Humboldt-Universität zu Berlin
Mathematisch-Naturwissenschaftliche Fakultät I
Institut für Biologie
Bachelor thesis
„Cellular localisation of Cytochrome P450 CYP-33E2 and CYP-29A3 in the
nematode Caenorhabditis elegans using GFP fusion constructs”
„Zelluläre Lokalisierung von Cytochrom P450 CYP-33E2 und CYP-29A3 des
Nematoden Caenorhabditis elegans mittels GFP Fusionskonstrukten“
presented by
Alexandra Bell
born 15.04.1984 in High-Wycombe, England
matriculation number: 507610
realized in the Group of Freshwater and Stress Ecology
at the Department of Biology
Berlin, May 2009
1.
Abstract
Alike mammals, the nematode Caenorhabditis elegans possesses the capacity to produce
cytochrome P450-dependent (CYP) eicosanoids by metabolizing arachidonic acid (AA) and
eicosapentaenoic acid (EPA). Whereas these signal molecules play an important regulatory
role in numerous vascular, renal and cardiac functions in mammals, their biological functions
within C. elegans, an animal lacking a blood and cardiovascular-system, are still unknown.
The purpose of this study was to localize the gene expression of the two CYP isoforms CYP29A3 and CYP-33E2 mostly contributing to eicosapentaenoic acid (EPA) metabolism in C.
elegans. Moreover, a first estimation concerning the biological roles of the CYP-33E2- and
CYP-29A3-derived eicosanoids was hoped to be gained. In order to study the gene expression
of these two cyp genes, transgenic hermaphrodites were exposed to fenofibrate and βnaphthoflavone, two well-known xenophobic cytochrome P450 inducers.
The localization of cyp gene expression was revealed employing GFP (green fluorescent
protein) fusion constructs. Therefore the promoter region of cyp-29A3 and cyp-33E2,
respectively, was fused with the GFP reporter, which was included in the target vector
pDW20.20 used for the ballistic transformation. The influence of the two xenobiotics on cyp
gene expression was tested by analysing GFP staining after the treatment of worms with
fenofibrate and β-naphthoflavone, respectively, after 24 and 48 h.
A localisation of cyp-29A3 gene expression was not possible, since the transformation was not
successful. However, the transgenic C. elegans line with the plasmid containing the cyp-33E2
promoter and the GFP gene, showed cyp-33E2 gene expression in the cells of the pharynx and
the anterior intestine. The cyp promoter driven GFP production was slightly induced by the
two xenobiotics, yet a statistically significant induction of cyp-33E2 gene expression was only
proven for β-naphthoflavone after the 24 hour treatment (p < 0.05).
The results suggest that cyp-33E2 gene expression occurs in the pharyngeal muscle cells and
that the eicosanoids are most likely to play a crucial role in the pharyngeal pumping activity.
Moreover, they may contribute to muscle contraction by modulating the activity of various
ion channels and thus may also influence feeding behaviour.
Zusammenfassung
Der Nematode Caenorhabditis elegans besitzt wie Säugetiere die Fähigkeit mit Hilfe von
Cytochrom P450 Enzymen (CYP) aus Arachidonsäure (AA) und Eicosapentaensäure (EPA)
Eicosanoide zu bilden. Diese Signalmoleküle regulieren in Säugetieren vielfältige Gefäß-,
Herz-, und Nierenfunktionen, wohingegen ihre biologische Funktion in C. elegans, ein Tier
ohne Blut- und Herz-Kreislauf-System, noch völlig unbekannt ist. Das Ziel dieser Arbeit war
die Lokalisierung der Genexpression von denen im Wesentlichen an der Metabolisierung von
EPA beteiligten zwei CYP Isoformen CYP-33E2 und CYP-29A3 in C. elegans. Weiterhin
sollte eine erste mögliche Einschätzung der biologischen Funktionen der Metabolite erfolgen.
Um die Genexpression der cyp Gene zu untersuchen, wurden transgene Hermaphroditen den
zwei bekannten xenobiotischen Cytochrom P450 Induktoren Fenofibrat und β-Naphthoflavon
ausgesetzt.
Die Lokalisierung der cyp Genexpression erfolgte mittels GFP Fusionskonstrukten. Hierfür
wurde jeweils die Promotorregion der beiden cyp Gene in einen GFP tragenden Vector,
pDW20.20, ligiert. Die Transformation von C. elegans erfolgte mittels ballistischer
Transformation. Die Auswirkung der beiden Xenobiotika wurde anhand der GFP Färbung
nach jeweils einer Behandlungsdauer von 24 und 48 Stunden bestimmt.
Eine Lokalisierung der Genexpression von cyp-29A3 konnte nicht erfolgen, da die ballistische
Transformation nicht erfolgreich war. Die Würmer, welche mit dem Plasmid, das mit dem
cyp-33E2 Promoter und dem GFP Gen ligiert war, transformiert wurden, zeigten dagegen
cyp-33E2 Genexpression im Pharynx und im vorderen Teil des Darms. Es erfolgte eine
leichte cyp-Promoter induzierte GFP Produktion nach Zugabe der beiden Xenobiotika. Eine
statistisch signifikante Induktion konnte allerdings nur durch β-Naphthoflavon nach einer
Behandlungszeit von 24 Stunden nachgewiesen werden (p < 0.05).
Die Ergebnisse deuten darauf hin, dass die Genexpression von cyp-33E2 in den Muskelzellen
des Pharynx stattfindet und die von CYP-33E2 gebildeten Eicosanoide wohlmöglich
regulierend an der Pumpaktivität des Pharynx beteiligt sind. Ferner könnten diese Eicosanoide
durch die Modifikation von diversen Ionenkanälen an der Kontraktion der Muskelzellen
beteiligt sein und wohlmöglich auch an der Regulierung des Verhaltens bei der
Nahrungsaufnahme mitwirken.
Table of Contents
1
Abstract
2 Introduction
3
1
2.1 Caenorhabditis elegans
1
2.2 Cytochrome P450
2
2.3 PUFA and eicosanoids
3
2.4 DNA transformation of C. elegans by gene bombardment
5
2.5 Aim of the study
5
Methods and Material
3.1 Nematodes
7
7
3.1.1 Strain
7
3.1.2 Cultivation and Storage
7
3.2 Bacteria
7
3.3
8
Types of growth media
3.4 Vectors
9
3.5
9
Polymerase Chain Reaction (PCR)
3.5.1 Primer design for amplification of cyp-33E2 and cyp-29A3 promoter sequences
3.5.2 PCR
3.6
Molecular Cloning
10
11
3.6.1 Plasmid preparation (Mini-preparation)
11
3.6.2 Restriction with Endonucleases
12
3.6.3 DNA-Ligation
13
3.6.4 TOPO cloning reaction
13
3.7
Agarose gel electrophoresis
14
3.8
Rapid purification and concentration of DNA fragments and plasmids
15
3.9
Measurement of DNA concentration and quality
15
3.9.1 Determination of volume and concentration using Quantity One
15
3.9.2 Quantification and quality determination of DNA using a spectral
photometer
16
3.10 Transformation of worms by gold particle bombardment
4
16
3.10.1 Preparation
16
3.10.2 Ballistic particle bombardment – Transformation
17
3.10.3 Treatment of worms after transformation
18
3.10.4 Screening procedures
18
3.10.5 Single worm PCR
18
3.10.6 Expression Tests
20
3.10.7 Fluorescence Microscopy
20
3.10.8 Analysis of fluorescence intensity
20
Results
21
4.1 Amplification of cyp promoter sequence – PCR
21
4.2 Plasmid preparation – pGEM-T::cyppr
22
4.3 Restriction analysis from pGEM-T::cyppr
24
4.4 Preparative Restriction of plasmids for ligation of promoter DNA from
pGEM-T with pDW20.20
5
26
4.5 Plasmid preparation – pDW20.20::cyppr
26
4.6 Restriction analysis with pDW20.20::cyp33E2pr and pDW20.20::cyp29A3pr
27
4.7 Ballistic Transformation
28
4.7.1 Screening procedure by pBX plasmid
28
4.7.2 Expression of cyp-29A3 and cyp-33E2 promoter driven GFP
29
4.7.3 Single worm PCR for the pDW20.20::cyppr strains
32
4.7.4 Expression test with xenobiotics
33
Discussion
35
5.1 Ballistic transformation of C. elegans
35
5.2 CYP-29A3
36
5.3 Induction of gene expression by xenobiotics
37
5.4 Gene expression of cyp-33E2 in C. elegans
37
5.5 Perspective
40
Appendix
41
I
Acknowledgment
41
II
List of Abbreviation
42
III
C. elegans life cycle
42
IV
Vectors
43
V
Overview of promoter sequences
44
VI
Restriction analysis from pGEM-T::cyppr
45
VII
Data from internet tool (expression test)
47
VIII Chemicals
50
IX
Laboratory equipment
51
X
Software
51
XI
Reagents and stock solutions
52
XII
Media
54
XIII
References
55
XIV Affidavit
59
2 Introduction
2.1
Caenorhabditis elegans
In 1963 Sydney Brenner `felt very strongly that most of the classical problems of molecular
biology had been solved and that the future lay in tackling more complex biological
problems`[http://elegans.swmed.edu/Sydney.html]. He soon introduced the soil nematode
Caenorhabditis elegans (shown in Fig. 1) as a more complex animal that could be handled
with the same experimental convenience as a single-cell organism. This small transparent
animal grows to about 1 mm in length, reproduces under optimal conditions with a short life
cycle of about 3 days and has a life
span around 2 to 3 weeks. Further
on, these animals have the advantage
of being unproblematic concerning
propagation and maintenance, as
they can be cultivated in large
numbers and are fed by bacteria, such as Escherichia coli. The nematode exists in two sexes,
as male (XO) and as hermaphrodites (XX, Fig. 1). Wild type hermaphrodites, the most
common sex found in nature, consists of exactly 959 cells, all showing a constant position.
