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1
Jansen, Yvette. "Characterisation of a high copy number mutant pAL5000 origin of replication." Thesis, Stellenbosch : Stellenbosch University, 2001. http://hdl.handle.net/10019.1/52159.
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Thesis (MScMedSc)--Stellenbosch University, 2001.<br>ENGLISH ABSTRACT: The plasmid pAL5000 is a mycobacterial plasmid isolated from Mycobacterium
fortuitum. It is a low copy number plasmid, which replicates in both fast growing (e.g.
M. smegmatis) and slow growing (e.g. M. bovis BCG) mycobacteria. Most
mycobacterial-E. coli shuttle vectors utilise the pAL5000 origin of replication. The
minimum replicon consists of ORF1 (RepA), ORF2 (RepB) and the origin of
replication.
Dr W.R. Bourn created an E. coli-mycobacterial vector based on the pAL5000 origin
of replication (pORI) and then subjected it to semi-random mutagenesis. A high copy
number mutant was identified (pHIGH) and the causative mutation was tentatively
identified as a 3bp deletion situated just upstream of repB. This work describes the
further characterisation of the mutant plasmid.
Firstly, it was shown by retransforming M. smegmatis with both the original and
mutant plasmids (pORI and pHIGH), that the mutation causing the increased copy
number was plasmid-encoded and not on the chromosome. Following this, it was
demonstrated by simple subcloning of the region that carries the 3 bp deletion, that
other pAL5000-based vectors could be converted to high copy number. In addition to
this, the subcloned region was sequenced and the nature of the mutations was
confirmed. The subcloning experiment confirmed that the 3bp deletion caused the
high copy number phenotype.
Following this, the exact copy number of pHIGH and the relative increase in copy
number was determined. From this, the copy number of pORI could also be
determined. The plasmid pHIGH has a copy number of approximately 54, compared
to the 8 of pORI (a relative increase by a factor of 7).
Because it is important for researchers to know the characteristics of the vectors that
they use, especially the influence it will have on its host, stability tests and growth
curves were also performed. It was seen that the higher copy number did not
markedly increase the stability, however, this is because pORI is already extremely, and unexpectedly, stable in the host M. smegmatis. According to the growth curves,
the increased copy number has little effect on the growth of the host M. smegmatis.
Possible mechanisms for the increased copy number were then investigated. By using
a promoter probe vector, the possible existence of a promoter situated between the
two open reading frames of pAL5000 (repA and repB) was investigated. It was
thought that the mutation might have created, or changed an existing promoter,
situated between repA and repB. The results showed, however, that in both pORI and
pHIGH there might be a very weak promoter upstream of repB, but the mutation did
not cause any change that was measurable by the method that was used.
A further possibility was that the mutation caused a change in the RNA secondary
structure, which might then have an effect on the translational efficiency of RepB. It
was found that the 3bp deletion in pHIGH causes a change in the local RNA
secondary structure around the ribosomal binding site and the start codon, when
compared to pORI (wild type). This change may cause the translation initiation rate of
RepB to be different between pHIGH and pORI. Ultimately it would lead to a
different ratio of RepA and RepB in the cell.<br>AFRIKAANSE OPSOMMING: Die plasmied pAL5000 is ‘n mikobakteriele plasmied wat vanuit Mycobacterium
fortuitum gei'soleer is. Dit is ‘n lae kopie-getal plasmied wat in beide vinnig groeiende
(bv. M. smegmatis) en stadig groeiende (bv. M. bovis BCG) mikobakteriee kan
repliseer. Die meeste mikobakteriele-E. coli shuttle vektore gebruik die pAL5000
oorsprong van replisering. Die minimum replikon bestaan uit ORF1 (RepA), ORF2
(RepB) en die oorsprong van replisering.
Dr. W.R. Bourn het ‘n E. coli-mikobakteriele vektor gemaak wat gebaseer is op die
pAL5000 oorsprong van replisering (pORI), en dit onderwerp aan semi-random
mutagenese. ‘n Hoë kopie-getal mutant is gei'dentifiseer (pHIGH) en die mutasie
hiervoor verantwoordelik was tentatief gei'dentifiseer as ‘n 3bp delesie, net stroomop
van repB. Die projek beskryf die verdere karakterisering van die mutante plasmied.
Eerstens, deur M. smegmatis te hertransformeer met die plasmied DNA (pORI en
pHIGH), is dit bewys dat dit mutasie wat die toename in kopie-getal veroorsaak, deur
die plasmied gekodeer word, en dat dit nie ‘n mutasie op die chromosoom is nie.
Hierna is dit deur eenvoudige subklonering bewys dat die gedeelte wat die 3bp delesie
dra, ander pAL5000-gebaseerde vektore ook kan verander in ‘n hoër kopie-getal. Die
sub-klonerings eksperiment het ook bewys dat die 3 bp delesie die oorsaak is vir die
hoë kopie-getal fenotipe.
Volgende is die presiese kopie-getal van pHIGH en die relatiewe toename in kopiegetal
bepaal. Die kopie-getal van pORI kon vanaf hierdie data bepaal word. Die
plasmied pHIGH het ‘n kopie-getal van ongeveer 54 in M. smegmatis, in vergelyking
met die 8 van pORI (‘n relatiewe toename met ‘n faktor van 7).
Aangesien dit vir navorsers belangrik is om die eienskappe van die vektore wat hulle
gebruik, te ken, en veral die invloed wat dit op die gasheer sal hê, is stabiliteits toetse,
en groeikurwes gedoen. Die hoër kopie-getal het nie die stabiliteit werklik verbeter
nie, maar dit is omdat pORI alreeds uiters stabiel is in die gasheer M. smegmatis. Volgens die groeikurwes het die toename in kopie-getal ‘n minimale effek op die
groei van die gasheer M. smegmatis.
Moontlike meganismes vir die hoër kopie-getal is ook ondersoek. Die moontlike
bestaan van ‘n promoter tussen die twee oop-leesrame van pAL5000 (repA en repB)
is ondersoek deur gebruik te maak van ‘n “promoter probe” vektor. Die mutasie kon
moontlik ‘n promoter geskep het, of ‘n bestaande een tussen repA en repB verander
het. Die resultate het gewys dat daar in beide pORI en pHIGH moontlik ‘n baie swak
promoter stroomop van repB is, maar die mutasie het nie enige veranderinge
veroorsaak wat meetbaar was met die metode wat gebruik is nie.
‘n Verdere moontlikheid was dat die mutasie ‘n verandering in die RNA sekondere
struktuur kon veroorsaak het, en dit mag ‘n effek hê op die translasie effektiwiteit van
RepB. Daar is gevind dat, in vergelyking met pORI, het die 3bp delesie in pHIGH ‘n
verandering in die lokale RNA sekondere struktuur rondom die ribosomale bindings
posisie en die begin-kodon veroorsaak. Die verandering mag veroorsaak dat die
translasie inisiasie tempo van RepB verskillend is vir pORI en pHIGH. Uiteindelik sal
dit lei tot ‘n heeltemal ander verhouding van RepA en RepB in die sel.
2
Udo, Edet Ekpenyong. "Characterisation and molecular studies of plasmids from Nigerian staphylococci." Thesis, Curtin University, 1991. http://hdl.handle.net/20.500.11937/1845.
