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P Upcroft

Publications and source records attributed to P Upcroft.

72 records · Page 4Linked to original sources
PubMed1988

Entrapment of recombinant plasmids in SeaPlaque agarose plugs and their rapid purification from recircularised vectors.

A simple method is described which permits both the separation and concentration of circular recombinant plasmids from smaller plasmid vectors that are an undesirable by-product of a ligation reaction. SeaPlaque agarose plugs are used to entrap open-circular forms of recombinant plasmids during electrophoresis. In the example described over 98% of supercoiled, open-circular and linear forms of the 2.9-kb Bluescript plasmid vector, as well as the equivalent dimer forms, pass through the 1.4% SeaPlaque plug. Circular recombinant plasmids greater in length than the vector dimer are entrapped within the plug. By increasing the concentration of SeaPlaque, recombinants smaller than the vector dimer are retained in the trap, but with a concomitant increase in contamination by open-circular vector dimers. For most library constructions the high ratio of insert to vector used during the ligation reaction reduces the formation of vector dimers and makes this level of contamination inconsequential. The recombinant plasmids can be extracted readily from the SeaPlaque plug by excising it, melting the agarose and extracting with phenol. Alternatively, the excised plug can be melted and the recombinant plasmids used to transform bacteria, or mammalian cells, directly in the agarose. The procedure should be valuable for cloning large inserts for 'jumping' and 'linking' libraries, for large inserts in general where recircularisation is a low-frequency event, e.g., minichromosomes, for pulsed-field gel electrophoresis applications, and for hosts and vectors where genetic selection of the recombinant is not possible.

Cloning, Molecular↗
PubMed1987

Transformation of mammalian cells with recombinant DNA directly from Seaplaque agarose.

Transfection of African green monkey kidney cells directly with recombinant DNA excised from, but still present in, Seaplaque agarose after electrophoresis, is described. Efficiencies of transfection increased by 30% when the gel was present compared with transfection in the absence of the agarose. Extraction of the DNA from the gel was not necessary, thereby obviating a purification step and the concomitant losses. To generate recombinant molecules bacterial plasmid sequences are not necessary, thereby reducing considerably the size of the recombinant molecule and removing extraneous and deleterious sequences, e.g., "poison sequences." Linear or circular DNA molecules could be transfected in the melted and diluted agarose with the same ease as in its absence. Hence linear partial ligation products can be excised from the gel after electrophoresis to generate recombinant DNA molecules directly in mammalian cells.

Animals↗
PubMed1987

Giardia intestinalis antigens expressed in Escherichia coli.

cDNA and genomic DNA of Giardia intestinalis have been cloned in pUC vectors and used to express Giardia antigens in Escherichia coli. Several expression libraries have been produced and positive clones identified by immuno-colony assays with antisera raised against whole parasites and partially purified antigen(s). Those clones which express G. intestinalis antigens have been used to raise antisera in mice and the antisera used in immunofluorescence assays. The proteins expressed by the clones have been shown to represent a 32 kDa protein of the flagellae and axonemes, a protein associated with the spiral part of the ventral disc, proteins covering the surface of the trophozoite or associated with the coat, and other proteins associated with axonemes of posterolateral flagellae, kinetosomes and funis, and the anterolateral axonemes. mRNA was purified from G. intestinalis and translated in a cell free lysate. A rabbit antiserum raised against trophozoites immunoprecipitated several translation products while an antiserum raised against a purified 32 kDa protein only immunoprecipitated this protein. G. intestinalis rRNA subunits also were examined in the course of mRNA purification. Two rRNA species were evident, the small rRNA and the post-transcriptionally processed large rRNA.

Animals↗
PubMed1987

Rapid and efficient method for cloning of blunt-ended DNA fragments.