They can self-fertilize, which allows them to produce about 300 to 350 genetic identical
progeny. They generate male animals by spontaneous non-disjunction during meiosis in the
hermaphrodite germ line, with whom they can then mate. A further advantage is due to the
transparency of the animals that makes it possible to follow the development and life cycles of
nematodes [cf. app. Fig. I], beginning from the embryonic stage and followed by the four
larval stages and adulthood.
Ever since Sydney Brenner introduced C. elegans as a new and more complex model
organism, a sophisticated knowledge infrastructure has developed. Numerous research
methods, protocols and all of the results are published as central data (www.wormbase.org),
leading to a well-established model organism for example in developmental biology and
ecology. Moreover, C. elegans was the first multi-cellular animal with a completely
sequenced genome, which was entirely sequenced at the end of 1998 [1]. Since then it has
also become a helpful tool especially for microbiological studies. Notably is the similarity of
the C. elegans genome to that of humans (40% homologous), wherefore C. elegans has
become a popular instrument for the study of human diseases. Providing an example,
-1-
mitochondrial mutants have been used to study human mitochondrial-associated diseases,
ranging for example from obesity, neurodegeneration, cardiomyopathy, diabetes and also
aging [2, 3].
Besides, since the discovery of RNAi [4], C. elegans has been frequently used for screening
genes involved in fat regulation [5, 6], in particular because abnormalities in fat accumulation
can produce pathological states in vertebrates. For instance, obesity can increase the risk of
heart disease, hypertension and other diseases [7, 8]. Hence, the search to identify genes that
control the development, differentiation and function of fat storage has intensified. As data
has revealed, C. elegans contains homologies of genes encompassing a wide range of
components of the mammalian fat regulatory cascade, wherefore this animal seems to
function perfectly as model for fat regulation research. For example, fat storage pathways are
regulated in both mammals and worms by neuropeptide, serotonergic and insulin signalling
pathways [9-11].
Further on, C. elegans is more frequently used for research studies regarding the function of
cytochrome P450 (CYP). The C. elegans genome contains 80 cytochrome P450 genes, whose
individual functions are largely unknown. So far, C. elegans has been used in toxicological
studies for examining the effect of chemicals regarding CYP enzymes [12, 13, 14]. Latest
research studies have revealed that C. elegans possesses similar to mammals a microsomal
monooxygenase system constituted by specific CYP isoforms and a NADPH-CYP reductase
(CPR) component, which produces eicosanoids through converting eicosapentaenoic acid
(EPA) and arachidonic acid (AA) [15]. Hence, C. elegans may also serve as model organism
for elucidating further biological functions of the CYP enzymes and their metabolites, namely
the eicosanoids.
2.2
Cytochrome P450
Cytochrome P450 (CYP) is a superfamily of heme-thiolate enzymes, which are ubiquitous
common in all organisms. In prokaryotes P450s are to be found as water-soluble compounds
in the plasma, whereas in eukaryotes the cytochrome P450s are primarily located in the
endoplasmatic reticulum (ER) or in the inner membrane of mitochondria as membraneassociated proteins. These enzymes were first reported as pigments in liver cells in 1958 [16,
17] and received, due to the characteristic absorption maximum of their carbon monoxide
adduct at 450 nm, the term P450. P450 enzymes play a significant role in the metabolism of
drugs and xenobiotics (biotransformation) and are especially important during phase I of the
-2-
biotransformation. Phase I reactions generally modify lipophilic xenobiotics by adding a
functional group. P450s in general introduce a hydroxyl group or epoxy group for the
activation of these exogenous substances. The reactive group then enables Phase II enzymes
to subsequently increase the solubility of the modified xenobiotics through conjugative
reactions in water. CYP enzymes require oxygen, the cofactor NADPH and also the
cytochrome P450 reductase (CPR) for the reactions mentioned above. Together they form the
P450-containing monooxygenase system. Further on they play a key role during numerous
endogenous processes, as the cholesterol biosynthesis pathway, the synthesis of steroids and
vitamin D, and during the metabolism of unsaturated fatty acids.
2.3
PUFA and eicosanoids
Polyunsaturated fatty acids (PUFA) have a variety of biological functions in organisms [18].
They play a role as energy supplier, as important component of the cell membranes and they
also serve as precursor of biologically active signalling molecules. In mammals, these socalled eicosanoids can prevent hypertension and coronary heart diseases due to their
antithrombotical, anti-inflammatory and antiarrhythmical properties [19]. They are converted
from arachidonic acid (AA) and eicosapentaenoic acid (EPA) through hydroxylation and
epoxylation reactions by cyclooxygenases, lipooxygenases and also cytochrome P450s in
mammals. Up to now no obvious orthologs of mammalian cyclooxygenases and
lipoxygenases have been found within C. elegans. Yet, recent studies have identified a
microsomal monooxygenase system, consisting of specific CYP isoforms and a CPR
component, which converts AA and EPA through hydroxylation and epoxylation reactions to
CYP-dependent eicosanoids [15].
In general, AA and EPA are converted from the unsaturated fatty acids linoleic acid (n-6) and
alpha-linolenic acid (n-3). Whereby linoleic acid and alpha-linolenic acid are only accessible
to mammalian organisms through nutrition, C. elegans possesses the ability to synthesise n-6
and n-3 polyunsaturated fatty acids themselves, since they contain the required set of
desaturase (fat-1 till fat-4) and elongase (elo-1 and elo-2) genes for the biosynthesis of
arachidonic acid, eicosapentaenoic acid and related PUFAs [18, 20]. A lot of research work
has already been carried out in this area and the biosynthesis of PUFAs within C. elegans has
been as far as possible sophisticated [20]. Mutations in these genes lead to osmosensory,
mechanosensory and olfactory deficits [21] [22] assuming that they possess a vital role in
different regulatory processes, including various behavioural and developmental capacities of
C. elegans [23] [24].
-3-
However, little is known about the CYP-dependent metabolism of the PUFAs in this
nematode. A recent study by Kulas et al. [15] identified CYP-29A3 and CYP-33E2 as the
major CYP isoforms involved in EPA metabolism, the predominant polyunsaturated fatty acid
of this nematode. Interestingly these two CYP-enzymes show partially a high degree of
homologous amino acid sequences to members of the CYP enzymes, which catalyze the
metabolism of EPA and AA in mammals. These include CYP-4A and CYP-4F enzymes,
which catalyze the hydroxylation reactions, and CYP-2C and CYP-2J enzymes, which
catalyze the epoxidation reactions of n-6/-3 polyunsaturated fatty acids [25-29]. Providing an
example, in this study by Kulas et al. [15] CYP-29A3 and CYP-4V2 shared in an overlap of
507 amino acids 52% conserved positions and 34% sequence identity.
Further on, three main EPA metabolites in C. elegans were identified during this study, 17,18hydroxyeicosatetraensäure (17,18-DHETeTR), 19/20-hydroxyeicosapentraensäure (19/20OH-EPA) and 17,18-epoxyeicosatetraensäure (17,18-EETeTr) [15]. Yet, the biological
functions of the CYP-dependent eicosanoids within C. elegans are questionable in this animal
lacking a blood and cardiovascular-system, since CYP-dependent eicosanoids, as for
instances the AA-dependent eicosanoids epoxyeicosatrinoic acid (EETs), 19- and 20hydroxyeicosatetraenoic acid (20-HETE), regulate numerous of vascular, renal and cardiac
functions within mammals [30, 31]. In particular their regulation of different ion channels
plays an extraordinary role in these mechanisms [32, 33]. For example, CYP-dependent EETs
have been identified as activator of cardiac and vascular ATP-sensitive (KATP) channels in rats
[34].