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Fifty three Staphylococcus aureus isolates were obtained from three centres, two hospitals and a private pathology laboratory, and studied for susceptibility to bacteriophages, resistance to antimicrobial agents and plasmid contents.Results of bacteriophage typing revealed that they belonged to a variety of phage types. Eighteen were untypable by any of the International Set of Phages, 16 belonged to phage group 111, nine to group I, four to group 11, two to group IV and two to the miscellaneous group.The isolates were resistant to one or more of methicillin (Mc), benzyl penicillin (Pc), gentamicin (Gm) , kanamycin (Km) , neomycin (Nm) , streptomycin (Sm) , trimethoprim (Tp), sulphonamides (Su), tetracycline (Tc), minocycline (Mn), chloramphenicol (Cm), novobiocin (Nb) and fusidic acid (Fa). Resistance to Pc was due to the production of beta-lactamase (Bla). No resistance to vancomycin, spectinomycin and erythromycin was detected. Resistance was also found to heavy metals such as cadmium (Cd), mercury (Hg), phenyl mercuric acetate (Pma), arsenate (Asa) and to the nucleic-acid binding compounds propamidine isethionate (Pi) and ethidium bromide (Eb).All but one of the isolates harboured plasmids. The number of plasmids the isolates carried varied from one to six and their sizes ranged from < 1.0 kb to c.48 kb.Location of the resistance determinants was ascertained by curing and transfer experiments. Loss of resistance was tested after growth at 43.5°C and transfer of resistance determinants was attempted by transduction, mixed-culture transfer and conjugation. The results revealed that resistance to Mc, Gm, Tp, Mn and Fa was chromosomal in all the resistant isolates and in some isolates Bla and resistance to Sm and Cd were chromosomal as well as plasmid encoded. In the majority of cases Bla and resistance to Km, Nm, Sm, Tc, Cin, Cd, Hg, Asa, Pma, Pi and Eb was encoded by plasmids.Conjugation experiments led to the isolation of three unique conjugative plasmids which have not been found to confer resistance to antimicrobials or to produce haemolysins or diffusible pigment (Dip). The three plasmids, pWBG620, pWBG637 and pWBG661, were indistinguishable by restriction endonuclease analysis and DNA-DNA hybridisation. However pWBG620, unlike pWBG637 and pWBG661, was not detected in the cytoplasm of its host and was only detected in transconjugants after it mobilised a non-conjugative Sm-resistance (SmR) plasmid. Further analysis indicated that it is integrated into the chromosome of its host, excises during conjugation and mobilises the SmR plasmid.These plasmids were studied further using pWBG637 as a representative. It was compared with representatives of the two groups of conjugative plasmids which have been reported in the staphylococci. These are the plasmids which encode resistance to Gm, Km and Nm and those which code for the production of diffusible pigment. The three types of conjugative plasmids were compared by restriction endonuclease analysis and DNA- DNA hybridisation and were found to be different. A preliminary restriction map of pWBG637 has also been constructed.However since pWBG637 has no resistance phenotype direct selection for it was not possible in transfer experiments and for incompatibility (Inc.). To study it further it was necessary to construct resistant derivatives which could be selected for in transfer experiments. This was achieved by labelling pWBG637 with resistance transposons to generate two conjugative plasmids, pWBG636 carrying an insert of Tn3851 (Gm- resistance) and pWBG642 carrying an insert of Tn551 (hn- resistance). It was found that transposon labelling had not changed the incompatibility of pWBG637 and therefore pWBG636 and pWBG642 were used in further experiments in place of pWBG637. Inc. tests with the pWBG637 derivatives revealed that the pWBG637 type of plasmid is not only different from the other two types of conjugative plasmids but is different from any of the described staphylococcal Inc. groups and therefore the pWBG637 type of plasmids represent a new Inc. group 15. The pWBG637 type of plasmids were studied further using plasmids pWBG636 and pWBG642. They were able to transfer conjugatively to a capsulated S.aureus strain either by the polyethylene glycol method or on filter membranes. They also transferred by conjugation to S. epidermidis and Streptococcus faecalis and were able to transfer back from these strains to S.aureus indicating that they also replicate in these hosts. Consequently they have been used to mobilise non-conjugative plasmids from S.epidermidis and non phage typable S.aureus. Both plasmids failed to transfer conjugatively to Bacillus subtilis and Escherichia coli.pWBG637 transferred non-conjugative plasmids by mobilising them in a manner similar to mobilisation (donation) in E.coli or by recombining with them to form new resistance plasmids. In one case, pWBG628 which encodes Bla and resistance to Cd, Km, Nm and Sm and has no homology with pWBG637 recombined with it during conjugation to produce three new conjugative plasmids pWBG629, pWBG630 and pWBG631 carrying resistance determinants from pWBG628. One of these plasmids, pWBG629, was found to be pWBG637 which had acquired a 4.5 kb element encoding resistance to Km, Nm and Sm. This element was shown to be transposable in both rec+ and rec- backgrounds and has been designated Tn3854. It expressed Sm resistance in E.coli and differs on this account from the Gram-negative transposon Tn5 which expresses resistance to Km, Nm and Sm in non-enteric bacteria but only resistance to Km and Nm in E. coli.Where possible the non-conjugative plasmids encoding resistance to antimicrobial agents were compared with phenotypically similar plasmids isolated from other parts of the world. It was found that the Tc and Sm resistance plasmids were closely related to other plasmids with the same phenotype whereas the Cm resistance plasmids were different.Although the majority of the Bla plasmids belonged to Inc. group 1 they demonstrated significant restriction enzyme fragment length polymorphism when compared with other Bla plasmids.This study has provided the first data on the genetics of antimicrobial resistance in Nigerian S.aureus. Although many of the plasmids studied were found to be similar to those previously described the isolates also contained some unique and previously undescribed plasmids.
3
Seyler, Richard W. "Plasmid stability of pUB110 and pUB110-derived plasmids in Bacillus sphaericus 2362." Thesis, This resource online, 1991. http://scholar.lib.vt.edu/theses/available/etd-03022010-020139/.
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4
Hirst, Jonathan Michael. "Plasmids in thermoactinomyces." Thesis, Heriot-Watt University, 1991. http://hdl.handle.net/10399/826.
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5
Udo, Edet Ekpenyong. "Characterisation and molecular studies of plasmids from Nigerian staphylococci." Curtin University of Technology, School of Biomedical Sciences, 1991. http://espace.library.curtin.edu.au:80/R/?func=dbin-jump-full&object_id=15648.
