiology and evolution of Mycobacterium tuberculosis requires understanding of the dynamics of transposition. In laboratory model systems, it has been shown that transposition is context-sensitive, i.e. it is influenced by the nature of the site in which the insertion sequence is presented. Stimulation of transposition by activation of an adjacent promoter supports the hypothesis that transposition occurs more readily from transcriptionally active locations. In addition, it has been shown that transposition can be enhanced by the expression of the transposase in trans. These findings imply that the frequency of transposition will vary substantially between different strains of M. tuberculosis, and furthermore that a hitherto stable strain may develop more rapid variation due to transposition into an active site. The use of IS6110 fingerprinting for the analysis of longer-range relationships between M. tuberculosis isolates therefore needs to be interpreted with care.

Research paper thumbnail of The use of alkaline phosphatase to prevent vector regeneration

Methods in molecular biology (Clifton, N.J.), 1985

Linearized plasmid vector molecules produced by restriction enzyme digestion can be reformed into... more Linearized plasmid vector molecules produced by restriction enzyme digestion can be reformed into circles by the action of DNA ligase. This is the most favorable reaction even when foreign DNA fragments are present. Thus, unless a direct selection technique is available, the reformed parental molecule will also give transformants and hence reduce the overall cloning efficiency. Usually most of the recircularized parental plasmids can readily be distinguished from recombinants by the absence of insertional inactivation; nevertheless, this may involve screening large numbers of transformants for each recombinant.

Research paper thumbnail of Preparation and assay of phage lambda

Methods in molecular biology (Clifton, N.J.), 1985

Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensiti... more Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensitive cells, known as the lytic and lysogenic cycles. In the lytic cycle, after the lambda DNA enters the cells, various phage functions are expressed that result in the production of a large number of mature phage particles and cell lysis. In the lysogenic mode, which normally occurs in only a small proportion of the infected cells, the phage forms a more or less stable relationship with the host bacterium; this stable state is known as lysogeny. In a lysogenic cell, phage DNA is normally incorporated into the chromosomal DNA via specific attachment sites on both the phage DNA and the host chromosome. Replication of lambda DNA then occurs only during replication of the host chromosome, and the phage genome is inherited by each daughter cell at cell division. The phage is maintained in this prophage state through the action of a repressor protein, coded for by the phage gene cl. This repres...

Research paper thumbnail of Preparation of Chromosomal DNA from E. coli

Methods in molecular biology (Clifton, N.J.), 1985

This chapter describes a simple and rapid way of extracting and purifying chromosomal DNA from E.... more This chapter describes a simple and rapid way of extracting and purifying chromosomal DNA from E. coli and many other species of bacteria. This procedure is essentially a simplified version of that described by Marmur in 1961 (1). The cells are lysed by treatment with a detergent and the mixture is deproteinized by phenol-chloroform extraction. Further purification can be achieved by treatment with ribonuclease and proteinase K. The resulting DNA, free of protein and RNA contamination, is sufficiently pure to be used for restriction digestion and cloning, e.g., in the preparation of gene libraries.

Research paper thumbnail of Plasmid DNA isolation (sheared lysate method)

Methods in molecular biology (Clifton, N.J.), 1985

The cleared lysate method (see Chapter 25 ) is not usually very effective for isolation of plasmi... more The cleared lysate method (see Chapter 25 ) is not usually very effective for isolation of plasmids larger than about 20 kb. Recovery of plasmid DNA is often poor, presumably because high molecular weight plasmids are removed by the clearing spin. An alternative procedure, therefore, is to load the complete cell lysate onto a cesium chloride-ethidium bromide gradient that will separate the plasmid DNA from the chromosomal material and also from other cell components.

Research paper thumbnail of Immunological detection of gene expression in recombinant clones

Methods in molecular biology (Clifton, N.J.), 1985

After ligation and transformation, a number of clones will (it is hoped) be obtained that can be ... more After ligation and transformation, a number of clones will (it is hoped) be obtained that can be identified as recombinants by the occurrence of insertional inactivation, by analysis of small-scale plasmid preparations, or more specifically by in situ hybridization. An additional strategy that is superficially attractive, but not without considerable problems, is to look directly for expression of the cloned gene(s). This may be applicable in primary cloning if no specific DNA probe is available, but is more generally used in subsequent studies of expression of specific foreign genes.

Research paper thumbnail of Plasmid screening using single colony lysates

Methods in molecular biology (Clifton, N.J.), 1985

There are a number of methods available for screening either potential recombinant clones or natu... more There are a number of methods available for screening either potential recombinant clones or natural isolates for the possession of plasmids. The method described in this chapter is based on that described by Sherratt (1) and is the simplest and most rapid of these. The name arises from the fact that the technique was devised to yield results using a single colony that can be taken directly from an appropriate agar plate. Although this procedure works best for high copy number plasmids, it can also be used to detect plasmids in environmental isolates, for example.

Research paper thumbnail of Identification of recombinant phages by plaque hybridization

Methods in molecular biology (Clifton, N.J.), 1985

Phage vectors are often used rather than plasmids, particularly for the production of gene "... more Phage vectors are often used rather than plasmids, particularly for the production of gene "banks" or "libraries." The plaques produced can be screened for the presence of specific DNA sequences by hybridization using a procedure similar to that used for colony hybridization (see Chapter 42 ). Plaque hybridization offers some advantages over colony hybridization, largely because the area of the filter to which the DNA is bound is smaller and more defined. A higher density of plaques can therefore be used, which in turn means that more plaques can be screened in a single hybridization-several thousand on an ordinary (8.5 cm) petri dish; using larger containers such as baking dishes or trays the number can run into hundreds of thousands in a single hybridization. In addition, since the location of the plaques is not disturbed by the process, nor are they smudged in the way that colonies can be, several replicates can be taken from the same plate. This means that th...

Research paper thumbnail of Use of an arrayed promoter-probe library for the identification of macrophage-regulated genes in Mycobacterium tuberculosis

Microbiology (Reading, England), 2002

The survival of Mycobacterium tuberculosis within the human host after infection, especially with... more The survival of Mycobacterium tuberculosis within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes. To identify such genes, a promoter-probe library was constructed in which fragments of M. tuberculosis H37Rv DNA were inserted upstream of a lacZ reporter gene, using an Escherichia coli-mycobacterial shuttle vector. Mycobacterium bovis Bacille Calmette-Guérin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions. From preliminary screening, 41 clones were selected that exhibited upregulation of lacZ expression when subjected to acidified sodium nitrite. Subsequent sequence analyses identified 26 of these clones as containing potential promoters. After measuring lacZ expression in BCG clones recovered from a THP-1 macrophage cell line, three gene...