However, little is known about the biological functions of CYP/EPA-dependent
eicosanoids, primarily because research studies have focused up to now on the predominant
PUFA within mammals, namely AA. Yet, studies have revealed that these metabolites also
seem to play a role in vasodilatation and cardiac functions and that an improvement of
vascular function and protection against cardiac arrhythmia may rely in part on a shift of
CYP-dependent eicosanoids from n-6 to n-3 PUFA derived metabolites. Providing an
example, Barbosa et al. [35] demonstrated that 17,18-epoxy-EPA is a highly potent activator
of calcium-dependent potassium (BK) channels and hence an efficient vasodilator, suggesting
also that the CYP2C-dependent generation of physiologically active eicosanoids from AA- to
EPA-derived metabolites may be shifted through n-3 PUFA-rich diets [36].
In conclusion, since C. elegans does not possess a blood and cardiovascular-system in
contrast to mammals, the CYP-derived eicosanoids may have other biological functions than
in mammals. Moreover, it might even be possible to elucidate evolutionary conserved
mechanisms of these signal molecules by employing C. elegans as research object.
-4-
2.4
DNA transformation of C. elegans by gene bombardment
One intention of this study was to transform C. elegans with the GFP fusion constructs.
DNA transformation is an essential tool for C. elegans research [37]. Up to now,
transformations were typically performed by microinjection into the hermaphrodite germ line,
a proven but also a laborious and technically difficult method of introducing DNA into the
worm [38]. An increasing popular alternative technique to microinjection is the new technique
of gene bombardment, which was also used in this study to produce transgenic worms.
Particle bombardment was originally invented for the engineering of plant species [39] and is
a well established method for genetic manipulations in this area. Hereby the DNA is bound to
gold particles which are then shot into worms by means of a helium beam using a biolistic
bombardment instrument or gene gun. Moreover, studies have demonstrated that
microparticle bombardment can induce integrative transformation in C. elegans, whereby
single- and low-copy chromosomal insertions are generated [40]. Besides, most of the
integrated transgenes do not fail to express due to germ line silencing, like conventional
extrachromosomal arrays produced by microinjection [37]. Hence, germ line expression can
remain stable and low-copy integrated lines can be used to express transgenes in the C.
elegans germ line [37, 40].
2.5
Aim of the study
The aim of this study was the localisation of cyp-33E2 and cyp-29A3 gene expression in C.
elegans and a first estimation concerning the biological roles of the CYP-33E2- and CYP29A2-derived eicosanoids, which are up to now unknown within these animals. Moreover, a
hint concerning potential evolutionary conserved mechanisms of these signal molecules was
hoped to be obtained by employing C. elegans as research object. So far, experiments have
revealed that PUFAs play essential roles in a variety of biological cascades and that most
behavioural capacities of C. elegans require intact pathways of PUFA biosynthesis [23]. C.
elegans mutants with deficiency in the PUFA synthesis show developmental and neurological
defects, as also behavioural and sensorial deficits [21, 23, 41]. For example, fat-3 mutants
lack Delta6 desaturase activity and therefore fail to produce C20 PUFAs. These mutants
showed in recent studies behavioural and developmental effects, ranging from slow growth,
an altered body shape, less spontaneous movement and a reduced progeny number, and could
be rescued by C20-PUFA feeding [23]. Further on, PUFA also play a role during the sperm
recruitment to the spermatheca, which is controlled by certain signals. PUFAs function in the
-5-
oocytes as precursor of these signals [42]. Other data indicate that PUFAs are essential for
efficient neurotransmission in C. elegans [22]. Considering these result, cyp-33E2 and cyp29A3 gene expression was suspected within muscle cells, neurons and/or intestine cells of C.
elegans.
In order to localise the gene expression of the two CYP isoforms, the promoter region of the
two cyp-genes cyp-29A3 and cyp-33E2, respectively, was fused with the GFP reporter, which
was included in the target vector pDW20.20. Thermosensitive pha-1(e2123ts) mutants from
the C. elegans wild type strain Bristol N2 were transformed with the plasmid/promoter
constructs and pBX, carrying the pha-1(e2123ts) wild type gene, via ballistic bombardment.
The rescue of transgenic animals due to the pBX plasmid was used as primarily screening
procedure. In addition, the localisation of the gene expression was detected by visualising
additional GFP gene expression as a green fluorescent light under a fluorescent microscope.
Further on, the influence of the two xenobiotics β-naphthoflavone and fenofibrate regarding
the gene expression of the two cyp genes were investigated.
-6-
3
Materials and Methods
3.1
Nematodes
3.1.1
Strain
In this study the mutant pha-1 from the C. elegans wild type strain Bristol N2 was used. This
mutant is named after the organ-specific mutation pha-1(e2123ts), which causes temperature
sensitive embryonic lethality. This embryonic lethal mutation causes pha-1(e2123ts) worms
to grow normally at 15°C. However, at 25°C the pharynx fails to undergo late morphogenesis
and differentiation, what results in a 100% embryonic lethality at this temperature. Therefore,
pha-1(e2123ts) was used as a selectable marker for the gene transfer, allowing selecting and
maintaining the transgenic strains easily [43, 44].
3.1.2
Cultivation and storage
The nematodes were maintained on NGM (Nematode Growth Medium) agar plates by using
standard procedures [45]. The NGM plates were inoculated with Escherichia coli strain OP50
as a food source and incubated, unless otherwise noted, at 15°C.
From the successfully transformed strains a master stock was stored in a freezer (-80°C). This
step was important to preserve the new transgenic strain. For this purpose, several plates of
worms were used, which had just exhausted the E. coli OP50 lawn and that contained lots of
L1 – L2 animals. These worms were placed into a freezing solution, which contained to the
same amount M9 buffer and freezing buffer. Because the worms survive a graduate cooling to
-80°C better, the freezer vials were placed in a styrofoam box with slots for holding vials at 80°C, which allowed a 1°C decrease in temperature per minute [46].
3.2
Bacteria
Bacteria were maintained and grown either on LB agar plates, in LB liquid medium or
conserved by frozen storage.
As previously mentioned, E. coli strain OP50 was used as food source. OP50 is an uracilrequiring mutant whose growth is limited on NGM plates, depending on the amount of uracil
in the medium [45]. The bacteria food source was prepared by adding 20 µl bacteria from
frozen conserves to 200 ml LB liquid medium and allowing the cultures to grow overnight in
-7-
a incubator at 37°C while shaking at 240 rpm (revolutions per minute). 200 µl bacteria
solution were then used for seeding the NGM plates. To enable the bacteria lawn to grow, the
plates were incubated for 8 hours at 37°C. The seeded NGM plates and the liquid culture were
stored at 4°C.
Chemical competent E. coli TOPO 10F [Invitrogen] was used for cloning and thus also for the
preparation of plasmids. The transformed strains were conserved by mixing grown culture
medium with 300 µl SOC medium and 200 µl glycerol in an Eppendorf tube. The tubes were
immediately stored in a -80°C freezer.
3.3
Types of selective growth media
Selective growth media is commonly used to inhibit the growth of certain microbes and to
provide general information regarding the bacteria that are able to grow on this specialised
agar. In this study media with Ampicillin were used for selecting E. coli cells that were
successfully transformed with the vector carrying the β-Lactamase gene as a marker.
Ampicillin was added to the media (50 µg/ ml LB) after autoclaving and cooling down to
approximately 55°C using sterile procedures.
Blue-white screening was used in some cases for the detection of successful vector and gene
ligations. This molecular mechanism is based on the disruption of the lacZ gene containing
the multiple cloning site (MCS). The LacZ gene encodes the α subunit of β-galactosidase
enzyme while the chromosome of the host strain encodes the remaining Ω subunit. Only when
the two fragments are associated they form a functional enzyme which can metabolize
galactose to lactose and glucose. If the MCS gets cleaved by restriction enzymes and the
foreign DNA is inserted within the LacZ gene, the production of functional β-galactosidase is
disrupted. In this study X-gal (5-bromo-4-chloro-3-indolyl-[beta]-D-galactopyranoside) was
used as substrate for this screening mechanism. β-galactosidase can cleave this colourless
modified galactose sugar into galactose and 5-bromo-4-chloro-3-hydroxyindole. 5-bromo-4chloro-3-hydroxyindole then is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo. This
insoluble product is bright blue and thus functions as an indicator for colonies containing
relegated, empty vectors. Colonies containing an insertion remain white. IPTG (Isoproyl-β-Dthiogalactopyranoside), which functions as an inducer of the lac operon, was used to induce
gene expression [47].
-8-
3.4
Vectors
Vectors are DNA molecules into which a foreign DNA fragment can be inserted. If the
resulting product is introduced into living cells, the vectors are capable of replicating the
insert.