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Fifty three Staphylococcus aureus isolates were obtained from three centres, two hospitals and a private pathology laboratory, and studied for susceptibility to bacteriophages, resistance to antimicrobial agents and plasmid contents.Results of bacteriophage typing revealed that they belonged to a variety of phage types. Eighteen were untypable by any of the International Set of Phages, 16 belonged to phage group 111, nine to group I, four to group 11, two to group IV and two to the miscellaneous group.The isolates were resistant to one or more of methicillin (Mc), benzyl penicillin (Pc), gentamicin (Gm) , kanamycin (Km) , neomycin (Nm) , streptomycin (Sm) , trimethoprim (Tp), sulphonamides (Su), tetracycline (Tc), minocycline (Mn), chloramphenicol (Cm), novobiocin (Nb) and fusidic acid (Fa). Resistance to Pc was due to the production of beta-lactamase (Bla). No resistance to vancomycin, spectinomycin and erythromycin was detected. Resistance was also found to heavy metals such as cadmium (Cd), mercury (Hg), phenyl mercuric acetate (Pma), arsenate (Asa) and to the nucleic-acid binding compounds propamidine isethionate (Pi) and ethidium bromide (Eb).All but one of the isolates harboured plasmids. The number of plasmids the isolates carried varied from one to six and their sizes ranged from < 1.0 kb to c.48 kb.Location of the resistance determinants was ascertained by curing and transfer experiments. Loss of resistance was tested after growth at 43.5°C and transfer of resistance determinants was attempted by transduction, mixed-culture transfer and conjugation. The results revealed that resistance to Mc, Gm, Tp, Mn and Fa was chromosomal in all the resistant isolates and in some isolates Bla and resistance to Sm and Cd were chromosomal as well as plasmid encoded. In the majority of cases Bla and resistance to Km, Nm, Sm, Tc, Cin, Cd, Hg, Asa, Pma, Pi and Eb was encoded ++<br>by plasmids.Conjugation experiments led to the isolation of three unique conjugative plasmids which have not been found to confer resistance to antimicrobials or to produce haemolysins or diffusible pigment (Dip). The three plasmids, pWBG620, pWBG637 and pWBG661, were indistinguishable by restriction endonuclease analysis and DNA-DNA hybridisation. However pWBG620, unlike pWBG637 and pWBG661, was not detected in the cytoplasm of its host and was only detected in transconjugants after it mobilised a non-conjugative Sm-resistance (SmR) plasmid. Further analysis indicated that it is integrated into the chromosome of its host, excises during conjugation and mobilises the SmR plasmid.These plasmids were studied further using pWBG637 as a representative. It was compared with representatives of the two groups of conjugative plasmids which have been reported in the staphylococci. These are the plasmids which encode resistance to Gm, Km and Nm and those which code for the production of diffusible pigment. The three types of conjugative plasmids were compared by restriction endonuclease analysis and DNA- DNA hybridisation and were found to be different. A preliminary restriction map of pWBG637 has also been constructed.However since pWBG637 has no resistance phenotype direct selection for it was not possible in transfer experiments and for incompatibility (Inc.). To study it further it was necessary to construct resistant derivatives which could be selected for in transfer experiments. This was achieved by labelling pWBG637 with resistance transposons to generate two conjugative plasmids, pWBG636 carrying an insert of Tn3851 (Gm- resistance) and pWBG642 carrying an insert of Tn551 (hn- resistance). It was found that transposon labelling had not changed the incompatibility of pWBG637 and therefore pWBG636 and pWBG642 were used in further experiments in place of pWBG637. Inc. ++<br>tests with the pWBG637 derivatives revealed that the pWBG637 type of plasmid is not only different from the other two types of conjugative plasmids but is different from any of the described staphylococcal Inc. groups and therefore the pWBG637 type of plasmids represent a new Inc. group 15. The pWBG637 type of plasmids were studied further using plasmids pWBG636 and pWBG642. They were able to transfer conjugatively to a capsulated S.aureus strain either by the polyethylene glycol method or on filter membranes. They also transferred by conjugation to S. epidermidis and Streptococcus faecalis and were able to transfer back from these strains to S.aureus indicating that they also replicate in these hosts. Consequently they have been used to mobilise non-conjugative plasmids from S.epidermidis and non phage typable S.aureus. Both plasmids failed to transfer conjugatively to Bacillus subtilis and Escherichia coli.pWBG637 transferred non-conjugative plasmids by mobilising them in a manner similar to mobilisation (donation) in E.coli or by recombining with them to form new resistance plasmids. In one case, pWBG628 which encodes Bla and resistance to Cd, Km, Nm and Sm and has no homology with pWBG637 recombined with it during conjugation to produce three new conjugative plasmids pWBG629, pWBG630 and pWBG631 carrying resistance determinants from pWBG628. One of these plasmids, pWBG629, was found to be pWBG637 which had acquired a 4.5 kb element encoding resistance to Km, Nm and Sm. This element was shown to be transposable in both rec+ and rec- backgrounds and has been designated Tn3854. It expressed Sm resistance in E.coli and differs on this account from the Gram-negative transposon Tn5 which expresses resistance to Km, Nm and Sm in non-enteric bacteria but only resistance to Km and Nm in E. coli.Where possible the non-conjugative plasmids encoding resistance to ++<br>antimicrobial agents were compared with phenotypically similar plasmids isolated from other parts of the world. It was found that the Tc and Sm resistance plasmids were closely related to other plasmids with the same phenotype whereas the Cm resistance plasmids were different.Although the majority of the Bla plasmids belonged to Inc. group 1 they demonstrated significant restriction enzyme fragment length polymorphism when compared with other Bla plasmids.This study has provided the first data on the genetics of antimicrobial resistance in Nigerian S.aureus. Although many of the plasmids studied were found to be similar to those previously described the isolates also contained some unique and previously undescribed plasmids.
6
Ophel, Kathleen Margaret. ""Agrobacterium" : plasmids and biovars /." Title page, contents and summary only, 1987. http://web4.library.adelaide.edu.au/theses/09PH/09pho61.pdf.
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7
Pinder, David. "Illegitimate recombination in plasmids." Thesis, University of Edinburgh, 1996. http://hdl.handle.net/1842/11260.
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Illegitimate recombination mechanisms are important for genetic change within an organism. They are also the cause of many instability problems in biotechnology and have been associated with certain human genetic disorders and cancers. The original aim of this work was to construct a deletion (illegitimate recombination) resistant cosmid based, cloning system, for the cloning of unstable human DNA. Two 'mutant plasmids' pMS5 and pMS7 were isolated. The plasmids were derived from pUC18 and appeared to stabilise the propagation of a long DNA palindrome. The basic concept was to construct a new cosmid using pMS7 as part of the backbone. The construction of two new cosmids cDRII (Deletion Resistant) and cDRIII is described. However, they are unlikely to contain a mutation which stabilises unstable sequences. This thesis also describes the search for the 'mutation' in pMS7 by, single-strand conformation polymorphism and fragment swap analysis. This work led to the isolation of a novel mutation composed of both direct and inverted repeats, which I have called DIR. The presence of DIR in pAC2 (a derivative of pUC18, with the same DNA palindrome as pMS7), supports the absence of a stabilising 'mutation in pMS7. The structure of the DIR mutation is analysed in detail and a hypothesis for its formation is proposed. Finally, the behaviour of four long DNA palindromes (other than that cloned in pMS7), is investigated when ligated into pM<SUP>*</SUP> (a derivative of pMS7).
8
Maia, Mauricio Silva. "Plasmids of Azotobacter vinelandii." Thesis, North Texas State University, 1986. https://digital.library.unt.edu/ark:/67531/metadc798298/.
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Nineteen laboratory strains of Azotobacter vinelandii and two organisms of the same species isolated from water samples were screened for the presence of plasmid deoxyribonucleic acid (DNA). Three laboratory strains and both organisms isolated from water samples contained one plasmid each. The migration distances of the plasmids in agarose gel electrophoresis were different molecular weights. The plasmids were cured by SDS or ethidium bromide treatment of the cultures.
9
Chilley, Paul Morris. "Leading regions of enterobacterial plasmids." Thesis, University of Leicester, 1995. http://hdl.handle.net/2381/34394.
