Biomedical subjects

M J Casadaban

Publications and source records attributed to M J Casadaban.

At least 19 recordsLinked to original sources

PubMed1995

A chromosome integration system for stable gene transfer into Thermus flavus.

We have developed a chromosomal integration system for gene transfer into the extreme thermophile Thermus flavus. The system relies on integration at the site of leuB (3-isopropylmalate dehydrogenase) which was cloned from T. flavus. The leuB gene was insertionally inactivated in vitro with a thermostable kanamycin-resistance gene and transformed in single-copy into the chromosome of T. flavus on a plasmid vector. Gene replacement strains required leucine for growth, were stably kanamycin-resistant and could grow in the presence of kanamycin at temperatures up to 55 degrees C.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

Molecular cloning of the pyrE gene from the extreme thermophile Thermus flavus.

Mutants of the extreme thermophile Thermus flavus in the pyrimidine biosynthetic pathway (Pyr-) were isolated by resistance to 5-fluoroorotic acid. The pyrE gene, which codes for the orotate phosphoribosyltransferase, was cloned by recombination with one of the isolated Pyr- T. flavus mutant strains. It was subcloned by complementation of an Escherichia coli pyrE mutant strain and was sequenced. The deduced polypeptide sequence extends over 183 amino acids. Several independent Pyr- mutations were mapped within the pyrE locus by recombination with fragments of the cloned gene.

Amino Acid Sequence↗

PubMed1992

Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus.

The region of the Escherichia coli K-12 chromosome encoding the enzymes responsible for the synthesis of responsible for the synthesis of the lipopolysaccharide (LPS) core has been cloned in vivo by using a mini-Mu vector. This region, formerly known as the rfa locus, comprises 18 kb of DNA between the markers tdh and rpmBG. Results of in vitro mutagenesis of this region with MudII1734 indicate the presence of at least 17 open reading frames or genes, a number considerably higher than expected on the basis of genetic and biochemical studies. Specific insertions in different genes have been recombined into the chromosome, and the mutations have been phenotypically characterized. Complementation analysis indicates that these genes are arranged in three different operons transcribed in opposite directions. A detailed physical map of this region has been constructed on the basis of complementation analysis, fusion protein data, and phenotypic characterizations. Additionally, the role of some genes in the synthesis of LPS has been defined by complementation analysis with known Salmonella typhimurium LPS mutants. The genetic organization of this locus seems to be identical in E. coli K-12 and S. typhimurium.

Chromosome Mapping↗

PubMed1991

Genome mapping and protein coding region identification using bacteriophage Mu.

Transposons such as bacteriophage Mu provide a means to clone bacterial genes as alternatives to using standard recombinant DNA technologies. A DNA-cloning and gene-expressing system has been developed with a bacteriophage Mu (DNA capacity of 38 kb) vector that combines the Mu transposition capabilities and a specialized promoter from bacteriophage T7. Genes cloned with this vector can be identified by transcription in vivo with T7 RNA polymerase and subsequent host translation. This system, illustrated with the characterization of a 35-kb region of the Escherichia coli K-12 chromosome, is applicable to other Enterobacteriaceae, which are hosts for Mu phage, and is potentially applicable to other bacteria, including Pseudomonas aeruginosa, which have Mu-like phage, and to other organisms for which high-frequency transposons are available.

Bacteriophage mu↗

PubMed1991

Analysis of the host ranges of transposon bacteriophages Mu, MuhP1, and D108 by use of lipopolysaccharide mutants of Salmonella typhimurium LT2.

The lipopolysaccharide receptors for the mutator bacteriophages Mu, MuhP1, and D108 were investigated with lipopolysaccharide mutants of Salmonella typhimurium LT2. Mu adsorbed only to mutants lacking the terminal O antigen but retaining the main chain sugars of the core; the side chain N-acetylglucosamine was not required. MuhP1 and D108 adsorbed partially to cells with the same receptors but adsorbed well only to cells with shorter lipopolysaccharides of the Rc and Rd1 chemotypes.

Acetylglucosamine↗

PubMed1991

Specificity of mini-Mu bacteriophage insertions in a small plasmid.

Target site selection for bacteriophage Mu transposition was studied in pools of over 10(7) independent mini-Mu insertions in pUC9, selected by transduction of the plasmid. Insertions in both orientations were clustered in three regions and, within these, at preferred sites.