Certain thermostable polymerases, such as the Hot Star Taq-Polymerase used in this study,
generate PCR products with a single deoxyadenosine by adding them to the 3`end. However,
when cutting vector pDW20.20 with restriction enzymes, ends with specific overhangs are
produced. Hence, prior to ligation compatible ends had to be constructed. Therefore the
pGEM®-T vector [cf. app. IV.I, Fig. II] was used to enable the cloning of the promoter
fragments into the pDW20.20 vector [cf. app. IV.II, Fig. III] including the GFP gene, by
generating the restriction sites from pGEM-T in order to insert the fragments exactly in front
of the GFP gene in pDW20.20. The pGEM-T system was suitable for this task, as the vector
was prepared by adding 3`terminal deoxythymidine to each end. These deoxythymidine ends
at the insertion site greatly improved the efficiency of ligation from the PCR products into the
plasmid. On the one hand by preventing recircularization of the vector and on the other hand
by providing a compatible overhang for the PCR products possessing deoxyadenosine
overhangs.
pDW20.20 served as target vector for the ballistic transformation. This vector included the
GFP gene, which was used as a reporter of cyp-29A3 and cyp-33E2 promoter driven gene
expression. Therefore the promoter DNA was ligated upstream of the GFP gene with
pDW20.20. Considering the ballistic transformation was successful, GFP was produced in
cells where the gene was expressed.
3.5
Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) is a technique widely used to exponentially amplify
target DNA sequences, by using single-stranded DNA as a template and DNA primers to
determine the target gene sequence. Heat-stable DNA polymerases are normally employed to
assemble a new DNA strand using nucleotides, whereby the primers serve as starting points.
In this study the method was used to perform the PCR reaction for the cyp-33E2 and cyp29A3 promoter regions and to proof the presence of the GFP gene and the promoters in
transgenic worms [single worm PCR, cf. 3.10.5].
-9-
3.5.1
Primer design for amplification of cyp-33E2 and cyp-29A3 promoter sequence
www.wormbase.org was used to determine the promoter sequences from the two genes cyp29A3 and cyp-33E2, which were supposed to be cloned and used for the ballistic
transformation. Promoter fragments with a size more than 1000 bp were chosen. They were
assembled selecting at least 1000 bp from the nucleotides located exactly in front of the gene
and the following 50 bp, which were positioned at the beginning of the coding gene. The
Clone Manager Professional Version 8 Software was used to design primers, which were
complementary to the ends of the promoter sequences. Two primers were designed for each
cytochrome P450 gene and used to amplify the promoter DNA during the PCR. Table 1
shows the primer pairs used for the amplification of the promoter sequence of cyp-29A3 and
cyp-33E2. For the whole promoter sequence please see appendix [cf. app. V].
Table 1. Primer pairs used for the amplification of the promoter sequences.
Primer description
cyp-33E2-7 (sense primer)
cyp-33E2-6 (antisense primer)
cyp-29A3-7 (sense primer)
cyp-29A3-6 (antisense primer)
3.5.2
Sequence
5’ GTGCAAATTGCGGGTTCTAC 3’
5’ TCTGAAAAGAGAAAATTAAAAAAAAATTTC 3’
5’ AACCAGGGCTGTGCGACATC 3’
5’ TTTGATGATCTAACTTTATCAAAC 3’
PCR
The PCRs for amplifying the promoter regions, were performed as described in the HotStar
HiFidelity PCR Handbook [Protocol: Amplification Using HotStar HiFidelity DNA
Polymerase; QIAGEN Sample and Assay Technologies, 2008] using the HotStar HiFidelity
Polymerase Kit and a thermal cycler [FPROGO50, Techne]. The annealing temperature
depends on the primer pair and is typically about 1°C below the melting temperature of the
primers. The melting temperature depends on the percentage of cytosine and thymine and the
primer length. Following mathematical formula was used to estimate the melting temperature:
(Formula 1)
Tm = 69.3 + 0.41 (GC%) – 650/n
n = amount of binding bp
GC% = percentage of guanine and
cytosine of the primer
The reaction mix was prepared according to Table 2. Since HotStar HiFidelity DNA
Polymerase is inactive at room temperature it was not necessary to keep the reaction vessel on
ice. The tubes were then placed in the thermal cycler and the PCR cycling program was
performed as shown in Table 3.
- 10 -
Table 2. PCR components (reaction mix and template DNA):
Reaction
mix
Component
Volume/reaction
5x HotStar HiFidelity PCR Buffer
(contains dNTPs)
Primer A
Primer B
HotStar HiFidelity DNA Polymerase
(2.5 units/ µl)
RNase-free water
Template DNA
4 µl
Template
DNA
Total volume
Final
concentration
1x
0.2 µl
0.2 µl
0.3 µl
1 µM
1 µM
10.3 µl
5 µl
0.1 ng – 50 ng
20 µl
-
Table 3. PCR cycling protocol.
Reaction step
Time
Initial activation step
Denaturation
Annealing
Extension
Final extension step
End of PCR cycling
5 min
15 sec
1 min
2 min
10 min
indefinite
3.6
Optimized
temperature
95°C
95°C
52°C
70°C
72°C
10°C
Number of cycles
1x
30 x
1x
Molecular Cloning
DNA cloning is an important cornerstone of molecular biology. It is the art of isolating a
defined DNA sequence and obtaining multiple ‘clonal’ copies in vivo. The procedure involves
restriction with endonucleases, ligating the DNA sequence with a vector that enables the
resulting construct to be introduced into a cell and plasmid preparation.
3.6.1
Plasmid preparation (Mini-preparation)
The mini preparation was performed according to Del Sal et al. [48]. The selected colonies
were inoculated in 2 ml LB medium and incubated at 37°C overnight. 1.5 ml bacterial culture
was transferred from the overnight cultures into 1.5 ml Eppendorf tubes and centrifuged for
ten minutes by room temperature and 4000 rpm. The medium was completely removed and
the bacterial pellet resuspended in 0.2 ml STET and 10 µl Lysozym-stock solution (lysis), and
incubated for 5 minutes at room temperature. To denaturize the proteins, the bacterial lysate
was heated for 90 seconds at a temperature of 95°C in an incubator and centrifuged at 14800
rpm for 10 minutes. The pellet was then entirely removed with a sterile toothpick. Afterwards
- 11 -
a chelating agent was used to remove disruptive polysaccharides and proteins from the
supernatant. For this reason 8 µl CTAB was added. This mixture was vortexed and
centrifuged for 5 minutes at room temperature (14800 rpm). After this step, the supernatant
was discarded and the pellet was resuspended in 300 µl 1.2 M NaCl. Then 750 µl of 100%
ethanol was added and the composite material was mixed over head. The mixture was
centrifuged for 10 minutes at 14800 rpm. Thereupon the supernatant was discarded and the
pellet was washed with 250 µl 70% ethanol and again centrifuged for one minute at 14800
rpm. The residual liquid was removed completely and the air-dried pellet was solved in 20 µl
TE-Puffer and 0.05 µl RNase, incubated for 5 minutes at room temperature and then
incubated for 20 minutes at 37°C. After this last step the solution was vortexed, shortly
centrifuged and stored at -20°C.
3.6.2
Restriction with Endonucleases
Restriction endonucleases are enzymes which cleave DNA at a specific nucleotide sequence
called recognition sequence, producing blunt ends or ends with overhangs. In this study these
enzymes were used to capture the promoters from cyp-29A3 and cyp-33E2 from the
recombinant DNA vector construct, to prepare DNA and plasmids for ligation (preparative
restriction) and to prove, if the correct recombinant DNA-vector constructs were present
(restriction analysis). The Clone Manager Professional Version 8 software was used to create
the plasmid/promoter construct, to display their restriction maps and to select specific
restriction endonucleases with suitable recognition sites. The fragments were virtually
generated and their sizes were noted for later comparison.
The restriction cutting mix was prepared according to Table 4, vortexed, shortly centrifuged
and then incubated for 90 minutes at 37°C. After the restriction the digests were “run out” on
an agarose gel (0.7% agarose) to determine the size of the fragments generated and then
compared with the fragment sizes generated with the help of the cloning manager. A 1 kb
ladder was used as DNA standard marker for the evaluation of the fragment sizes.
Table 4. Restriction cutting mix.
Components
Plasmid DNA
Concentration
[restriction analyse]
3 µl
Concentration
[preparative restriction]
10 µl
10x buffer
1.5 µl
5 µl
Enzymes
Each 0.2 µl
Each 2 µl
RNase free water to a final volume of
15 µl
50 µl
- 12 -
3.6.3
DNA-Ligation
T4 DNA Ligase was used to ligate the promoter DNA from cyp-33E2 and cyp-29A3 into the
vector DNA (pGEM-T and pDW20.20). This enzyme catalyzes the formation of a
phosphodiester bond between the 5’-phosphat and the 3’-hydroxyl groups of adjacent
nucleotides in either a cohesive-ended or blunt-ended configuration.
