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Genomic and Cellular Integration in the Tripartite Nested Mealybug SymbiosisHUSNÍK, Filip January 2017 (has links)
The PhD thesis is composed of three publications on genomic, metabolic, and cellular integration between the host and its symbionts in the tripartite nested mealybug system. The articles revealed a path to an intimate endosymbiosis that can be compared to what we think happened before (and to some extent after) bacterial ancestors of key eukaryotic organelles, mitochondria and plastids, became highly integrated into their host cells. I argue that these much younger symbioses may tell us something about how the mitochondria and plastids came to be, at the very least by revealing what types of evolutionary events are possible as stable intracellular relationships proceed along the path of integration.
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Adenovirus endocytosis and adenoviral gene transfer in cardiovascular and dermatologic disease modelsRauma-Pinola, T. (Tanja) 10 September 2004 (has links)
Abstract
Adenoviral gene transfer is a valuable tool in molecular biology
research. In order to be an efficient and safe vector, adenovirus
structure and infection mechanism as well as molecular biology of the
used transgene need to be well studied. The aim of this study was to
evaluate the role of adenovirus as a gene transfer vector from several
perspectives. Adenovirus uses receptor-mediated endocytosis in order to
enter the target cell. The effect of Rab5 GTPase on adenovirus entry and
gene transfer efficiency was examined first. Next, adenovirus was used
as an investigatory tool in the cardiovascular research, focused on
clarifying the role of adrenomedullin (AM) in heart and vascular
remodeling. Finally, a model of adenoviral gene transfer into skin
fibroblasts was used.
The role of Rab5 GTPase in the adenovirus endocytosis was examined
in HeLa cells using Cy3-labeled adenovirus, and gene transfer efficiency
using β-galactosidase encoding adenovirus. Rab5 increased both
adenovirus uptake and gene transfer, whereas dominant negative Rab5S34N
decreased both endocytosis and gene transfer. The data indicate that
Rab5 is needed in mediating the adenovirus uptake into the target
cell.
In the rat heart, adenovirus-mediated AM gene transfer transiently
improved systolic function both in vivo and
in vitro. AM caused activation of translocation of
protein kinases C ε and δ, whereas phosphorylation of p38
mitogen activated protein kinase was decreased in the left ventricle. AM
significantly attenuated the development of angiotensin II-induced
cardiac hypertrophy. In rats with myocardial infarction, AM enhanced
dilatation of left ventricle and thinning of anterior wall. The role of
AM in neointima formation was evaluated in rat artery after endothelial
injury. Intravascular AM gene transfer decreased neointimal growth and
increased neointimal myofibroblasts apoptosis. These results show that
AM regulates left ventricular systolic function and remodeling in the
heart, and plays a role in pathological vascular remodeling.
Adenovirus-mediated lysyl hydroxylase (LH) gene transfer into skin
fibroblasts of type VI Ehlers-Danlos syndrome patient and rat skin
increased functional LH production, elevated LH activity, and human LH
mRNA production both in vitro and in
vivo. LH gene replacement therapy may thus lead to
possibilities to improve skin wound healing in Ehlers-Danlos syndrome
patients.
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Surgical organ perfusion method for somatic gene transfer:an experimental study on gene transfer into the kidney, spleen, lung and mammary glandParpala-Spårman, T. (Teija) 04 February 2000 (has links)
Abstract
The progress in recombinant DNA technology has made possible
the introduction of exogenous genetic material into cells. Gene
therapy aims to correct a defective gene, introduce a therapeutic exogenous
gene or a counteracting gene into somatic cells without modification
of the germ-cell line. The most important technical interests in
the field of gene therapy research have pertained to the development
of safe and effective vectors and suitable methods for the delivery
of the exogenous gene carrying vectors into the target cells. The
aim of this study was to evaluate surgical methods used for gene
delivery and to develop an effective gene transfer method for organ-specific
gene transfer, primarily into the renal glomeruli.
There are genetic and acquired diseases that are candidates
for gene therapy. Alport syndrome is an X-chromosome-linked disease
caused by a mutation in the type IV collagen α5 chain gene,
which causes a defect of the glomerular basement membrane in the
kidney, leading to progressive renal failure in males. This manifestation
could theoretically be prevented by the transfer of a normal α5 chain
gene into the renal glomerular cells. Cystic fibrosis and α1-antitrypsin
deficiency are examples of pulmonary diseases and genetic lysosomal
storage diseases that are candidates for splenic gene transfer.
The gene transfer strategies used so far have proved relatively
ineffective. Recombinant adenovirus, retrovirus, adeno-associated
virus and liposomes have been previously used as vectors. Direct
injection, intra-arterial, intravenous and intratracheal delivery
of vectors have been the most extensively studied methods.