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Gram-negative bacterial conjugation is a specialised replicative event that increases the population size of a plasmid during its horizontal transfer between organisms. Work reported in this thesis has focused on the genetic structure of the leading regions of enterobacterial plasmids. A leading region is the first segment of a plasmid to enter the recipient cell during conjugation. Some leading regions carry conserved loci. Examples are ssb encoding a single-stranded DNA binding protein; psiB which functions to inhibit induction of the bacterial SOS response; ardA which encodes antirestriction activity, and hok, a gene that contributes to plasmid maintenance. The objective of this work was to determine the distribution of these genes on enterobacterial plasmids and their arrangement relative to the origin of conjugative transfer (oriT). Sequences homologous to ardA were found on plasmid representatives of five (FV, B, I, K and N) out of 23 incompatibility groups tested. Mapping data shows that the ardA homologues are leading region genes, and subcloning experiments coupled with phenotypic assays provide evidence that the IncB and IncFV ardA homologues are functional antirestriction genes. Sequence data reveal that the ardA gene family has diverged by at least 60% at the nucleotide sequence level when compared to the prototype ardA gene of ColIb-F9. A non-homologous antirestriction gene, ardB, was only detected on IncN plasmids. Conserved ssb and psiB genes are known to be present on a number of enterobacterial plasmids representing nine different incompatibility groups (B, com9, I1, FI, FII, FIV, K and Y). It is reported that the leading region of an IncFV plasmid, F0lac, also carries conserved ssb-psiB genes. In common with previous findings, the genes are separated by a spacer of ~2.5kb, indicating their presence in a conserved module. Genes of the hok family of killer genes are found on the leading region of FI and FII plasmids. Southern hybridisation experiments revealed that IncI1 plasmids lack a member of the hok subfamily but contain a member of the pnd subfamily. Functional and hybridisation tests localised pnd to the trailing region, defined as the last segment of a plasmid to enter the recipient during conjugation. Hybridisation data also localised the pnd genes of B and K plasmids to be outside of the leading region. The finding that the killer genes are scattered is consistent with the concept that the ecological role of the hok gene family is in plasmid maintenance rather than in conjugation. The results show that while enterobacterial plasmid leading regions collectively contain some conserved genes, there is considerable heterogeneity in their combination and arrangement on different plasmids with respect to oriT. It is postulated that the difference reflects independent acquisition of modules during the evolution of the different plasmid types. An important finding is that leading region genes are each orientated such that the transcribed strand is the same as the transferred strand. This arrangement holds for plasmids ColIb (IncI1), F (IncFI), pKM101 (IncN) and F0lac (IncFV) and could be important for the expression of these genes. One possibility is that the genes are expressed from the incoming plasmid strand before it is converted into duplex DNA.
10
McKibben, Ann Laura. "Characterization of plasmids in Gluconobacter." Thesis, Virginia Tech, 1992. http://hdl.handle.net/10919/44232.
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11
McKibben, Laura Ann. "Characterization of plasmids in Gluconobacter /." This resource online, 1992. http://scholar.lib.vt.edu/theses/available/etd-08142009-040503/.
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12
Cook, Marisa Anne. "Replicons derived from endogenously isolated plasmids used to classify plasmids occurring in marine sediment bacteria." Thesis, Georgia Institute of Technology, 2001. http://hdl.handle.net/1853/25736.
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13
Asghari, Abdolkarim. "Physical Characterization and Restriction Mapping of the Sal Plasmid From Pseudomonas Putida." Thesis, North Texas State University, 1987. https://digital.library.unt.edu/ark:/67531/metadc798475/.
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Physical and restriction mapping of the salicylate catabolic plasmid SAL from Pseudomonas putida strain PpG 2119 was carried out by standard multiple restriction analysis and by cross hybridization studies using radioactively labeled restriction fragment probes. The total numbers of fragments produced, their respective sizes, the arrangement of the restriction fragments on the plasmid and the map locations of the enzyme recognition sites for Hpal, Xhol, Dral and Smal are given.
14
Zainuddin, Zainul Fadziruddin. "Mycobacterial plasmids and related DNA sequences." Thesis, University of Surrey, 1988. http://epubs.surrey.ac.uk/843201/.
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An investigation into the presence of plasmids in Mycobacterium tuberculosis was conducted. No extrachromosomal DNA was found although some evidence for their presence were detected. Plasmid-related sequences were however found in the genomic DNA of M, tuberculosis. Isolation of plasmid-related sequences from a strain of M, tuberculosis resulted in two different DNA probes which showed high specificity for M, tuberculosis and related species. One of these probes gave banding patterns which suggest the presence of restriction fragment polymorphism in M, tuberculosis. The other probe gave banding patterns which were unique for each strain tested. The use of these probes for epidemiological studies is suggested. An E. coli plasmid, pIJ666, was successfully introduced into Mycobacterium smegmatis ATCC607 and stably maintained through several subcultures. One of the selective markers, a chloramphenicol resistance gene from Tn9, was efficiently expressed in M. smegmatis ATCC607. The development of a transformation system in M. smegmatis from this initial study is indicated.
15
Walters, John Anthony. "Replication of plasmids of Staphylococcus aureus." Thesis, University of Oxford, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315767.
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16
Winson, Michael Kenneth. "Molecular biological studies on catabolic plasmids." Thesis, Bangor University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305927.
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17
Thomas, Nicholas Simon. "The molecular biology of chlamydial plasmids." Thesis, University of Southampton, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295857.
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18
Saw, Howard Thien Hui. "The biology of antibiotic resistance plasmids." Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/6083/.
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Plasmids confer genes encoding clinically relevant antibiotic resistance. It was hypothesised that the AcrAB-TolC multidrug resistance efflux pump was required for clinically relevant levels of carbapenem resistance. However, carbapenemase-producing \(Salmonella\) TolC mutants were less susceptible to carbapenems. In the presence of the efflux inhibitor phe-arg- β-naphthylamide (PAβN), wildtype bacteria and 36/86 non-replicate clinical isolates of carbapenem-producing Enterobacteriaceae were ≥4-fold less susceptible to ertapenem. Experimental data suggested that OmpF repression conferred the increased carbapenem MICs. Two blaKPC-encoding plasmids have been isolated in the UK; pKpQIL-UK was found in K. \(pneumoniae\), but its variant, pKpQIL-D2 was also found in other species. Therefore, it was hypothesised that a region of pKpQIL-D2 either conferred a broader plasmid host range and/or a fitness benefit to the host bacterium. Fitness studies measuring growth rates, ability to form biofilm, conjugation frequency and plasmid persistence showed that both plasmids affected the host bacterium but in different ways. Compared to pKpQIL-UK, pKpQIL-D2 did not confer a significant fitness advantage to its host under the conditions tested. RNAsequencing showed both plasmids affected a different set of genes related to metabolism. The understanding of the factor(s) contributing to the persistence and dissemination of successful plasmids may help to control antibiotic resistance.
19
Taylor, James Andrew. "The plasmids of Rhodococcus aetherivorans I24." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613971.
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20
Coons, Terry M. "Restriction mapping and expression of recombinant plasmids containing the arsenic resistance genes of the plasmid R45." PDXScholar, 1986. https://pdxscholar.library.pdx.edu/open_access_etds/3597.
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The trivalent (arsenite) and pentavalent (arsenate) forms of arsenic are introduced into the environment through the use of arsenic in herbicides, pesticides, fertilizers, and the smelting of arsenic-bearing ores. Bacteria resistant to arsenic are readily isolated from surface waters, sewage, and clinical infections. Although some bacterial resistance is provided by inducible phosphate transport systems that discriminate against arsenate, marked resistance is carried on bacterial plasmids.