Ligations were performed using the protocol for pGEM-T® and pGEM-T® Easy Vector
Systems [Promega]. When cloning a fragment into a plasmid vector, it is recommended to use
a 3:1 molar ratio of insert DNA termini to vector DNA. The following equation was used for
the conversion of molar ration to mass ration and to calculate the appropriate amount of insert
to include in the ligation reaction:
(Formula 2)
ng of vector x kb size of insert x insert:vector molar ratio = ng of insert
kb size of vector
The reaction shown in Table 5 was assembled in a sterile microcentrifuge tube.
Table 5. Ligation reaction.
Components
2x Rapid Ligation Buffer, T4 DNA Ligase
Vector (50ng)
PCR product
T4 DNA Ligase (3 Weiss units/ µl)
Deionized water to a final volume of
Standard reaction
5 µl
1 µl
X µl
1 µl
10 µl
The reactions were mixed by pipetting and incubated at room temperature for one hour. The
reactions were then incubated overnight at 4°C to increase the number of transformants.
3.6.4
TOPO cloning reaction
This cloning reaction was performed using One Shot Chemically Competent E. coli
[Invitrogen]. 200 µl of the competent cell suspension were placed in a chilled microcentrifuge
tube and kept on ice for cooling. 2 µl of the ligation reaction were added to the competent E.
coli cells, cautiously mixed by flicking the tube with the finger and incubated on ice for 30
minutes. The cells were then heat shocked for 90 seconds at 42°C and immediately placed
back on ice. 250 µl SOC medium was added to the cells and the tubes were incubated for 1
hour at 37°C, shaking them at 200 rpm. The cells were then spun for five minutes at 3000
- 13 -
rpm. 350 µl of the supernatant was removed from the tube and the cells were resuspended by
pipetting them up and down in the remaining 100 µl media.
Using a sterile glass spreader, 8 µl of IPTG and 40 µl of X-Gal were spread on an LB plate
containing 50 µg/ml of Ampicillin. When dried, 100 µl of the cells were spread on the LB
plates and incubated overnight at 37°C. On the next day positive colonies, obvious through
growing good on the Ampicillin-plate and remaining white, were selected and overnight
cultures were prepared. Therefore 2 ml LB-medium were given into a test tube and with a
sterile tooth picker a colony from the overnight agar plate was selected and added to the test
tube. Several colonies were selected and incubated overnight at 37°C.
3.7
Agarose gel electrophoresis
The agarose gel electrophoresis is commonly used to isolate DNA-fragments and plasmids
according to their size. This method is based on the use of an electric field, which influences
the migration velocity of the molecules according to their size. The migration velocity of
linear molecules is reverse proportional to their molecular weight. Ethidium bromide, which
interacts with DNA and then fluoresces under UV-light, was used to visualize the DNAfragments. Due to the fact that the intensity of the fluorescence depends on the amount of the
DNA-fragments, one can roughly estimate the amount of DNA in a sample by comparing the
fluorescence with the fluorescence of marker bands. A gel documentation system [Geldoc
200, Bio-Rad], which was connected to a computer, was used to visualize the gel bands. The
software Quantity One 4.2.1. was used for imaging and analyzing the electrophoresis gels.
Basically the volumes tools were used for the quantification of data in the image [cf. 3.9]. A
photo was taken from every gel electrophoresis performed.
In this study the agarose gel electrophoresis was used to separate PCR products, to estimate
the size of plasmids and DNA molecules following restriction enzyme digestion and for the
separation of restricted plasmid DNA prior to the following cloning. The DNA sample plus 6x
loading dye was separated in a 0.7% or 1% agarose gel including 7.5 µl/ml ethidium bromide
and using 1x TE buffer. The electrophoresis was usually run for 30 min at 100 V.
When performing agarose gel electrophoresis with DNA-fragments, a 1-kb-ladder marker was
used as DNA-standard for estimating the sizes of the fragments. Plasmids with similar sizes
were used as standards and therefore for comparison, when running plasmids with an
electrophoresis.
- 14 -
3.8
Rapid purification and concentration of DNA fragments and plasmids
The illustraTM GFXTM PCR DNA and Gel Band Purification Kit [GE Healthcare, 28-903470] was used for the rapid purification and concentration of DNA fragments from TAE
agarose gel bands and restriction digestions. The isolations were performed using the Protocol
for purification of DNA from TAE and TBE agarose gels described in the product booklet.
The elution step was modified by repeating the step once using in each case 10 µl of elution
buffer.
Plasmid samples were purified according to the PureYield Midiprep System [Promega].
3.9
Measurement of DNA concentration and quality
It was important to know the DNA concentrations in order to calculate the amount of insert
and plasmid required for the ligation and the transformation.
3.9.1
Determination of volume and concentration using Quantity One
Prior to ligation, a gel electrophoresis was performed with a 1000 bp mass standard (100 ng/
µl) to estimate the concentration of the insert solutions and the fermented plasmid solutions.
For a successful estimation, three mixtures with different concentrations were prepared with a
mass standard for the gel electrophoresis. The pipetting scheme is shown in Table 6. The
Volume Tools of Quantity One were used to quantify and compare the intensity data from the
image of the gel electrophoresis. Here a volume is defined as the sum of the intensities of the
pixels within the volume boundary x pixel area. A volume object was designed by using the
volume rectangle to draw a box around the specific DNA band that should be quantified. The
volume objects were then classified as unknown and standards and compared using the
Volume Regression Curve features. A regression curve of all volumes was displayed, marking
the concentrations and the unknown volumes of the samples.
Table 6. Pipetting scheme for gel electrophoresis.
DNA sample
Employed volume
A. dest.
6x loading dye
cyp-33E2pr
3 µl
2 µl
1 µl
cyp-29A3pr
3 µl
2 µl
1 µl
50 ng (m. s.)
0.5 µl
4.5 µl
1 µl
100 ng (m. s.)
1 µl
4 µl
1 µl
200 ng (m. s.)
2 µl
3 µl
1 µl
- 15 -
3.9.2
Quantification and quality determination of DNA using a spectral photometer
Prior to the ballistic transformation, the concentration and the quality of the plasmid samples,
sustained through the mini preparations, had to be determined. For this reason the DNA was
quantified in a spectral photometer. DNA absorbs light maximal at 260 nm so that light of 260
nm passing 1 cm through DNA at 50 µg/ml concentration has an absorbance of 1.0. Hence,
the DNA concentration in the samples was determined using following equation:
(Formula 3) (DNA) = OD260 nm * 50 µg/ml * dilution factor [100/200]
The DNA purity was estimated by the A260/A280 ratio. The absorbance at 280 nm is
commonly used as indicator for protein contamination, since tyrosine residues absorb strongly
at this wavelength. If the solution contains pure DNA, the absorption at 260 nm is twice that
at 280 nm. A ratio between 1.8 and 2.0 generally represents a high-quality DNA sample.
3.10
Transformation of worms by gold particle bombardment
The ballistic transformation was performed following the protocol from Wilm et al. [49].
Alterations of the procedure improving the method were withdrawn from the “Protocol for
transformation of worms by gold bombardment” by Ralf Schnabel (5/2000) and realized in
the following way.
3.10.1 Preparation
Preparation of shooting plates
Per shot one 60 x 10 mm NGM plate was required. Altogether three shots were run for each
target gene, so six NGM shooting plates had to be prepared. The shooting plates were seeded
one day before the shot with 50 µl E. coli [OP50] with a diameter of 10 mm in the centre of
the plates and incubated overnight at 37°C. They were placed on ice before use.
Preparation of worms
This method required young adult worms with a few eggs inside. Two methods were used
during this study to obtain synchronous populations. Either a hypochlorite treatment was
carried out to obtain the eggs or filter with a pore diameter of 10 µm were used to select L1
worms. When most of the worms were adults and contained a few eggs inside, they were
washed off the plates with M9 buffer and pooled in 50 ml tubes. They were washed twice
with 50 ml M9 buffer and were then allowed to settle down by gravity. The settled worms
- 16 -
were mixed with half the volume of M9 buffer [e.g. 100 µl of worms + 50 µl of M9 buffer].
About 100 µl of the resuspended worms were placed onto the OP50 colony in the centre of
the NGM plates [cf. preparation of shooting plates] which were cooled on ice to stop the
worms from moving around. The worm plates were ready for the ballistic transformation
when the liquid had been taken up by the agar plates and the worm spot was dry.