We describe a system to generate cDNA or genomic libraries from DNA segments that have blunt termini. Background and rearrangement levels are low, but efficiencies are high and the procedural times very short. T4 ligase in the presence of polyethylene glycol produces high Mr oligomers of vector and insert. These concatemers are reduced to vector-insert monomers at a high frequency by subsequent cleavage with a restriction endonuclease, which recognises the insert rarely, if at all, and the vector once. The monomers are recircularised under standard ligation conditions prior to transformation. Thus insertion conditions are optimised independently of those for recircularisation. All reading frames for expression libraries are generated by short BAL 31 cleavage followed by the blunt-end cloning procedure. Similarly, genomic expression libraries can be made by BAL 31 or mung-bean nuclease treatment after cleavage with DNase I is the presence of Mn2+. The technique is suitable for any DNA segment that is blunt-ended or can be made so. When the vector is treated with alkaline phosphatase, recombinants are generated at a frequency greater than 90% and have single inserts. Yields are 3-5 X 10(6) colony-forming units per micrograms of insert.

Cloning, Molecular↗
PubMed1987

Simian virus 40 infection is not mediated by lysosomal activation.

The uptake of simian virus 40 (SV40) virions to the nucleus, the site of viral replication, proceeds via engulfment at the cytoplasmic membrane and transport in monopinocytotic vesicles through the cytoplasm to the nuclear membrane. In the case of Semliki Forest virus and poliovirus which undergo primary endocytosis in a similar manner, neutralization of the acid pH in these vesicles abolishes viral infectivity. We have examined the effects of the lysosomotropic agents chloroquine and ammonium chloride on the uptake of SV40 and find that neutralization of the acid pH in cellular organelles has no effect on the progress of SV40 infection. Although the initial endocytotic pathway appears similar for the viruses, the vesicular transport of SV40 to the nucleus proceeds, therefore, via an alternative endocytotic compartment which is not inhibited by increasing the endosomal pH.

Ammonium Chloride↗
PubMed1986

Mammalian cell functions mediating recombination of genetic elements: topological restraints.

In extending the scope of our previous analyses of the functions that mammalian cells engender to recombine elements of genetic information (Upcroft, Carter and Kidson, 1980a and b), I describe the capacity of cells to recognise differentially combinations of open-ended and closed molecules. The frequency of successful recombination, as assayed by the generation of viable SV40 genomes from appropriate substrates, was shown to decrease with the loss of free termini capable of invasion of an homologous DNA duplex and to be influenced by the presence of non-homologous flanking sequences, the length of homology and whether the sequence was internal or terminal. The effect of stabilisation with long homologous regions on recombination over short homologous regions and inter-versus intra-molecular DNA sequence homologies was also investigated.

Animals↗
PubMed1980

Mammalian cell function mediating recombination of genetic elements.

Recombination of segments of the SV40 genome by a variety of mechanisms is described. These include the faithful joining of linear segments that have flush termini as opposed to previously described cohesive or resected termini. Lack of involvement of viral proteins has been demonstrated for recombination of segments with homologous overlapping termini, but probably applies also to the other joining reactions. Segments of the genome that have been cleaved in such a manner as to be unable to manufacture any known viral proteins are neutral elements of genetic information, incapable of selection by replication or biological function until recombined. These recombination functions presumably are available to the host cell and any element of genetic information that can be generated in that cell.

Animals↗
PubMed1980

Analysis of recombination in mammalian cells using SV40 genome segments having homologous overlapping termini.

Segments of SV40 DNA having homologous overlapping termini recombine to produce viable genomes in monkey cells. Frequencies of recombination on either side of a deletion marker are non-random; replication and palindromes do not appear to be essential. Since recombination involves host enzymes, a suitable system has been devised for analysing host cell recombination functions.

Animals↗
PubMed1978

Transduction of a bacterial gene into mammalian cells.