This preclinical experimental work for marker gene transfer
into the kidney, spleen, lung and mammary gland was done by using
rabbits, pigs and goats as test animals. The adenoviral vector carrying
a β-galactosidase reporter gene was first infused in the
renal artery of rabbits and pigs in vivo with
or without pharmacological agents. This did not result in any remarkable
gene transfer into the kidney. Next, the incubation time between
the vector and the target cells was prolonged by ex vivo perfusion of explanted kidneys
for 12 hours. Perfusion at room temperature did not improve gene transfer.
When the perfusion temperature was raised to 37°C, improved
and mostly glomerular gene transfer was observed, with up to 80% of
the glomeruli showing β-galactosidase expression in four ex vivo experiments.
A closed-circuit organ perfusion method for in vivo gene transfer was developed
in this study. The surgical perfusion experiment was tested successfully
in ten in vivo perfusions of
the kidney, eight of the spleen and eight of the lung in a porcine
model. This method led to effective, up to 75% gene transfer
into the renal glomeruli as assessed after four days. In the spleen,
the perfusion method resulted in relatively effective gene transfer
into perifollicular splenic cells, mostly macrophages and endothelial
cells. Lung perfusion yielded transgene expression in alveolar epithelial
cells, bronchiolar epithelial cells and, to a lesser extent, arteriolar
endothelial cells and alveolar macrophages. Perfusion of the goats
mammary gland using a retroviral vector in three experiments resulted
in growth hormone secretion into the milk.
The gene transfer operation was well tolerated by the animals,
and no clinical signs of inflammation were observed. No remarkable
humoral immunological response against adenovirus or β-galactosidase
was elicited in the kidney experiments, but histological signs of
inflammation as mononuclear cell clusters in the kidney and lung
were seen four and seven days after the experiments. The spleen
showed no macroscopic or microscopic pathologic alterations after
the perfusion.
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Evolutionary Status of Mitochondrial Ribosomal Protein Genes rps19 and rpl2 and their Transfer to the Nucleus in GrassesAtluri, Sruthi January 2015 (has links)
Massive mitochondrial gene transfer to the nucleus occurred very early during eukaryotic evolution following endosymbiosis, and is still ongoing in plants. Tracking recent gene transfer events can give us insight into the evolutionary processes by which a transferred gene becomes functional in the nucleus and how its protein gets targeted back into the mitochondrion, where it is needed. Rps19 and rpl2 are two such ribosomal protein genes that are known to have been transferred to the nucleus, many times independently during flowering plant evolution. My research project focusses on determining the status and expression of rps19 and rpl2 in the mitochondrion and nucleus of selected grasses and in particular brome (close relative to agronomically important crops such as wheat, rye and barley). My results at the level of DNA and RNA (PCR and RT-PCR, respectively) show that the mitochondrial brome rpl2 copy is a pseudogene while its functional gene is in the nucleus. The brome mitochondrial genome has a copy of rps19 which is transcribed and C-U edited. Surprisingly, the brome nuclear genome also has functional copies of rps19.The targeting sequence for the nuclear rps19 gene was acquired from duplication of mitochondrial targeting heat shock protein (hsp70) presequence. Comparative analysis strongly suggests that a functional rps19 gene was transferred to the nucleus before rice and maize lineages split and now that brome rps19 has been found to be present in both compartments, this implies a transition stage of about 60 million years. Oats was found to have a functional rps19 copy in the nucleus and has a novel presequence due to lineage specific rearrangements and exon shuffling. Functional paralogous copies were found in wheat, and maize while barley lost one of the copy. Thus, following transfer, duplication of rps19 gene must have occurred in the ancestor of barley and wheat clade. Maize might have had a recent duplication or gene conversion events along its lineage as its paralogous copies are very similar to each other. More information is needed to determine if this duplication event extends to wheat-brome, wheat-oats or even before rice and maize split. Barley was also found to have a recent independent DNA mediated transfer in addition to the common transfer, as it possesses an unedited nuc-mt rps19 in its nuclear genome. This suggests that barley must also have had a transition stage for ~60MY and lost its mitochondrial copy very recently.