Preparation of gold particles
1 mg of gold powder was weighed in a 1.5 ml Eppendorf tube and 100 µl of 50 mM
spermidin solution were added. Spermidin was used for loading the gold particles with DNA,
as spermidin binds and precipitates DNA. The mixture was vortexed and then set into an ultra
sonic bath for 30 seconds. 8 µg pBX (carrying the pha-1 wild type gene) and 8 µg of the
target plasmid DNA were added and incubated for 10 minutes at room temperature. During
this period the Eppendorf tube was flicked with the finger a few times. The volume was
adjusted with deionized water to 360 µl, vortexed and incubated for another 10 minutes at
room temperature while flicking the tube every now and then. After incubation, 100 µl CaCl2
were added drop wise to avoid clumping and left at room temperature for precipitation.
Afterwards the solution was spin down for 30 second at 13000 rpm and the supernatant was
removed except of 10 µl. The gold particles were carefully mixed with the remaining
supernatant and then washed three times with 1 ml absolute ethanol. Finally the suspension
was vigorously resuspended in 200 µl PVP-solution. In this study three different volumes of
the solution were used for the shots, aiming to improve the efficiency of the experiment. Per
gene three shots were carried out using 20, 25 and 30 µl of the solution, respectively, per shot.
For transformation the solution was loaded on to the steel grid of the filter holder used for the
ballistic bombardment.
3.10.2 Ballistic particle bombardment – Transformation
For every DNA sample one set of glands and filter holders were required. The filter holder
were disposed for a few hours in 70% ethanol and wrapped into tissues at least 15 minutes
before use in order to dry. They were built together shortly before the bombardment and the
bombardment device was calibrated by two shots at a filter paper placed at shooting distance.
Directly before the shot, the filter holder with the steel grid carrying the DNA-loaded gold
particles was placed in the chamber. The worm plates were placed in the centre of the target
area and the lid was removed just before closing the chamber.
- 17 -
Following parameters were used for the bombardment:
Helium pressure:
8 bar
Puls time:
10-30 ms
Vacuum:
< 0.5-0.6 bar
Distance plate-filter holder: 12 cm
3.10.3 Treatment of worms after transformation
After the shot, the agar of each plate was cut into six pieces and every piece was placed on a
fresh 90 mm enriched NGM plate using sterile procedures. The plates were incubated at 15°C.
3.10.4 Screening procedures
To facilitate the identification of transgenic animals, two types of screening were employed in
the experiment. GFP constructs were used to visualize and monitor gene expression of the
target genes, which was distinguished in a fluorescent microscope as a green fluorescent light
produced by the folded protein.
The second screening system was employed in addition. It involved selection of transformed
animals by rescue of the temperature-sensitive lethal mutation pha-1(e2123ts). For this reason
the gold particles were also loaded with the pBX plasmid, containing the pha-1(e2123ts) wild
type gene that rescues the temperature-sensitive lethality. For this screening method, the
worms were shifted to 25°C two days after the ballistic transformation and the rescue of pha1(e2123ts) was indicated by the appearance of next generation larvae on the plates. About 100
L1 larvae per target gene transformation were tested for stable transformation by separating
them from the other worms and cultivating them each on separate small NGM [35 x 10 mm]
plates at 25°C.
3.10.5 Single worm PCR
Single worm PCRs were carried out to proof the presence of the GFP gene and the cyp29A3pr and cyp-33E2pr promoter fragments, respectively, in an apparently stable transgenic
strain.
For every single worm PCR performed, one adult worm was picked from a plate and
transferred to a 2 µl drop of single worm lysis mix in a PCR tube. A microscope was used to
check, whether the worm was in the drop of lysis buffer. Then the tubes were frozen for 30
minutes at -80°C. Two drops of mineral oil were added to the tubes and the samples were
- 18 -
heated to 60°C for 1.5 hours followed by 95°C for 15 minutes. Thereafter the samples were
kept on ice and 23 µl PCR mix were added to the bottom phase of the PCR tube. Only the
bottom aqueous phase was pipetted twice up and down. In order to prove the presence of the
promoter DNA and the GFP gene in the transgenic strains, three samples were run in the PCR
for either CYP gene using specific primer pairs shown in Table 7. Negative single worm PCR
controls were performed with C. elegans pha-1(e2123ts) worms. The PCR cycling protocol is
shown in Table 8 and the expected PCR fragment sizes from the single worm PCRs are
shown in Table 9.
Table 7. Overview of the single worm PCRs which were performed with the strain pha-1(e2123ts)
(control) and the transgenic worms from the ballistic transformation with pDW20.20::cyp33E2pr and pDW20.20::cyp-29A3pr. Column two till three display the specific primers
and their sequences used for the single worm PCRs.
C. elegans strain
first sample
sample
pDW20.20::cyp33E2pr
GFPpr1/GFPpr2
pha-1(e2123ts)
pDW20.20::cyp29A3pr
5’CACTGGAGTTG
TCCCAATTC3’/
5’GTGTAATCCCA
GCAGCTGTT3’
Sense primer/antisense primer
second sample
cyp-33E2-7 (sense
primer)/GFPpr2
5’GTGCAAATTGCGGGTTCTA
C3’/
5’GTGTAATCCCAGCAGCTGT
T3’
[Proof of GFP gene]
[proof of cyp-33E2pr::GFP]
third
cyp-29A3-7 (sense
primer)/
GFPpr2
5’AACCAGGGCTGT
GCGACATC3’/
5’GTGTAATCCCAGC
AGCTGTT3’
[Proof of cyp29A3pr::GFP]
Table 8. Single worm PCR Cycling Protocol.
Reaction steps
Initial activation step
Denaturation
Annealing
Extension
Final extension step
End of PCR cycling
Time
3 min
45 sec
45 seconds
3 min
10 min
indefinite
Temperature
95°C
95°C
54 °C
72°C
72°C
10°C
Cycles
1
35 cycles
1 cycle
Table 9. Overview of the expected sizes from the single worm PRC products.
C. elegans strain
pDW20.20::cyp-33E2pr
pha-1(e2123ts)
pDW20.20::cyp-29A3pr
GFPpr1/GFPpr2
820 bp
-
820 bp
- 19 -
cyp-33E2pr::GFP
2030 bp
-
cyp-29A3pr::GFP
2915 bp
3.10.6 Expression Tests
These tests were performed to find out, whether the expression of the two cytochrome P450
genes was inducible through pollutants. In this study β-naphthoflavone and fenofibrate were
tested. For this experimental study 6 NGM-plates [90 x 10 mm] had to be prepared and
inoculated with OP50 and the two inductors. Therefore a sterile glass spreader was used to
spread fenofibrate (4 mg) and β-naphthoflavone (0.088 mg) each with 200 µl OP50 onto the
NGM-plates, which were then incubated overnight at 37°C. For each test, one well covered
NGM-plate was washed off and half of the worms were placed on a plate with βnaphthoflavone and the other half was placed on the plate with fenofibrate. A negative control
was performed without inductor. The worms were observed after 24 and 48 h using a
fluorescent microscope. For the analysis of the fluorescent intensity about 20 worms were
picked from every plate, placed on a spot agarose on an object slide and immobilized by
tetramizole.
3.10.7 Fluorescent Microscopy
A fluorescent microscopy [Nikon Eclipse E200], which was equipped with a digital camera
[Canon PowerShot A95], was used to visualize and image the transgenic worms. For this
purpose the transgenic and GFP stained C. elegans strains were either placed on a slide or the
worms were directly observed on their NGM-plates.
3.10.8 Analysis of fluorescence intensity
As mentioned above, 20 worms per inductor plate were picked for the quantification of the
GFP staining. Pictures were taken with the digital camera and the GFP staining was quantified
by an image internet tool (http://imagetool.lsmod.de/farbquant.cgi). Before, Adobe Photoshop
6.0 was used to adjust the size of the images, moreover, the fluorescing part of the worm was
selected and the background was set in blue, as these pixels are ignored by this image tool.
PNG files were required and the images had to be scaled below 1000x1000 pixels. The
internet tool analyzes the images for content of brightness, whereby black gives 0 and pure
white 1. Further on, the median and the average of brightness of all pixels from the image are
provided by this tool. The statistical software program SigmaStat 3.5 [SPSS] was used to test
the statistically significant difference between the different inductor treatments and the control
group by performing a one-way analysis of variance [ANOVA] with the medians of the
images if possible. Otherwise a Kruskal-Wallis One Way Analysis of Variance on Ranks was
performed.
- 20 -
4
Results
4.1
Amplification of cyp promoter sequence – PCR
The promoter sequences were used for a promoter driven gene expression in C. elegans. For
this reason, PCRs were performed at the beginning of the study, due to the amplification of
the promoter sequences of the two genes cyp-33E2 and cyp-29A3.
Two samples of genomic C. elegans DNA with different dilutions were available at the
laboratory. These genomic DNA samples were used as template DNA and tested for
efficiency: genomic DNA sample 3 in the concentrations 1:10 and 1:100; genomic DNA
sample 2 in the concentration 1:100. The test set-up and the expected PCR product lengths are
shown in Table 10. After the PCR a gel electrophoresis was run with the PCR samples. Figure
2 shows the image of the gel electrophoresis.