Bacteriophage mu↗

PubMed1990

The upstream activating sequence for L-leucine gene regulation in Saccharomyces cerevisiae.

The upstream activating sequence (UAS) conferring leucine-specific regulation of transcription in Saccharomyces cerevisiae was identified by analysis of the LEU2 promoter and by comparison to other genes regulated by leucine. The UAS was localized with deletions and cloned synthetic DNA. Point mutations and sequence rearrangements were used to identify important basepairs and to construct an improved UAS with increased regulation and expression. The improved UAS contains a core ten basepair, GC-rich, palindromic sequence, which is sufficient to confer minimal levels of activation and regulation, within a 36 basepair palindromic sequence which confers maximal activation and regulation. Deletions downstream of the UAS indicated that the UAS must act in conjunction with at least one other site, perhaps a TATAA region, in order to confer high levels of activation. Tandem copies of the UAS in front of LEU2 increased expression and regulation. Tandem UAS elements in trans on a multi-copy 2 mu-based plasmid decreased expression and regulation. These results are consistent with a model that the UAS serves as the DNA-binding site for diffusible activation factor(s), possibly the LEU3 gene product.

3-Isopropylmalate Dehydrogenase↗

PubMed1990

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 require pili and surface growth for adsorption.

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 were found to require pili for infection. Seventy mutants of P. aeruginosa PAO selected by resistance to D3112 or B3 were also resistant to the phage not used in the selection and suggested that the receptors of these two phages are identical. Of five resistant mutants examined, all were defective in the production of pili and did not adsorb either phage. P. aeruginosa PAK strains altered in pilus expression, such as hyperpiliated or nonpiliated mutants, adsorbed the phage but were not productively infected, implying that an additional host function was required for infection. The cell-associated lipopolysaccharide was not required for D3112 or B3 infection, since mutants deficient in O side-chain and core biosynthesis were still capable of adsorption and productive infection. This is in contrast to Escherichia coli mutator phages Mu and D108, which are dependent on lipopolysaccharide for adsorption. The P. aeruginosa phages adsorbed only to cells grown on solid media or in liquid media supplemented with agents that increase the macroviscosity, such as polyvinylpyrrolidone. Adsorption time course studies of D3112 and B3 using cells grown in solid media revealed similar but not identical adsorption patterns. These studies suggested that expression of the D3112 and B3 cell receptor is induced by growth on solid media.

Adsorption↗

PubMed1989

Nucleotide sequences required for Tn3 transposition immunity.

The Tn3 transposon inserts at a reduced frequency into a plasmid already containing a copy of Tn3, a phenomenon known as transposition immunity. The cis-acting site on Tn3 responsible for immunity was mapped by deletions from each side to be within the terminal 38-base-pair sequence that is inversely repeated at the ends of Tn3. Two palindromic sequences are present in the essential part of this region. Some deletions conferred only partial immunity, and others conferred negative immunity. Multiple copies of partially immune ends conferred additional immunity. No other part of Tn3 was necessary for immunity.

Base Sequence↗

PubMed1989

Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa.

Small bacteriophage D3112 transposable elements deleted for most of the phage-lytic functions while retaining the sites required for transposition and packaging were constructed to facilitate genetic studies in Pseudomonas aeruginosa. These mini-D derivatives were constructed with the terminal 1.85 kilobases (kb) of the phage left end and 1.4 kb of the phage right end and either the Tn5 kanamycin resistance or the pSC101 (pBR322) tetracycline resistance determinant. Thermally induced lysates of strains lysogenic for both a mini-D element and D3112 cts (temperature-sensitive repressor) transduced P. aeruginosa PAO recipients to drug resistance at frequencies of between 10(-4) and 10(-5)/PFU of the helper phage. As for the parent plaque-forming D3112 phage, the mini-D171 element could insert itself into many different sites in the chromosome but the frequency of insertion into particular genes varied widely. Among 1,000 insertions, none resulted in auxotrophy but 10 resulted in pigment production. Insertions were also selected in a cloning plasmid with a transduction scheme. At least eight different insertion sites were found to have been used among 10 individual insertions. Transductants harboring these mini-D elements were immune to infection by D3112, since they contained the D3112 repressor gene in the left 1.85-kb terminal fragment. Chromosomal genes were transduced in a generalized fashion 100 to 1,000 times more frequently by the mini-D-D3112 cts lysates than by the D3112 cts phage alone. Mini-D171-D3112 cts lysates also yielded some transductants that retained the drug resistance marker of the mini-D element and which were unstable for the chromosomal transduced marker. This is consistent with the miniduction properties of Mu whereby transduced genes are flanked by two mini-D elements in the same orientation.