A 6.9 kilobase fragment previously derived from one such plasmid, R45, and containing the genes for inducible resistance to arsenite and arsenate was ligated into the cloning vectors puce and pUC9 in opposite orientations and transformed into Escherichia coli JM 105. Insertion into the multiple cloning site of the pUC vectors places the inserted fragment under the inducible control of the lac operon promoter. An attempt was made to determine the direction of transcription in the fragment by growth in 10-3 M isopropyl-β-D-thiogalactoside prior to challenge with arsenite.
21
Lazdins, Alessandro. "Development of plasmids that can displace antibiotic resistance plasmids from bacteria in the human and animal gastrointestinal tract." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/7990/.
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Antibiotic resistance is one of the greatest scientific and medical challenges of the 21st century. However, our arsenal to combat this surge is limited to a small number of antimicrobial drugs. Plasmids are major contributors to the problem, yet specifically targeting them to reduce the burden of resistant infections has not been fully exploited. It is possible to repress plasmid’s replication and eliminate them from a population by harnessing their innate replication and maintenance functions. This concept has been applied in this study to construct conjugative broad-host range plasmids aimed at curing either IncF or IncK plasmids based on IncPα RK2 and pUB307 backbones. pUB307- based pCURE gave a stronger curing phenotype as tested by a novel simple curing experiment and long-term invasion assays. The curing potentiation was investigated and found to be due to the absence of iteron 10 in oriV and a resulting higher copy number. The clinical potential of the technology was demonstrated by successfully curing a novel resistance plasmid, pEK499 and by in vivo mice experiments where pCURE conjugated into and cured target plasmids from E. coli established within the gastrointestinal tract. With further work to ensure long-term curing and biosafety, pCURE could improve the treatment options for multi-drug resistant infections.
22
Catchpole, Ian R. "Studies of small plasmids of Staphylococcus aureus." Thesis, University of Oxford, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.253298.
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23
Harrison, Peter William. "Bacterial chromids are neither chromosomes nor plasmids." Thesis, University of York, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556257.
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In addition to the main chromosome, many bacterial genomes have a second element, often called a "second chromosome" or "megaplasmid". It has become clear that, while they carry some essential genes, these replicons do not have the properties normally expected of chromosomes. In this thesis a systematic analysis of all publicly available sequenced bacterial genomes demonstrates that these large secondary replicons represent a single class of elements with a distinct and consistent set of properties. The term 'chromid' is proposed to distinguish them from both chromosomes and plasmids. Chapter Two utilises compositional measures to demonstrate that chromids are present in approximately one in ten sequenced genomes, and found in many bacteria of medical, agricultural' and environmental importance. Moreover, the nucleotide composition and codon usage of chromids is very similar to that of the chromosomes they are associated with, despite possessing plasmid replication and partitioning systems. Chapter Three determines that chromids possess a number of orthologous genes held on chromosomes in other species, as well as being particularly rich in genus-specific genes. This, combined with chromids of different genera having unrelated replication and maintenance systems, suggests that the creation of a new chromid may be associated with the origin of a genus. Chapter Four demonstrates that only genes held on the chromosome maintain high levels of phylogenetic consistency, and Chapter Five highlights the complexity of the interaction between gene composition, expression and function, investigating differences between the three classes of replicon. No large-scale study incorporating all published data has previously been conducted. Yet the identification and characterisation of this component provides an important framework for understanding the evolution and organisation of bacterial genomes. By proposing a clear definition, this thesis seeks to encourage consistent annotation and discussion of the evolution and functional biology of this distinct and common type of replicon.
24
Chak, K.-F. "Expression and regulation of Col E plasmids." Thesis, University of East Anglia, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370379.
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25
Lyle, Keenan Harris. "Comparison of plasmids from clinical Lactobacillus strains." University of the Western Cape, 2018. http://hdl.handle.net/11394/6397.
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Magister Scientiae - MSc (Biotechnology)<br>The vaginal mucosa is dominated by Gram positive, rod shaped lactobacilli which serve as a
natural barrier against infection. In both healthy and BV infected women Lactobacillus
crispatus and Lactobacillus jensennii has been found to be the predominant Lactobacillus
species. Many studies have been conducted to assess factors influencing lactobacilli dominance
in the vaginal microbiome. However, no study has evaluated the impact of plasmids on the
vaginal lactobacilli. In the present study two plasmids, pLc17 and pLc4, isolated from vaginal
Lactobacillus species of both healthy and BV infected women were characterized. pLc4 was
present in both Lactobacillus crispatus and Lactobacillus jensennii while pLc17 was only
present in Lactobacillus crispatus. pLc17 (16663 bp in size) encoded a ribonucleotide
diphosphate reductase (RNR), a filamentation induced by cAMP-like (FIC-like) protein and
numerous mobile elements. The FIC-like protein may assist pLc17 to persist within the
bacterial population, while RNR is commonly associated with phages and may indicate phage
infection. pLc4 (4224 bp in size) encodes for a replication initiator protein and a plasmid
partitioning protein. The replication protein on pLc4 shows 44% identity with the replication
initiation protein of pSMQ173b_03. On further phylogenetic and sequence analysis with other
Rolling Circle Replication (RCR) plasmids, pLc4 appears to be novel as the plasmid shows a
low degree of similarity to these RCR plasmids. pLc17 appears to carry both a RCR replicon as
well as a theta replicon, similar to pIP501, the broad-host-range plasmid from Bacillus subtilis.
The relative Plasmid Copy Number (PCN) for pLc4 and pLc17 was analysed using quantitative
polymerase chain reaction (qPCR) for the healthy state relative to the disease state from twentyeight
vaginal swab samples obtained from the National Institute for Communicable Diseases
(NICD). The relative PCN for pLc4 and pLc17 had a fold increase of ~2.803 and ~1.693,
respectively in the healthy patient samples relative to BV infected patient samples. However,
there were not found to be significant differences when taking the standard error into account
Due to the novelty of these plasmids further analysis and characterisation is required for both
plasmids, to establish what role they may play in the health of the vaginal milieu.
26
Pinto, Beatriz Lázaro. "Broad host range plasmids in estuarine environments." Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/3515.
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Mestrado em Biotecnologia<br>A transferência horizontal de genes permite a adaptação microbiana a nichos
especiais, dos quais dois bons exemplos são a camada superficial do mar e a
coluna de água. Os plasmídeos com gama alargada de hospedeiros (BHR),
responsáveis pelo fluxo de material genético entre cromossomas bacterianos
(inclusivamente entre microorganismos muito afastados filogeneticamente) têm
um papel essencial na evolução das comunidades microbianas. Entre eles, os
plasmídeos pertencendo ao grupo de incompatibilidade IncP-1 têm um
interesse especial por causa da sua extraordinária flexibilidade na iniciação da
replicação e da estabilidade na sua manutenção numa ampla gama de
hospedeiros. Estes elementos genéticos móveis, além de constituírem
ferramentas úteis na engenharia genética, representam uma grande fonte de
genes que codificam para características tão significativas como a resistência
a antibióticos e a degradação de xenobióticos. No entanto, a diversidade nesta
família de plasmídeos BHR tem sido subestimada até agora: actualmente
sabe-se da existência de cinco subgrupos divergentes, mas embora alguns
plamídeos modelo tenham sido estudados ao detalhe, ainda há muito para
investigar.