Table 10. PCR samples performed using different genomic DNA as template DNA and expected
size of the promoter sequences.
Cytochrome
P450 gene
1 sample
2 sample
3 sample
cyp-33E2
Genomic DNA 2
(1:100 dilution)
Genomic DNA 2
(1:100 dilution)
Genomic DNA 3
(1:100 dilution)
Genomic DNA 3
(1:100 dilution)
Genomic DNA 3
(1:10 dilution)
Genomic DNA 3
(1:10 dilution)
cyp-29A3
Expected size
of PCR
product
1030 bp
1948 bp
Figure 2. Image of the gel electrophoresis with the PCR samples; slots 1 and 4 with genomic DNA 2
(sample 1); slots 2 and 5 with genomic DNA 3 (1:100; sample 2); slots 3 and 6 with
genomic DNA 3 (1:10; sample3); M: 1 kb ladder.
- 21 -
The PCR reactions which were performed using the sample one (genomic DNA 2) and
sample two (genomic DNA 3, dilution 1:100) as template, resulted in a successful
amplification. In both cases DNA fragments with the correct size were amplified and
observed as gel bands. However, according to the image of the gel electrophoresis, sample
three (genomic DNA three, dilution 1:10) did not result in an amplification of the promoter
sequence. Hence, sample three was too concentrated for a successful PCR and more diluted
samples seem to work better as template DNA.
Prior to ligation (cf. 3.9.1 and 3.6.3), the DNA products were purified from the agarose gel as
described in the product booklet of the illustra GFX PCR DNA and Gel Band Purification Kit.
Due to cloning, the ligation samples were then used for the transformation of chemical
competent E. coli and the bacteria was cultured overnight.
4.2
Plasmid preparation – pGEM-T::cyppr
Plasmid preparations were performed to gain the pGEM-T::cyppr plasmids from the overnight
cultures. This step was necessary for the restriction of the promoter sequences from pGEM-T
and their further ligation in pDW20.20.
To screen for positive colonies, nine colonies were picked from both pGEM-T::cyppr
transformations and subsequent to their separate cultivation, mini preparations were
performed with all colonies (cf. 3.6.1). A gel electrophoresis was performed with the mini
preparations using pGEM 35A4 and pGEM K10 as control plasmids to estimate the plasmid
sizes. Table 11 represents the expected plasmid sizes and Figure 3 shows the image of the gel
electrophoresis run with the mini preparation solutions.
Table 11. List of plasmids and their expected bp sizes.
Plasmid
Size
pGEM 35A4 (control)
4532 bp
pGEM K10 (control)
5042 bp
pGEM-T::cyp33E2pr
4059 bp
pGEM-T::cyp29A3pr
4947 bp
- 22 -
Figure 3.
Gel electrophoresis with plasmids from mini preparation; slots 1-9 represent plasmids
from colonies from transformation reaction with pGEM-T::cyp-33E2pr; slots 13-21
represent plasmids from colonies from transformation reaction with pGEM-T::cyp29A3pr; slots 11, 22: control plasmid pGEM 35A4; slots 12, 24: control plasmid pGEM
K10.
Figure 3 indicated that the transformation with pGEM-T::cyp-33E2pr was successful. Positive
colonies originating from the transformation reaction with pGEM-T::cyp-33E2pr were found
(slots 1-5, 8). The comparison of their plasmid sizes (1059 bp insert + 3000 bp pGEM-T) with
the size of the control plasmids (4532 bp and 5042 bp) indicated that the required plasmid was
present. However, the colonies shown in slots 6, 7, and 9 did not possess the desired plasmid.
The colony from slot 8 (pGEM-T::cyp-33E2pr) was selected for the further accumulation of
the plasmid, which was required for the ligation of the promoter with pDW20.20.
Figure 3 also indicates that the transformation with pGEM-T::cyp-29A3pr was not successful.
No colony possessed the required plasmid (4947 bp). Hence, all steps were repeated with this
cyp-gene from the very first. Figure 4 shows the image of the gel electrophoresis performed
with the mini preparations after the second transformation attempt. The comparison of the
plasmid size of pGEM-T::cyp-29A3pr (4947 bp) with the control plasmids indicated that the
correct plasmid was present in all colonies.
- 23 -
Gel electrophoresis with mini preparations (slots 1-4, 9-14; pGEM-T::cyp-29A3pr) from
different colonies after second transformation attempt.
Figure 4.
4.3
Restriction analysis from pGEM-T::cyppr
Due to the deoxyadenosine 3’ ends of the promoter sequences and the desoxythymidine 3’
ends from pGEM-T, the promoter sequences were able to ligate in two different orientations
with pGEM-T [cf. app. VI, Fig. IV – VII]. To determine the insert orientation within the
pGEM-T vector, a restriction analysis was performed. Restriction enzymes with either one,
two or three restriction sites in the plasmid were chosen. The expected fragment lengths for
both possible orientations were predicted using the clone manager suite and are displayed
with the employed enzymes in Table 12 (pGEM-T::cyp-33E2pr) and 13 (pGEM-T::cyp29A3pr). Orientation b was required for a correct ligation of cyp-33E2pr into the GFP
carrying vector pDW20.20 and orientation a for a correct ligation of cyp-29A3pr in
pDW20.20.
Table 12. Enzymes used for restriction analyse of pGEM-T::cyp-33E2pr and the expected fragment
sizes due to their orientation in the plasmid after restriction with the specific enzymes.
Enzyme
NdeI
PvuII
PstI + HindIII
Table 13.
Orientation
a
972, 3135 bp
438, 1060, 2564 bp
313, 3749 bp
b
199, 3863 bp
324, 1174, 2564 bp
797, 3265 bp
Enzymes used for restriction analyse of pGEM-T::cyp-29A3pr and the expected fragment
sizes considering the insert orientation (columns two and three).
Enzyme
AccI
PvuII + BamHI
PstI + HindIII
Orientation
a
122, 4828 bp
423, 1963, 2564 bp
1710, 3240 bp
- 24 -
b
1880, 3070 bp
541, 1845, 2564 bp
288, 4662 bp
The comparison of the expected fragment sizes from the restriction analysis of pGEM-T::cyp33E2pr with the image of the gel electrophoresis performed after the restriction analysis
(Figure 5) showed that the plasmid with the correct insert orientation (b) was present in
colony 8.
Figure 5. Restriction analyse of colony 8 from pGEM-T::cyp-33E2pr
transformation.
The colonies 1, 2 and 8 from the transformation with pGEM-T::cyp-29A3pr were selected for
restriction analyses (Fig. 6). The image of the gel electrophoresis from the restriction analysis
showed that the colonies 1 and 8 featured the correct insert orientation a. The bacterial culture
1 was used for the further enrichment of the pGEM-T::cyp-29A3pr plasmid, required for the
following experimental work.
Figure 6.
Restriction analyse with pGEM-T::cyp-29A3pr; slots 1-3: colony 1; slots 5-7:
colony 2 and slots 8-10 colony 8. PstI + HindIII: slots 1, 5, 8; PvuII + BamHI:
slots 2, 6, 9; AccI: slots 3, 7, 10.
- 25 -
4.4
Preparative Restriction of plasmids for ligation of promoter DNA from pGEM- T
with pDW20.20
A preparative restriction of both plasmids (pDW20.20, pGEM-T::cyppr) was necessary to
obtain the inserts ligated with pGEM-T and to enable the ligation of the free insert with
pDW20.20. The restriction reaction was performed as described in 3.6.2 using the enzymes
SphI, XbaI and PstI for the restriction of pDW20.20 and the enzymes SphI and SpeI for the
restriction of the pGEM-T::cyppr plasmids.
Subsequent to the gel electrophoresis, a sterile scalpel and long wavelength UV were used to
cut the desired DNA bands with the restricted pDW20.20 plasmid and the insert DNA, and to
transfer the slice to a pre-weighed tube. Prior to ligation of the insert with pDW20.20, the
DNA was purified from the TAE agarose gel. As before, the ligation reactions were used for
the transformation of the One shot competent E. coli and carried out as described in 3.6.4.
4.5
Plasmid preparation – pDW20.20::cyppr
Plasmid preparations were performed with the overnight cultures to obtain pDW20.20::cyp33E2pr and pDW20.20::cyp-29A3pr for the ballistic transformation of C. elegans. From both
cyp-genes positive colonies were observed on the LB-plates and per gene ten colonies were
picked in order to cultivate liquid bacteria cultures. These were then used for the mini
preparations.