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Inferring a Network of Horizontal Gene Flow among Prokaryotes Using Complementary ApproachesSengupta, Soham 08 1900 (has links)
Horizontal gene transfer (HGT), a mechanism that facilitates exchange of genetic material between organisms from different lineages, has a profound impact on prokaryotic evolution. To infer HGT, we first developed a comparative genomics-based tool, APP, which can perform phyletic pattern analysis using completely sequenced genomes to identify genes are unique to a genome or have sporadic distribution in its close relatives. Performance assessment against currently available tools on a manually created 18-genome dataset and 2 benchmarking datasets revealed the superior accuracy of APP over other methods. We then utilized a parametric method to construct a gene exchange network. The composition-based method, Jenson-Shannon Codon Bias (JS-CB), groups genes into clusters based on similar codon usage bias. These clusters were analyzed using APP and examined for the enrichment HGT associated marker genes, then annotated as of native or alien origin based on these multiple lines of evidence. Intergenome clustering enabled identification of genes mobilized across alien components of the genomes (alien-alien transfer) and from native components of donor genomes to the recipient genomes (native-alien transfer). Functional classification of alien gene clusters revealed that metabolism associated genes are most frequently mobilized, in concurrence with previous reports, and additionally, a large number of genes with yet unknown functions were found to have been horizontally transferred, a important finding that needs to be further investigated.
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Building a History of Horizontal Gene Transfer in E. ColiWilber, Matthew 01 January 2016 (has links)
Bacteria's ability to pass entire genes between one another, a process called Horizontal Gene Transfer (HGT), has a major impact on bacterial evolution. In an ongoing project at Harvey Mudd, computational methods have been used to catalogue the HGT events that have impacted a group of closely related bacteria.
This thesis builds on that project, by improving our ability to identify gene families --- groups of genes in different strains that are related. Previously, similarity was measured only by comparing two genes' DNA sequences, ignoring their positions on the organism's DNA. Here, we leverage genes' relative position to make a better measurement of gene similarity. These improved similarity measurements will improve the existing pipeline's ability to identify HGT events.
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The Contribution of Horizontal Gene Transfer to the Evolution of Fungi.Hall, Charles Robert 10 May 2007 (has links)
The genomes of the hemiascomycetes Saccharomyces cerevisiae and Ashbya gossypii have been completely sequenced, allowing a comparative analysis of these two genomes, which reveals that a small number of genes appear to have entered these genomes as a result of horizontal gene transfer from bacterial sources. One potential case of horizontal gene transfer in A. gossypii and 10 potential cases in S. cerevisiae were identified, of which two were investigated further. One gene, encoding the enzyme dihydroorotate dehydrogenase (DHOD), is potentially a case of horizontal gene transfer, as shown by sequencing of this gene from additional bacterial and fungal species to generate sufficient data to construct a well-supported phylogeny. The DHOD-encoding gene found in S. cerevisiae, URA1 (YKL216W), appears to have entered the Saccharomycetaceae after the divergence of the S. cerevisiae lineage from the Candida albicans lineage and possibly since the divergence from the A. gossypii lineage. This gene appears to have come from the Lactobacillales, and following its acquisition the endogenous eukaryotic DHOD gene was lost. It was also shown that the bacterially derived horizontally transferred DHOD is required for anaerobic synthesis of uracil in S. cerevisiae. The other gene discussed in detail is BDS1, an aryl- and alkyl-sulfatase gene of bacterial origin that we have shown allows utilization of sulfate from several organic sources. Among the eukaryotes, this gene is found in S. cerevisiae and Saccharomyces bayanus and appears to derive from the alpha-proteobacteria. / Dissertation
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Comparative genomic study for identifying gene acquisitions in Megavirales / Etude comparative génomique pour identifier les acquisitions de gènes à megaviralesJain, Sourabh 06 July 2017 (has links)
La découverte de virus géants avec une taille de génome géante et des caractéristiques génomiques surprenantes soulève différentes questions sur leur origine et leur évolution. De nombreuses études phylogénétiques ont souligné le rôle décisif des HGT et des échanges génétiques sur l'évolution des MV, mais la plupart d'entre eux sont basés sur des familles MV étroitement liées. Pour enquêter sur les événements HGT, nous avons déterminé la distribution des gènes et les phylogénies de gènes pour les 86 ORFomes MV complets classés dans 6 familles définies et 4 putatives, dans le cadre de leurs homologues d'autres domaines de la vie. À l'aide d'un flux de travail phylogénétique automatisé MimiLook, 4577 OG ont été détectés, dont 91% des OG ont été jugés spécifiques à la famille, alors que 9% sont représentés par des protéines de 2 familles MV ou plus. 414 OG ont été détectés comme événement HGT. Nous avons appliqué une procédure similaire aux 7 898 protéines non orthologues pour détecter les événements de transfert et identifié 259 HGT à partir de protéines non orthologues. Les cas de HGT révèlent la spécificité des donneurs. En