Em trabalhos anteriores na Ria de Aveiro (costa NW de Portugal), foi feita a
captura exógena de plasmídeos bem como o isolamento de bactérias
potencialmente hospedeiras de plasmídeos endógenos. Neste trabalho,
sequências de nucleótidos específicas foram amplificadas mediante reacções
em cadeia da polimerase para determinar a presença de plasmídeos BHR. Os
sete plasmídeos IncP-1 detectados, foram em primeiro lugarfilogeneticamente
estudados. O alinhamento das sequências de nucleótidos de 281 pb que foram
amplificadas e que correspondem a um fragmento do gene trfA (que codifica
para uma proteína do início da replicaçao) sugeriu o estabelecimento de dois
novos clusters situados filogeneticamente em dois subgrupos diferentes de
IncP-1: IncP-1β e o recentemente descrito IncP-1ε. Estes constituem os
primeiros replicões IncP-1 provenientes de ambientes estuarinos a serem
detectados e isolados. De seguida, uma comparação e caracterização
preliminar genética e fenotípica foi realizada com os plasmídeos purificados,
considerando a descrição já conhecida dos dois plasmídeos arquétipos
evolutivamente mais próximos, pB10 e pKJK5. Assim, as análises de
fragmentos de restrição, determinação da inibição do crescimento do
hospedeiro na presença de mercúrio e ensaios de resistência a diferentes
antibióticos ajudaram a compreender o elevado interesse que recai nestes
plasmídeos revelando a diversidade fenotípica e genotípica. Uma completa
descrição de qualquer destes novos plasmídeos pode ter una enorme
importância ecológica, evolutiva e biotecnológica, incrementada pela sua
procedência dum ambiente não clínico. Portanto este trabalho justifica um
estudo em maior profundidade destes replicões promíscuos.<br>The horizontal gene transfer allows microbial adaptation to special niches, from
which the sea-surface microlayer or the subsurface waters in estuarine
environments might be good examples. Broad host range plasmids,
responsible for the reshuffling of genetic material between bacterial
chromosomes (even amongst distantly related microorganims), play an
essential role on the evolution and diversity of microbial communities. Among
them, the incompatibility group IncP-1 plasmids have a special interest due to
their extraordinary flexibility in the replication initiation and stable maintenance
in such a wide spectrum of hosts. These mobile genetic elements, in addition to
the helpful genetic engineering tools they mean, represent a great source of
potentially useful genes encoding for traits as significant as antibiotic resistance
or xenobiotic degradation. Nevertheless, the diversity of this family of BHR
plasmids has been underestimated until recently: it is currently known to have
five divergent sub-groups, but although some prototype plasmids have been
studied in great detail, there is still much left to research.
In previous investigations exogenous plasmid capture was carried out as well
as putative endogenous plasmid bacterial hosts isolated in the Ria de Aveiro
lagoon (NW coast of Portugal). In this work, polymerase chain reactions were
developed to amplify specific nucleotide sequences and determine BHR
plasmids presence. From a bioinformatical approach, the seven IncP-1
plasmids detected were firstly phylogenetically studied. The alignment of the
amplified 281 bp nucleotide sequences corresponding to a fragment of the
replication initiation protein encoding gene trfA suggested the formation of two
novel clusters belonging to two different IncP-1 plasmid subgroups: IncP-1β
and the lately described IncP-1ε. Additionally, these represent the first
estuarine IncP-1 replicons to be detected and isolated. Then a preliminary
genetic and phenotypic comparison was performed with the purified plasmids,
by taking into account the known description of the evolutionary closest
models, pB10 and pKJK5. That way, restriction fragment analysis as well as
antibiotic and mercury resistance determination assays helped to comprehend
the high significance falling on the captured plasmids by revealing the genetic
and phenotypic diversity. A whole description of any of these novel plasmids
may have a huge ecological, evolutionary and biotechnological importance,
even more due to its precedence from a non-clinical environment. Therefore
this work justifies further studies on these promiscuous replicons.
27
El-Haffar, Mohammad I. A. "The survival of bacteria containing r-plasmids." Thesis, Aston University, 1985. http://publications.aston.ac.uk/12451/.
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28
Kawasaki, Haruhiko. "STUDIES ON PLASMIDS DETERMINING BACTERIAL HALOACETATE DEHALOGENASES." Kyoto University, 1985. http://hdl.handle.net/2433/78183.
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29
Bott, Martha Anne Brunner David P. "Growth inhibition mediated by E4 colicin plasmids." Normal, Ill. Illinois State University, 1986. http://wwwlib.umi.com/cr/ilstu/fullcit?p8626588.
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Thesis (Ph. D.)--Illinois State University, 1986.<br>Title from title page screen, viewed July 13, 2005. Dissertation Committee: David P. Brunner (chair), Herman E. Brockman, Arlan G. Richardson, H. Tak Cheung, Lynne Lucher. Includes bibliographical references (leaves 167-183) and abstract. Also available in print.
30
Sanders, Rhiannon. "Segregational stability of plasmids in Bacillus subtilis." Thesis, University of Warwick, 1986. http://wrap.warwick.ac.uk/109832/.
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The stability of plasmids in Bacillus subtilis in the absence of selective pressure under long term chemostat culture and under different conditions of nutrient limitation was investigated. Culture and sampling conditions were established to allow the detection of plasmid-free cells and samples were routinely screened in order to monitor the structural integrity of the resident plasmid. Initially the stability of the plasmid pHV14-F (carrying the repressor gene of phage 0105) in the Bacillus subtilis strain 3G18 was investigated. This plasmid was found to be segregationally and structurally highly unstable. The stability of the Staphylococcus aureus plasmids pC194 and pUB110 along with the Bacillus cereus plasmid pBC16 was examined in the Bacillus subtilis strain 168 trp in chemostat culture under carbon, magnesium and phosphate limitation. All three plasmids were found to be remarkably stably maintained through up to 100 culture generations of their host strain under all three of the nutrient limitations. Under enforced competition from plasmid-free cells at 1% of the chemostat inoculum all three plasmids were retained in the culture but in the presence of 50% plasmid-free cells in the inoculum plasmid-carrying cells were rapidly displaced from the culture. Phosphate limitation was found to exert a slightly greater stringency of selection for plasmid-free cells than either carbon or magnesium limitations (which had similar effects). A derivative of pC194 was isolated, pC194-Ki which was segregationally unstable, the Bacillus subtilis culture losing its plasmid within 30 generations. On restriction endonuclease digestion analysis the plasmid was revealed to be physically very closely related to pHV14 (pC194 and pBR322 ligated at their HindIII sites). Under magnesium limitation all Bacillus subtilis cultures after 2-3 days were predominantly composed of cells with an unusual helical morphology. The development of this morphology under the conditions employed and the actual form of the helical cells themselves was found to be different from previously reported instances of the appearance of helical cells in cultures of Bacillus subtilis.
31
Harris, Lyle Keenan. "Comparison of plasmids from clinical Lactobacillus strains." University of the Western Cape, 2018. http://hdl.handle.net/11394/6439.