Bacteriophages↗

PubMed1989

In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage.

The transposition properties of the Pseudomonas aeruginosa mutator bacteriophage D3112 were exploited to develop an in vivo cloning system. Mini-D replicon derivatives of D3112 were constructed by incorporating broad host range plasmid replicons between short terminal D3112 sequences. These elements were made with small replication regions from the RK2, Sa, and pVS1 plasmids and selectable genes for tetracycline, carbenicillin, kanamycin, and gentamicin resistance. Some of the mini-D replicons also contain the RK2 oriT origin-of-transfer sequence, which allows them to be mobilized by conjugation to many different species of gram-negative bacteria. These elements were used to clone DNA by preparing lysates from P. aeruginosa cells harboring an inducible D3112 cts prophage and a mini-D replicon plasmid. These lysates were used to infect sensitive P. aeruginosa recipients and select recombinant plasmids as drug-resistant transductant colonies. These transductants form a gene library from which particular clones can be selected, such as by their ability to complement specific mutations. This system was used to clone nine different genes from the PAO chromosome. The ability of this system to precisely identify a gene was demonstrated by isolating clones of the argF+ and cys-59+ genes. Restriction maps of clones of these genes, which have different amounts of flanking DNA, located the positions of these genes. The sizes of the chromosomal DNA segments from 10 individual clones examined ranged from 6 to 21 kilobases (kb), with an average of about 10 kb. This is consistent with the approximately 40-kb DNA-packaging size of the D3112 phage.

Bacteriophages↗

PubMed1988

Bacteriophage Mu sites required for transposition immunity.

Plasmids with bacteriophage Mu sequences receive additional Mu insertions 20-700 times less frequently than plasmids without Mu sequences. The Mu sites required for this transposition immunity were mapped near each end, either of which was sufficient. The left site was between 127 and 203 base pairs from the left end, and the right site was between 22 and 93 base pairs from the right end. These sequences include the innermost but not the outermost of the three binding sites for the Mu A transposition protein at each end of Mu. Transposition immunity was cis-acting and independent of its location on a target plasmid. An additional copy of an immunity site reduced transposition a factor of 10 further. Transposition immunity was seen both during full phage lytic growth, with all the bacteriophage Mu genes, and during normal cellular growth, with a mini-Mu element containing only the Mu c and ner regulatory and A and B transposition genes.

Bacteriophages↗

PubMed1987

Hybrid protein thymidine kinase gene fusions: plasmid vectors for the study of transcription and translation initiation signals.

The thymidine kinase (TK) gene (tk) from Herpes simplex virus type 1 has been used to form gene fusions encoding enzymatically active hybrid proteins. The promoter, translation initiation region, and the first three codons of the tk gene were removed and replaced with a series of DNA restriction sites. DNA fragments containing gene initiation regions were cloned into these sites and shown to synthesize enzymatically active proteins in Escherichia coli. These gene fusions were shown to complement an E. coli strain which is deficient in TK function. Gene initiation regions were used from the lac operon, the tnpR gene of Tn3, and the insA gene of ISl. TK synthesis was regulated by the control signals of the promoter fused to tk, and was dependent upon the phase alignment of the codons at the fusion joint. The size of the resulting protein was shown to be increased over the size of the original TK protein by the length of the coding region fused to TK. This demonstrated that the tk gene has non-essential N-terminal amino acids that can be replaced by other amino acid sequences with the retention of TK enzymatic activity. Such tk gene fusions are useful in situations where fusions with other genes cannot be conveniently selected or assayed.

Cloning, Molecular↗

PubMed1987

In vivo DNA cloning with a mini-Mu replicon cosmid and a helper lambda phage.