The transduction of an Escherichia coli gene into mammalian cells is described. A supressor tRNA gene was linked to a simian virus 40 (SV40) vector in vitro and the recombinant was used to transfect rat embryo cells and monkey kidney cells. The hybrid SV40 genome, SV40-su+ III, retained genetic information required for autonomous replication and cellular transformation and had a 1300-base-pair DNA segment in the late gene region (between the restriction endonuclease sits Hpa II at 0.735 and EcoRI at 0/1.0 on the SV40 genetic map) replaced by an 870-base-pair bacterial DNA segment containing the suppressor tRNA gene, su+ III (tRNATyrsu+III). The structure and fate of the SV40-su+III chimera were determined by DNA reassociation kinetic analysis and restriction enzyme cleavage of the total cellular DNA from transformed rat embryo cells and persistently infected monkey cells. Hybridization with radiolabeled probes specific for vector (SV40) or su+III DNA sequences revealed primarily nonintegrated or free hybrid genomes. In cloned lines of both cell types, the bacterial DNA segment was recovered intact, as judged by the length of the segment excised by restriction endonucleases and its ability to hybridize to the radiolabeled bacterial DNA probe and not to the SV40 probe.

Cell Line↗
PubMed1975

Characteristics of a Bacillus subtilis W23 mutant temperature sensitive for initiation of chromosome replication.

A temperature-sensitive mutant of Bacillus subtilis W23, dna-20 (Ts), has been isolated and shown to be defective in initiation of rounds of chromosome replication at the nonpermissive temperature. Upon transfer of dna-20(Ts) from 30 to 45 C, deoxyribonucleic acid synthesis, as measured by [3H]thymine incorporation, gradually ceases. The distribution of genetic markers among unreplicated and replicated deoxyribonucleic acid, isolated from dna-20(Ts) after a period at 43 C in a medium containing 5-bromouracil, and fractionated in a CsCl gradient, shows that the cessation of initiation at the higher temperature is immediate. On the other hand, ribonucleic acid and protein synthesis continues at elevated or unaltered rates for some time after the shift to 45 C. Marker frequency analysis shows that all rounds of replication in progress at the time of the temperature shift terminate rapidly (within 40 min), even when chromosomes are replicating dichotomously in rich media. dna-20(Ts) remains 100% viable for at least 2 h at 45 C. Over a 5-h period at 45 C the nuclear bodies remain compact; a small number (less than 5%) of deoxyribonucleic acid-less cells are produced, but there is no morphological distortion of the cells. When the cells are returned to 30 C after 2 h at 45 C, chromosome replication is initiated rapidly at the normal origin and then proceeds in the normal established sequence. However, a second round of replication is initiated soon after the first. dna-20(Ts) has been shown to map as a B-group mutation, the major class of initiation mutants identified in B. subtillus 168.

Autoradiography↗
PubMedDate not supplied

Diagnosis of Plasmodium vivax malaria using a specific deoxyribonucleic acid probe.

A deoxyribonucleic acid (DNA) probe which specifically distinguishes Plasmodium vivax from P. falciparum malaria has been derived from a P. vivax genomic DNA library. This probe, VPL101, consists of 3.2 kilobase pairs and does not hybridize with up to 6 micrograms of human or P. falciparum DNA. VPL101 contains at least two copies of a 205 base pair repeat sequence. The subcloned repeat probe, VPL101/5, reacted with 73 of 76 microscopically diagnosed P. vivax samples but not with any of 17 human DNA samples or any of 8 P. falciparum DNA samples from cultured parasites. It was possible to detect P. vivax in mixed infections in which only P. falciparum parasites were identifiable by microscopy. This P. vivax DNA probe provides a useful epidemiological tool for malaria control programmes.

Animals↗
PubMedDate not supplied

Mapping variation in chromosome homologues of different Giardia strains.