Der volle Inhalt der QuelleAnnotation:
Magister Scientiae - MSc (Biotechnology)<br>The vaginal mucosa is dominated by Gram positive, rod shaped lactobacilli which serve as a
natural barrier against infection. In both healthy and BV infected women Lactobacillus
crispatus and Lactobacillus jensennii has been found to be the predominant Lactobacillus
species. Many studies have been conducted to assess factors influencing lactobacilli dominance
in the vaginal microbiome. However, no study has evaluated the impact of plasmids on the
vaginal lactobacilli. In the present study two plasmids, pLc17 and pLc4, isolated from vaginal
Lactobacillus species of both healthy and BV infected women were characterized. pLc4 was
present in both Lactobacillus crispatus and Lactobacillus jensennii while pLc17 was only
present in Lactobacillus crispatus. pLc17 (16663 bp in size) encoded a ribonucleotide
diphosphate reductase (RNR), a filamentation induced by cAMP-like (FIC-like) protein and
numerous mobile elements.
32
MacLellan, Shawn R. Finan Turlough M. "Study of megaplasmid partitioning and replication initiation." *McMaster only, 2005.
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33
Gill, Santokh Singh Carleton University Dissertation Biology. "IncN group plasmids and plasmid regions determining the killing of Klebsiella pneumoniae and of immunity to killing." Ottawa, 1985.
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34
Williamson, Phillip C. "A Novel Mechanism for Site-Directed Mutagenesis of Large Catabolic Plasmids Using Natural Transformation." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc2828/.
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Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.
35
Beck, Kirsten. "Charakterisierung des Replikationsproteins ORF904 des archaealen Plasmids pRN1." kostenfrei, 2008. http://opus.ub.uni-bayreuth.de/volltexte/2009/545/.
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36
Magnusson, Terese. "Optimised plasmids for sustained transgene expression in vivo." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-123258.
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37
Meijer, Wilhelmus Johannes Jozef. "Replication and maintenance of plasmids in Bacillus subtilis." [S.l.] : [Groningen] : [s.n.] ; [University Library Groningen] [Host], 1995. http://irs.ub.rug.nl/ppn/141649046.
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38
Friis, Lorna M. "Campylobacter plasmids and their role in bacterial virulence." Thesis, University of East Anglia, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423504.
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39
Jackson, Robert Wilson. "Plasmids and virulence in Pseudomonas syringae pv. phaseolicola." Thesis, University of the West of England, Bristol, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389510.
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40
Owuama, C. I. "Genetic transformation of Saccharomyces cerevisiae with chimaeric plasmids." Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381362.
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41
Park, Seonhee. "High-throughput intracellular delivery of proteins and plasmids." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54888.
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Intracellular delivery of macromolecules is crucial for the success of many research and clinical applications. Several conventional intracellular delivery methods have been used for many years but are still inadequate for several applications because of the issues associated with toxicity, low-throughput, and/or difficulty to target certain cell types. In this study, we developed and evaluated new high-throughput intracellular delivery methods for the efficient delivery of macromolecules while maintaining high cell viability. First, we studied the feasibility of using an array of nanoneedles, with sharp tip diameters in the range of tens of nanometers, to physically make transient holes in cell membranes for intracellular delivery. Puncture loading and centrifuge loading methods were developed and assessed for the effect of various experimental parameters on cell viability and delivery efficiency of fluorescent molecules. In both methods, high-throughput intracellular delivery was feasible by creating transient holes in cell membranes with the sharp tips of the nanoneedles. The second physical intracellular delivery method we studied was a novel microfluidic device that created transient holes in the cell membrane by mechanical deformation and shear stress to the cell. We observed efficient delivery of fluorescent molecules and studied the effect of device design and flow pressure on the delivery efficiency compared to data in the literature. We accounted for cell loss and clogging in the microfluidic devices and determined the true loss of cell viability associated with this method. Lastly, we investigated the possibility of intracellular delivery using nanoparticles on a leukemia cell line. Among number of materials for nanoparticles tested, mesoporous silica/poly-L-lysine nanoparticles were selected for further intracellular delivery study based on cell viability and intracellular delivery capability. We demonstrated the co-delivery of protein and plasmid by encapsulating into and coating onto the surface of the nanoparticles, respectively, which would be advantageous for certain therapeutic strategies. In summary, this work introduced two new intracellular delivery methods involving nanoneedles and novel nanoparticles, and provided an early, independent assessment of microfluidic delivery, showing the strengths and weaknesses of each method. These methods can be further optimized for a number of laboratory and clinical applications with continued research.
42
Comeaux, Jay Louis. "Transfer of plasmids by genetically-engineered Erwinia carotovora." Thesis, Virginia Tech, 1989. http://hdl.handle.net/10919/45933.
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<p>The ability of a genetically-engineered <i>Erwirzia carotovora</i> subsp. <i>carotovora</I> (Ecc)
strain to transfer recombinant chromosomal DNA or plasmids to wildtype Ecc or
<i>Pseudomonas fluorescens</i> was tested on filters, within soil microcosms, and <i>in planta</i>. Ecc was engineered by chromosomal insertion of a disarmed <i>endo</i>-pectate lyase gene marked with a 1.4kb DNA fragment conferring kanamycin resistance. Plasmids RPI and
pBR322 were introduced separately into engineered Ecc clones. These strains served as
donors in genetic transfer experiments. No transfer of the inserted kan marker or of
pBR322 was observed under any experimental condition. In filter matings, RPI was
transferred to wildtype Ecc at a frequency of 3.6 X 10<sup>-2</sup> transconjugants per donor (TPD)
and to P. <i>fluorescens</i> at a frequency of 2.4 X 10<sup>-5</sup> TPD. In matings conducted in potato tubers inoculated using sewing needles, the respective frequencies were 4.0 X 10<sup>-4</sup> and 2.0
X 10<sup>-4</sup>, while matings on potato slices yielded frequencies of 4.7 X 10<sup>-2</sup> and 2.3 X 10<sup>-2</sup>.
In soil microcosms, the maximum transfer frequencies observed were 2.3 X 10<sup>-3</sup> and 8.4
X 10<sup>-5</sup> TPD.</p><br>Master of Science
43
Zubrzycki, Igor Z. "Correlation between physical and genetic maps of plasmids." Doctoral thesis, University of Cape Town, 1992. http://hdl.handle.net/11427/21986.
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The aim of this study is to establish a correlation between the physical maps of plasmid DNA (in the form of calorimetric profiles, thermal denaturation profiles and electron micrographs of partly melted DNA sequences) and genetic maps of these DNAs and thus deal with questions which were not answered by previous researchers. viz: Is there a correlation between base sequence function and a measurable physical property which can be assigned to biologically important units such as promoters or coding sequences? Is there a correlation between the denaturation of gene sequences and cooperative transitions observed in a given temperature interval? To answer these questions, a systematic study was initiated based on two families of plasmids with three different genes incorporated, namely the pGV 403 family which contains a Chloramphenicol resistance gene on one side and the pUC9 family which contains an Ampicillin and a Tetracycline resistance gene on the other side. Three different techniques were used to address the stated problems i.e. differential scanning calorimetry, high resolution thermal denaturation and electron microscopy. The reason for using three techniques instead of only one or two as in previous studies is that each technique gives specific results which can be supplemented by the other techniques and only in this way it will be possible to approach a deeper understanding of changes induced by perturbing the sequence based structural integrity by elevating temperature. In addition to measuring the experimentally observable parameters listed. a theoretical model was developed to predict the changes. This approach is termed local compositional complexity (LCC) analysis. The final goal of this investigation was to establish whether there is any correlation between the local compositional complexity and these selected genetic units. Based on the calorimetric experiments an improved table of thermodynamic data including the stacking energy for ten different combinations of basepairs is presented. The prediction of a melting curve based on primary structure information can be based on the enthalpy individual combination of basepairs[41.50.58]. The tables of the thermodynamic data published in the literature are given in Appendix D. In this thesis a slightly different approach to predict tm's was chosen (cf. p 67). The results obtained by this combined approach showed that there is indeed a correlation between the specific base sequence of a given plasmid DNA and its biologically important units (genes) and thus confirms that there is a semi-empirical correlation between genes and the observed cooperative melting units.