conclusion, une distinction claire peut être observée dans le mosaïque du génome des familles de Megavirale éloignées, où elles ont évolué par spécificité génomique et acquisitions de gènes spécifiques à la famille de leur créneau écologique respectif. Notre recherche systématique d'événements HGT d'origine non-mégavirale fournit la première estimation de la contribution totale de HGT dans le mosaïque du génome spécifique à la famille des Megavirales éloignés. / Discovery of giant viruses with giant genome size and surprising genomic features raises different question about their origin and evolution. Many phylogenetic studies have pointed out decisive role of HGTs and genetic exchanges on evolution of MVs, but, majority of them are based on closely related MV families. To investigate HGT events, we have determined gene distributions and gene phylogenies for the 86 complete MV ORFomes classified in 6 defined and 4 putative families, in context of their homologs from other domains of life. Using an automated phylogenetic workflow MimiLook, 4577 OGs were detected, out of which, 91% of OGs were found to be family specific, whereas, 9% are represented by proteins from 2 or more MV families. 414 OGs were detected as HGT event. We applied a similar procedure to the 7,898 non-orthologous proteins to detect transfer events and identified 259 HGTs from non-orthologous proteins. Instances of HGT were found to be depicting donor specificity, as viruses of vertebrates/invertebrates acquired genes from donors like Euteleostomii, Eutheria, Baculoviridae and proteobacteria; algal viruses and protozoan viruses were found to be acquiring genes from donors like Dictyostellium, Mammeillales, Firmicutes, Clostridiales. In conclusion, clear distinction can be seen in the genome mosaicism of distantly related Megavirale families, where they evolved via genome specificity and family specific gene acquisitions from their respective ecological niche. Our systematic search for HGT events of non-megavirale origin provides the first estimate of the total contribution of HGT in family specific genome mosaicism of distantly related Megavirales.
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The Impact of Horizontal Gene Transfer on the Evolution of New Functions in Salmonella entericaNazmi Muhamer, Nevin January 2021 (has links)
No description available.
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The potential for toxin and antitoxin gene pairs to display a post-segregational killing phenotype, with regards to the ecology of mobile elements.Coray, Dorien Skye January 2014 (has links)
Genes are able to replicate horizontally and vertically- a given gene
may be more successful on horizontally mobile elements than others. This
includes genes that exhibit a post-segregational killing (PSK) phenotype.
PSK is generated by expression of a toxin and antitoxin from a mobile
element, such that if a bacterium loses the element the toxin becomes
active in the cell and the cell dies. All PSKs described to date involve a
toxin and an antitoxin function, though within a given group of toxin and
antitoxin gene pairs only some are likely to exhibit this phenotype. Here, I
investigate what differentiates genes that induce PSK from biochemically
similar genes that do not.
One group of genes of which some are known to induce PSK is toxinantitoxin
(TA) systems, composed of a stable toxin and an unstable
antitoxin. I analyzed computational data on the distribution of type I TA
systems (RNA antitoxin), which appear to be less mobile than type II
TA systems (protein toxin). Data on validated TAs suggests a correlation
between distribution, mobility and the PSK phenotype. Differences in
phylogeny could be due to differences in tendency to exhibit PSK in different
environments. This connection between distribution and PSK was
explored by experimentally testing a computationally described operon,
plasmid_Toxin-ptaRNA1, that exhibited structural and distributional
similarities to a mobile type I TA system. Despite this, expression of the
predicted toxin ORFs did not reduce growth (as measured by saturation
density) in E. coli, and the operon did not induce PSK.
The conditions of PSK were further tested with the toxin (barnase)
and antitoxin (barstar), which are not known to have the phenotype. A
number of heterologous expression systems were developed with these
genes in E. coli to test their ability to exhibit PSK in a manner akin
to both type II TA systems, with a cytoplasmic toxin, and bacteriocins,which have a secreted toxin. I used equations of logarithmic decay to
model the necessary expression of the proteins in the cell and their rate
of decay after plasmid loss to enable PSK. My results suggest there is
likely to be an evolutionary trend toward TA systems with high expression
levels of very unstable antitoxins. Secreted barnase was also tested
experimentally for its ability to induce PSK similar to bacteriocins, which
exhibit a PSK-like phenotype in monoculture by driving maintenance of
the immunity encoding plasmid. Barnase did not induce PSK, possibly
due to its inability to cause antibiosis in our test system.
Structural similarities and biochemical similarities are not sufficient to
determine whether a given system will act as a PSK because numerous
contextual factors have an effect on whether the genes are addictive.
A given set of genes may have the phenotype in one species but not
another, under one set of environmental conditions but not another, or
on one replicon but not another. This is consistent with the competition
hypothesis, which states that genes will be selected for on mobile elements
due to their ability to increase horizontal reproductive success, depending
on the environmental conditions.
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