A landmark physical map of the 2-Mb chromosome of the Giardia duodenalis cloned line WB-1B, constructed using randomly cloned, chromosome specific markers, was used to compare the organisation and map order of the equivalent chromosome in other strains. A representative marker from each of the 13 NotI segments of the 2-Mb chromosome was hybridized to NotI cleavages of whole chromosomes of the other strains. Two strains, one isolated from a human, and one from a cat, had the same chromosome hybridization patterns as WB-1B. A strain isolated from a sheep, had one NotI chromosome 5 segment larger than WB-1B. Two additional strains isolated from a calf and a human had significantly different NotI cleavage patterns from the previous strains and shared no similar-sized chromosome NotI segment from their 2Mb chromosome homologues and only one in common with WB-1B. In one strain, two markers from the same WB-1B NotI segment did not hybridize suggesting deletion events have occurred. The order of some NotI segments within the 2Mb chromosome homologue was maintained, as determined from partial NotI chromosome cleavages, while in the most divergent of strains internal chromosome rearrangements and deletions were evident. All but one of the 2Mb WB-1B chromosome markers examined hybridized to a single chromosome band in all strains. Thus, while Giardia chromosomes vary in size, copy number and organisation, some linkage of markers is apparently maintained in isolates from disparate hosts and localities. We have therefore generated a genetic analysis system for Giardia with landmark maps using representative markers to replace the paucity of classical genetic markers and mutants. This approach is being extended to the complete genome.

Animals↗
PubMedDate not supplied

Two distinct varieties of Giardia in a mixed infection from a single human patient.

Twenty-five in vitro cultures of Giardia duodenalis derived from a Brisbane patient were established to assess the genetic heterogeneity of a population. Each of the established lines carried a predominance of one of two distinct varieties of Giardia. The two varieties were heterogeneous by four unambiguous criteria that were representative of the whole genome. These included restriction enzyme polymorphisms, hybridization with the cloned rDNA repeat and with a gene encoding a cysteine-rich surface protein, electrophoretic karyotyping and DNA fingerprinting. Differences between parasites derived from this patient were greater than have been seen between all other established G. duodenalis in vitro cultures from both humans and animals. The cultures were heavily selected such that a single Giardia line carried a predominance of one genotype and was not representative of the entire original population.

Animals↗
PubMedDate not supplied

A new rDNA repeat unit in human Giardia.

The rDNA repeat unit from a new human Giardia duodenalis strain shows significant differences from the previously reported G. duodenalis rDNA repeat. Twelve base-pair changes occurred in 490 bp of the SSrRNA gene and new restriction enzyme sites occurred in the LSrRNA gene. The overall length of the rRNA genes is the same but the spacer is 76 bp longer than previously reported. A boundary within the spacers of the two different rDNA units divides a region of 50% homology near the LSrRNA gene from a region of 80% homology toward the SSrRNA gene. This boundary region includes two copies of a 78 bp repeat.

Animals↗
PubMedDate not supplied

Keto-acid oxidoreductases in the anaerobic protozoa.

In anaerobes, decarboxylation of pyruvate is executed by the enzyme pyruvate:ferredoxin oxidoreductase, which donates electrons to ferredoxin. The pyruvate:ferredoxin oxidoreductase and its homologues utilise many alternative substrates in bacterial anaerobes. The pyruvate:ferredoxin oxidoreductase from anaerobic protozoa, such as Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica have retained this diversity in usage of alternative keto acids for energy production utilising a wide variety of substrates. In addition to this flexibility, both T. vaginalis and G. duodenalis have alternative enzymes that are active in metronidazole-resistant parasites and that do not necessarily involve donation of electrons to characterized ferredoxins. Giardia duodenalis has two oxoacid oxidoreductases, including pyruvate:ferredoxin oxidoreductase and T. vaginalis has at least three. These alternative oxoacid oxidoreductases apparently do not share homology with the characterized pyruvate:ferredoxin oxidoreductase in either organism. Independently, both G. duodenalis and T. vaginalis have retained alternative oxoacid oxidoreductase activities that are clearly important for the survival of these parasitic protists.

Anaerobiosis↗