44
Clark, Burton David. "Characterization of plasmids from Bacillus thuringiensis var. israelensis /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487330761220416.
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45
Donaldson, Pauline A. (Pauline Alison) Carleton University Dissertation Biology. "Nodulation and tumorgenesis by Agrobacterium carrying Rhizobium plasmids." Ottawa, 1985.
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46
Poulter, Melinda D. "Structural Analysis of the TOL pDK1 xylGFJQK Region and Partial Characterization of the xylF and xylG Gene Products." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc2270/.
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TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where binding of the first substrate effects the enzyme's affinity for the second substrate.
47
Hares, Douglas R. (Douglas Ryan). "Physical and Functional Characterization of the xy1XYZ Region From TOL Plasmid pDK1 and its Associated Downstream Regulatory Elements." Thesis, University of North Texas, 1998. https://digital.library.unt.edu/ark:/67531/metadc278036/.
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The nucleotide sequence for the pDKl TOL plasmid region encoding toluate-1,2-dioxygenase (Xy1XYZ, TO) was determined. TO is the first enzyme in the meta-cleavage operon, responsible for the conversion of toluates and benzoates to their carboxy-substituted diols. DNA sequence analysis revealed the presence of three open reading frames (ORF). The three ORFs correspond to xylX (1353 bp), xylY (486 bp) and xylZ (1008 bp), encoding predicted protein products of 51370 Da, 19368 Da and 36256 Da, respectively.
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Aylett, Christopher Herbert Stanley. "A structural study of the TubZRC plasmid partitioning system." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609711.
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49
Van, Zyl Leonardo Joaquim. "Analysis of the mobilization region of the broad host-range IncQ-like plasmid, pTC-F14, and its ability to interact with a related plasmid, pTF-FC2." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/70109.
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Thesis (MSc)--University of Stellenbosch, 2003.<br>ENGLISH ABSTRACT: The 14.2 kb plasmid pTC-FI4 was isolated from the moderately thermophilic (45°-
50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic bacterium
Acidithiobacil/us caldus and has a replicon that is closely related to the promiscuous,
broad host-range, IncQ-family of plasmids. The region containing the mobilization
genes was sequenced and encoded five Mob proteins and an origin of transfer, which
are related to the DNA processing (Tral) region of IncPI plasmids, rather than to the
three Mob protein systems of the IncQ-l-group plasmids (e.g. plasmids RSFIOIO or
R1162). Plasmid pTC-F14 is the third example of an IncQ family plasmid that has
five mob genes, with the others being pTF-FC2 and pRAS3.1. The minimal region
that was essential for mobilization included the mobA, mobB and the mobC genes as
well as the oriT. The mobD and mobE genes were non-essential, but together
enhanced the mobilization frequency by approximately 300-fold. The repB gene
increased the mobilization frequency but was not essential for mobilization.
Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally
integrated RP4 plasmid was more than 3500-fold less efficient than the mobilization
ofpTF-FC2. When both plasmids were co-resident in the same E. coli host, pTC-FI4
was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-FI4
mobilization frequency was due to the presence of a combination of the pTF-FC2
mobD and mobE gene products, the functions of which are still unknown. pTF-FC2
could mobilize the oriT of pTC-FI4 whereas pTC-F14 could only mobilize the pTFFC2
oriT if provided with some of the mobilization genes from the pTC-FC2
mobilization region. Unexpectedly either the mobEDC genes or the mobAB genes
would allow the mobilization of the pTF-FC2 oriT by pTC-F14 even though there
was no common gene between the two subsets. No evidence for any negative effect
on the transfer of one plasmid by the related, potentially competitive plasmid was
obtained.<br>AFRIKAANSE OPSOMMING: Die 14.2 kb plasmied, pTC-F14, is uit die matig termofiliese (45°C tot 50°C), hoogs
asidofiliese (pH 1.5 tot 2.5), chemolitooutotrofiese bakterium Acidithiobaci/lus caldus
geisoleer en beskik oor 'n replikon wat verwant is aan die vanaf die IncQ-familie van
plasmiede. Hierdie plasmiede is alom bekend vir hulle promiskuïteit tydens
konjugasie asook hul vermoë om in 'n groot aantal verskillende gasheer organismes te
kan repliseer. DNA volgorde analise van die mobiliserings area het 'n oordrags
oorsprong asook vyf oop leesrame onthul wat nader verwant is aan die DNA
prosseserings gene van die Tral area op die IncP 1 plasmiede, as die van die
mobiliserings stelsel van die IncQ-l-groep plasmiede. Plasmied pTC-Fl4 is die derde
voorbeeld, saam met pTF-FC2 en pRAS3.1, van 'n IncQ-tipe plasmied met 'n vyfgeen
mobiliserings sisteem. Die kleinste area op die plasmied nodig vir mobilisering
van pTC-Fl4 is bepaal, en het die mobA, mobB en mobC gene sowel as die oordrags
oorsprong ingesluit. Saam, was die mobD en mobE gene verantwoordelik vir 'n 300-
voud toename in die mobilisasie frekwensie van pTC-Fl4 alhowel die gene nie
absoluut nodig was vir mobilisering van die plasmied nie. Die repB geen het ook
bygedra tot die frekwensie waarteen die volledige plasmied gemobiliseer was, maar
hierdie geen was ook nie nodig vir mobilisering van die pTC-F14 plasmied nie.
Die frekwensie waarteen pTC-Fl4 tussen Escherichia coli rasse beweeg het tydens
konjugasie, terwyl gebruik gemaak is van 'n chromosomaal geintegreerde RP4
plasmied, was ongeveer 3500-voud laer as die van pTF-FC2. Indien beide pTC-Fl4
en pTF-FC2 in dieselfde E. coli gasheer aangetref word, word beide plasmiede teen
ongeveer dieselfde frekwensie gemobiliseer. Die verhoogde frekensie vir pTC-Fl4
was as gevolg van die teenwoordigheid van beide die mobD en mobE gene van die
pTF-FC2 plasmied, waarvan die funksies nog onbekend is. Plasmied pTF-FC2 kon
die oordrags oorsprong van pTC-Fl4 mobiliseer waarteenoor plasmied pTC-FI4 die
oordrags oorsprong vanafpTF-FC2 slegs kon mobiliseer indien een van twee dele van
die pTF-FC2 mobiliserings gene voorsien word (al was daar geen oorvleuling tussen
die twee nie). Alhoewel die plasmiede moontlik kon kompeteer op die vlak van
plasmied oordrag is geen negatiewe kompetesie waargeneem tussen dié twee
verwante plasmiede nie.
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Meakin, Stephanie Asalyn Carleton University Dissertation Biology. "The molecular biology of methanogens: cell lysis, plasmid survey and the characterization of a novel plasmid." Ottawa, 1992.
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