A mini-Mu bacteriophage, containing the cohesive-end packaging site (cos) from a lambda-phi 80 hybrid phage, a high-copy-number plasmid replicon, and a kanamycin-resistance gene for independent selection, was constructed to clone genes in vivo. This mini-Mu element can be derepressed to transpose at a high frequency. DNA segments that become flanked by copies of this mini-Mu element in the same orientation can be packaged by a helper lambda phage. The resulting lambda lysate can be used to infect recipient cells where the injected DNA can circularize by annealing at the cos termini. Drug-resistant transductants obtained carry the mini-Mu-replicon cosmid element with inserts of different nucleotide sequences. These are analogous to recombinant DNA clones generated in vitro with restriction endonuclease cutting and ligase joining reactions replaced by the Mu transposition process. Clones of particular genes were isolated by their ability to complement specific mutations. Both recA+ and recA- recipient cells can be used with equal efficiency. Clones obtained with a helper lambda phage require the presence of the cos site in the mini-Mu replicon. They carry larger inserts than those isolated with the same mini-Mu element and Mu as a helper phage. The mini-Mu replicon-cosmid bacteriophage contains a lac-gene fusing segment for isolating fusions of lac operon DNA to gene control regions in the cloned sequences. Independent clones of a particular gene can be used to prepare a restriction map of the gene and its flanking regions.

Bacteriophage lambda↗

PubMed1987

High frequency generalized transduction by miniMu plasmid phage.

Deletion derivatives of phage Mu which replicate as multicopy plasmids, and also transpose and package like Mu, have been developed for the in vivo cloning of bacterial genes. We show here that these miniMu plasmid phage are also efficient at generalized transduction and that both in vivo cloning and generalized transduction of a given gene can be accomplished in a single experiment.

Coliphages↗

PubMed1987

Cloning of genes from members of the family Enterobacteriaceae with mini-Mu bacteriophage containing plasmid replicons.

An in vivo cloning system that uses derivatives of the Escherichia coli bacteriophage Mu with plasmid replicons has been extended to five different species of the family Enterobacteriaceae. Mu and these mini-Mu replicon elements were introduced into strains of E. coli, Shigella flexneri, Salmonella typhimurium, Citrobacter freundii, and Proteus mirabilis by infection, by transformation, or by conjugation with newly constructed broad-host-range plasmids containing insertions of these elements. Lysates from these cells, lysogenic for Mu and mini-Mu elements, were used to infect sensitive recipient strains of E. coli, S. typhimurium, and C. freundii. Drug-resistant transductants had mini-Mu replicon elements with inserts of different DNA sequences. All of the lysogens made could be induced to yield high phage titers, including those coming from strains that were resistant to Mu and Mu derivatives. Clones of 10 particular genes were isolated by their ability to complement specific mutations in the recipient strains, even in the presence of the E. coli K-12 restriction system. Some of the mini-Mu replicon elements used contained lac gene fusing segments and resulted in fusions of the lac operon to control regions in the cloned sequences.

Citrobacter↗

PubMed1986

crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli.

The complete nucleotide sequences of the Salmonella typhimurium LT2 and Shigella flexneri 2B crp genes were determined and compared with those of the Escherichia coli K-12 crp gene. The Shigella flexneri gene was almost like the E. coli crp gene, with only four silent base pair changes. The S. typhimurium and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 77 changes, but the corresponding amino acid sequences presented only one amino acid difference. The nucleotide sequences of the crp genes diverged to the same extent as in the other genes, trp, ompA, metJ, and araC, which are structural or regulatory genes. An analysis of the amino acid divergence, however, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Comparison of codon usage in S. typhimurium and E. coli for all genes sequenced in both organisms showed that their patterns were similar. Comparison of the regulatory regions of the S. typhimurium and E. coli crp genes showed that the most conserved sequences were those known to be essential for the expression of E. coli crp.

Amino Acid Sequence↗

PubMed1986

Transposition of the gram-positive transposon Tn917 in Escherichia coli.

The streptococcal transposon Tn917 was demonstrated to transpose in Escherichia coli from the Bacillus subtilis-E. coli shuttle plasmid pHK1207 into an F' plasmid derivative. Subsequently, a second round of transposition from the F' plasmid into pACYC184 could be readily demonstrated. These results represent the initial demonstration of the transposition of a gram-positive transposon in a gram-negative bacterium at a relatively high frequency.

Bacillus subtilis↗