Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Heartwater is a major threat to livestock in Africa due to its high mortality rate. The
intracellular nature of the causative organism, Cowdria ruminantium, makes it difficult to
study, hence an effective and user-friendly vaccine has been extremely difficult to obtain.
Two C. ruminantium DNA libraries have recently been constructed, the lambda GEM11
bacteriophage DNA library and the lambda ZAPII bacteriophage DNA library, and this has
lead to a renewed search for protective genes that could be used as a vaccine against
heartwater.
In this study, several molecular techniques including PCR, cloning and sequencing were used
to identify genes in the lambda GEM11 bacteriophage DNA library that code for proteins,
which could be used as vaccines to protect susceptible animals against heartwater.
The lambda GEM11 library was screened with a rickettsial secretory protein gene sequence,
known as seeD. One positive colony was selected from which the bacteriophage DNA was
isolated. The C. ruminantium DNA was amplified from the bacteriophage DNA by using PCR
and C. ruminantium-specific primers. The C. ruminantium DNA was screened with
Mycoplasma, bovine and Cowdria DNA probes. The amplified DNA was subeloned into two
vectors and the clones were screened by restriction analysis to identify clones containing
inserts. The appropriate clones were sequenced and overlapping sequences matched, ordered
and aligned. Two sequences were continuous with a short sequence of unidentified bases in
between. Oligonucleotide primers were designed to amplify the DNA sequence between the
two contiguous sequences. This led to the identification of the entire sequence of the C.
ruminantium genome contained within the bacteriophage plaque. The single contiguous
sequence was analysed and the putative protein-coding sequences were obtained and
compared to DNA sequences of known organisms using the BLAST program. Five open
reading frames were identified with homology to genes encoding specific proteins in bacteria.
Two open reading frames showed homology to the genes encoding the transporter proteins,
FtsY and the ABC transporter, and three open reading frames were found to be homologous
to genes encoding the essential enzymes dethiobiotin synthetase, pro lipoprotein
diacylglycerol transferase and the putative NADH-ubiquinone oxidoreductase subunit. The
five open reading frames encode for genes, which are essential for the normal functioning of
the C. ruminantium organism. However, these open reading frames might not be effective for
use in a DNA vaccine since none of the open reading frames showed homology to obvious genes that could play a role in immunity and therefore confer protection. The open reading
frames can be used in mutagenesis studies to produce attenuated strains of the organism that
possess mutated versions of these proteins. These attentuated strains could be used for the
vaccination of cattle, and thereby confer protection against viable pathogenic C. ruminantium
isolates. / AFRIKAANSE OPSOMMING: Hartwater is 'n bedreiging vir vee in Afrika weens die hoë mortaliteitssyfer verbonde aan die
siekte. Die intrasellulêre aard van die organisme wat hartwater veroorsaak, Cowdria
ruminantium, bemoeilik navorsing aangaande die organisme. Dit het tot gevolg dat 'n
effektiewe en gebruikersvriendelike entstof moeilik bekombaar is. Daar is onlangs sukses
behaal met die konstruksie van twee C. ruminantium DNA genoteke, die lambda GEM11
bakteriofaag genoteek en die lambda ZAPII bakteriofaag genoteek. Dit het gelei tot 'n
herlewing in die soektog na beskermende gene, wat in 'n entstof teen hartwater gebruik kan
word.
In hierdie studie is verskeie molekulêre tegnieke insluitende PKR, klonering en
geenopeenvolging bepaling, gebruik om gene te identifiseer in die lambda GEM11
bakteriofaag genoteek wat kodeer vir proteïene wat in entstowwe gebruik kan word as
beskerming teen hartwater.
Die secD geen is gebruik om die lambda GEM11 bakteriofaag genoteek te sif. Een positiewe
plaak is gevind waarna die DNA uit die bakteriofaag plaak geïsoleer en die C. ruminantium
DNA vanuit die bakteriofaag plaak geamplifiseer is deur gebruik te maak van PKR en
spesifieke C. ruminantium inleiers. Die C. ruminantium DNA is gesif met Mycoplasma, bees
en Cowdria radioaktief gemerkte DNA peilers. Die C. ruminantium DNA is vervolgens in
twee vektore gekloneer. Die klone is gesif deur middel van restriksie analise. Die DNA
volgorde van die klone is bepaal en twee ononderbroke sekwense is geïdentifiseer met 'n
gaping in die middel tussen die twee sekwense. Oligonukleotied inleiers is daarna ontwerp om
die geenopeenvolging van die gaping tussen die twee sekwense te vul. Hierdeur kon die
volledige geenopeenvolging van die genoom van C. ruminantium wat in die lambda GEM 11
bakteriofaag plaak voorkom, bepaal word. Hierdie volledige geenopeenvolging is vervolgens
geanaliseer en die oop leesrame wat daarin voorkom geïdentifiseer. Vyf leesrame is gevind
om homologie met gene wat kodeer vir proteïene wat in bakterieë voorkom, te toon. Twee
leesrame het homologie met die gene wat kodeer vir transport proteïene, FtsYen die ABC
transporter getoon, en drie leesrame het homologie met gene wat kodeer vir die essensiële
ensieme detiobiotin sintetase, prolipoproteïen diasielgliserol transferase en die NADHubikinoon
oksidoreduktase subeenheid getoon. Dié vyf leesrame het die potensiaal om as
entstowwe gebruik te word aangesien al vyf leesrame kodeer vir gene wat 'n belangrike rol
speel in die oorlewing van die C. ruminantium organisme. Alhoewel die leesrame moontlik nie so effektief sal wees in 'n DNA entstof nie, toon dit potensiaal om in mutasieeksperimente
gebruik te word. Organismes wat die gemuteerde weergawe van die geen besit
sal nie-funksionele proteïene produseer, wat 'n invloed kan hê op die normale fisiologiese
funksies van die organisme en dus sal lei tot 'n minder virulente organisme. Die geattenueerde
organisme kan moontlik gebruik word om diere te immuniseer en daardeur immuniteit aan
diere lewer wat beskerming sal bied teen patogeniese C. ruminantium isolate.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53052 |
| Date | 12 1900 |
| Creators | Patience, Trudy |
| Contributors | Bellstedt, D. U., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 168 p. : ill. (some col.) |
| Rights | Stellenbosch University |
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