Studies on trypanosomatid flagellates with special reference to antigenic variation and kinetoplast DNA

Hajduk, Stephen Louis (1980) Studies on trypanosomatid flagellates with special reference to antigenic variation and kinetoplast DNA. PhD thesis, University of Glasgow.

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The results presented in this thesis concern two aspects of the biology of trypanosomatids. First, the process of antigenic variation in the African trypanosomes and second the structure and function of the kinetoplast DNA of trypanosomatids. Part I. Trypanosomes were cyclically transmitted by the insect vector, Glossina morsitans, and the expression of variable antigen types (VATs) in the metacyclic populations from the salivary glands and the first bloodstream populations in metacyclic initiated infections in mice were analysed. Tsetse flies were fed on the blood of mice containing any one of 5 VATs of Trypanosoma brucei of the ANTAR 1 serodeme. The VATS of the metacyclic trypanosomes subsequently detected in the flies' saliva probes vere investigated using monospecific antisera to AnTAK 1 VaTs in indirect immunofluorescence and trypanolysis reactions; these sera included 5 raised against AnTats 1.6, and 1.45, previously idnetified as components of the metacyclic population (M-VATs), and against the 5 VATs originally ingested by the flies. The percentage of metacyclics reacting with a particular M-VAT antiserum remained more or less constant (AnTat 1.6, 6.0-8.3% AnTat 1.30, 13.7-18.2%; AnTat 1.45, 2.0-8.0%), regardless of the age of the fly or the ingested VaT. AS these 3 VATS account for no more than one-third of the metacyclic population, the existence of at least one more VAT is envisaged. The ingested VAT could not be detected among the AnTAR 1 metacyclic trypanosomes. Metacyclic trypanosomes from the salivary glands of infected tsetse flies were also used to initiate infections in mice. Immunofluorescence and trypanolysis reactions employing 24 monospecific antisera were used to analyse the VATS present in the mice following cyclical transmission. Regardless of the VAT used to infect tsetse flies, the first VATS detectable in the bloodstream were those previously identified as M-VATS. These were present until at least 5 days after infection, at which time lytic antibodies against at least 2 of the M-VATs were detectable in the blood of infected mice. In mice immunosuppressed by X-irradiation the M-VATs were detectable in the bloodstream for longer periods, but the percentage of the population labelled with anti-metacyclic sera showed a decrease on day 5 as in non-irradiated animals. The VAT ingested by the tsetse was always detectable early during the first parasitaemia following cyclical transmission and was usually the first VAT detectable after the M-VATs. Neutralization of selected M-VATs before infecting mice resulted in elimination of the neutralized M-VAT from the first parasitaemia but had no effect on the expression of other VATs in the early infection. Part II. In studies on the structure and function of the kinetoplast DNA (KDNA) of trypanosomatids I have examined the KDNA structure and mitochondrial activity of two species of Herpetomonas and also a stock of T. brucei which has lost the ability to activate its mitochondrion during syringe passaging in laboratory rodents.The structure of the KDNA of Herpetomonas muscarum and Herpetomonas inrenoplastis was compared by electron microscopy, restriction endonuclease digestion and hydridization with cloned portions of the maxi-circle from T. brucei 427. The KDNA of both H. musearum and H.ingenoplasits has a buoyant density of 1 .69B g/cm3; however, the KDNA of H. ingenoplastis represents 31% of the total cellular DNA as compared with 8% for H. muscarum KDNA. The KDNA network of H. muscarum consists of thousands of mini-circles of 0.6 to 0.7 x 106 daltons and a few large circular molecules, maxi-circles, of 21 x 10 daltons. The mini-circles of H. muscarum show sequence heterogeneity while maxi-circles of H. muscarum have a unique nucleotide sequence. The KDNA of H. ingenoplastis completely lacks mini-circle size molecules and the network is composed 6 6 6 entirely of large circular molecules of 11 x 106, 15.5 x 106 and 24 x 106 daltons. The 11 x 106 and 15.5 x 106 dalton molecules show sequence heterogeneity and are the major component of the KDNA. Hybridization studies with cloned fragments of T. brucei maxi-circle suggest that the 24 X 106 dalton component of H. ingenoplastis kDNA is functionally equivalent to the maxi-circle of other trypanosomatids. It was concluded that the 11 x 106 and 15.5 x 106 dalton circles of H. in enoplastis are functionally similar to mini-circles of other tryoanosomatids and that the maxi-circles of H. ingenoplastis differ from those of T. brucei and H. muscarum in major nucleotide sequences. The structure and activity of the mitochondrion from H.ingenoplastis and H. muscarum have been studied by electron microscopy, respiration studies with different substrates and inhibitors, analysis of oligomycin- sensitive ATPase activity and low-temperature difference spectra of respiratory chain cytochromes. Certain differences in the two species can be correlated with alterations in the maxi-circle of H. ingenoplastis described in the preceding paper.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: The work presented in this thesis was supported by a research assistantship from the Overseas Development Ministry (Scheme R3338) and a short-term fellowship from the European Molecular Biology Organization.
Subjects: Q Science > QL Zoology
Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Supervisor, not known
Date of Award: 1980
Depositing User: Mrs Marie Cairney
Unique ID: glathesis:1980-38921
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 12 Nov 2018 08:33
Last Modified: 12 Oct 2021 15:15
Thesis DOI: 10.5525/gla.thesis.38921
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