Figures 7 (pDW20.20::cyp-33E2pr) and 8 (pDW20.20::cyp-29A3pr) show the images of the
gel electrophoresis. Observing the image of the gel electrophoresis performed with the mini
preparations of the pDW20.20::cyp-29A3pr colonies, one can recognise, that colony 8 didn’t
possess any plasmid. This was predictable, as no pellet was observed during the mini
preparation. Apart from this case, the images of the gel electrophoresis indicated that all other
colonies possessed a plasmid with the correct size (pDW20.20::cyp-33E2pr: 5369 bp;
pDW20.20::cyp-29A3pr: 6257 bp).
Figure 7.
Gel electrophoresis with mini preparations from transformed cells with pDW20.20::cyp33E2pr; C1: control plasmid, 4918 bp; C2: control plasmid, 5748 bp; 1-10 mini
preparations.
- 26 -
Figure 8.
Gel electrophoresis with mini preparations from transformed cells with pDW20.20::cyp29A3pr; C1: control plasmid, 4532 bp; C2: control plasmid, 5042 bp.
The colonies 4, 6 and 9 from the pDW20.20::cyp-33E2pr transformation and the colony 3
from the pDW20.20::cyp-29A3pr transformation were chosen for the further accumulation of
the plasmids, which was necessary for the ballistic transformation.
4.6
Restriction analysis with pDW20.20::cyp33E2pr and pDW20.20::cyp29A3pr
Prior to the ballistic transformation, a restriction analysis was performed with the colonies to
control the plasmid/promoter construct. The employed enzymes and the expected fragment
lengths after the restriction are presented in Table 14. Figure 9 shows the image of the gel
electrophoresis of the restriction analyse reaction with pDW20.20::cyp33E2pr. Figure 10
shows the image of the restriction analyse with pDW20.20::cyp-29A3pr.
Table 14.
Enzymes used for restriction analysis and fragment lengths after digest.
Enzyme Fragment lengths for Fragment lengths for
pDW20.20::cyp-33E2pr
pDW20.20::cyp-29A3pr
HindIII 322, 5047 bp
153, 1147,4957 bp
PvuII
377, 1224, 3768 bp
3116, 3141 bp
Figure 9. Gel electrophoresis of restriction analysis reaction with pDW20.20::cyp-33E2pr colonies.
- 27 -
Considering Fig. 9 (pDW20.20::cyp-33E2pr) all three selected plasmids were cut into the
desired fragments. Thus, the correct plasmid/promoter construct was present. The same result
was obtained for colony 3 from the pDW20.20::cyp-29A3pr transformation (Fig. 10). The
image of the gel electrophoresis performed with the restriction analysis reaction of
pDW20.20::cyp-29A3pr showed also the necessary fragments and therefore the required
plasmid.
Fig. 10. Gel electrophoresis of restriction analysis reaction
with pDW20.20::cyp-29A3pr colony 3.
4.7
Ballistic Transformation
C. elegans was transformed with the plasmids pDW20.20::cyp-29A3pr or pDW20.20::cyp33E2pr and pBX via ballistic bombardment. Three different volumes (20, 25 and 30 µl) of the
solution were used per shot, in order to examine the effect of the solution volume concerning
the transformation efficiency.
About half of the worms died due to the ballistic bombardment. Yet, there was no difference
recognisable between the three different shots using 20, 25 and 30 µl of the reaction mix,
regarding the mortality rate.
4.7.1
Screening procedure by pBX plasmid
The pha-1(e2123) mutation is a temperature sensitive embryonic lethal mutation that causes
embryonic lethality at 25°C. Hence, for the selection of transgenic worms the transformed
worms were shifted after about 3 days from the 15°C fridge to 25°C.
From every transformation reaction performed worms were observed, which were producing
descendants at 25°C. Nevertheless, there was a noticeable difference concerning the number
of worms producing descendants in relation to the shots with the different amount of solution.
More adult worms were sight on the plates from the shots with 20 µl and 25 µl solution,
which were producing descendants. Besides, the number of larvae produced from the isolated
- 28 -
worms varied. Very obvious was the difference in the number of larvae produced by the
worms from the shot with 30 µl, which was very small, to the other two shots. Although the
larvae number from the worms of the shots with 20 and 25 µl was also often very small, a
number of worms were found producing a typical amount of larvae. The descendant number
allowed a first evaluation considering the stable presence of transgenic worms. Worms with
many descendants (mainly from the two shots with 20 and 25 µl) were maintained by
cultivating young larvae on new NGM plates.
4.7.2
Expression of cyp-29A3 and cyp-33E2 promoter driven GFP
To localize cyp-29A3 and cyp-33E2 gene expression, the GFP fluorescence from transgenic
worms was observed under a fluorescence microscope.
cyp-33E2pr::GFP
The promoter driven expression of GFP was observed in the pharynx and the anterior part of
the intestine (Fig. 11) in worms originating from the transformation approaches with 20 and
25 µl of the solution. Fluorescence was observed in adult worms as well as in larvae and
varied between the worms. Some worms showed a more intense GFP fluorescence than other
worms and mosaic patterns of GFP expression were observed. In addition, GFP fluorescence
was especially intense in worms originating from the ballistic transformation shot with 20 µl
of the reaction mix.
Figure 11. Localisation of cyp-33E2pr::GFP. GPF fluorescent was detected in cells of the pharynx
and the intestine of adult worms and larvae by fluorescent microscopy. These images
show an adult worm. On the right the Normarski image, on the left the
corresponding GFP fluorescence image.
For the identification of the specific cells which could have shown GFP fluorescence, photos
and images from the pharynx and the intestine were selected from different websites, such as
from the database wormatlas. They were then used for the comparison with the photos taken
from transgenic animals with the cyp-33E2 promoter driven GFP expression. GFP
fluorescence most probably occurred in the muscle cells pm3 (procorpus), pm4 (metacarpus),
mp5 (isthmus), pm6, pm7 (posterior bulb) (Fig. 12 and Fig. 13) and the first ring of the
- 29 -
intestine (violet arrow in Fig. 12). Fluorescence could have also originated from muscle cells
pm1 and pm2 (Fig. 14 and Fig. 15). Please not that Figures 12, 15, 16 and 18 show images of
fluorescing worms taken during this study, whereby Figures 13, 14, 21, 23 – 25 were obtained
from different sources for the illustration of the results.
Figure 12. Image of pharynx; strain
marker: cyp-33E2pr::GFP
fluorescence in pm3 – pm7 and
anterior intestine.
Figure 13. Image of pharynx (corpus + isthmus)
Image source: R. Newbury.
Figure 14. Image of procorpus neuron pm1;
Figure 15. Image of GFP fluorescence in procorpus;
strain marker: F20.B10.1-GFP;
cyp-33E2pr::GFP; marked with green
strain source: Hacklei-Topper&Peles.
arrows: pm1; orange arrow: pm2.
In addition, GFP fluorescence could have as well derived from marginal cells. The fluorescing
parts in Fig. 16 can represent the three pm3 muscle cells or the three marginal cells from the
procorpus. Fig. 17 shows a cross section of the procorpus and how the marginal cells run
through the pharynx. When comparing the run of the marginal cells (mc) through the pharynx
with Fig. 18, it is possible that marginal cells were fluorescing. Yet, Fig. 18 suggests that
pm3, pm4, pm6 - pm8 were also fluorescing.
- 30 -
Figure 16. GFP fluorescence in pm3 or
marginal cells of procorpus.
Figure 17. Cross section of procorpus
(www.wormatlas.org).
Supplementary, it is also possible that neuronal cells were fluorescing. The fluorescing string
marked by the yellow arrows in Fig. 18, could also represent the motor neuron M1. Its cell
body is located in the terminal bulb and the single process runs anterior (Figure 19). The two
fluorescing strings underneath the marked string in the isthmus (Fig. 18; in many cases three
fluorescing strings were observed in the isthmus), could represent interneuron I4 (Fig. 20,
left) or motor neuron M2 (Fig. 20, right). Apart from this, the three strings could also embody
the three muscle cells from pm5.
Figure 18. GFP fluorescence in pharynx
Figure 19. Drawing of motor neuron M1
and intestine; fluorescing string,
(www.wormatlas.org).
pointed out by yellow arrows, could
represent the fluorescing neuron
M1 or mc.
Figure 20. Interneuron I4 (left) and motor neuron M2 (right); (www.wormatlas.org).
- 31 -
cyp-29A3pr::GFP
Several worms, especially from transformation approaches with shots using 20 and 25 µl of
the solution, were able to produce progeny. Yet, GFP expression was not observed very often
in worms from the pDW20.20::cyp-29A3pr transformation. When it was observed, the
fluorescence was very weak and only located at the beginning of the intestine (Fig. 21).
Figure 21. Localisation of cyp-29A3::GFP. The proteins were detected in cells of the intestine of
adult worms by fluorescent microscopy. The image shows an adult worm. On the right the
Normarski image, on the left the corresponding GFP fluorescence image.
Besides, no abnormality in the growth of both transgenic strains was observed.
4.7.3
Single worm PCR for the pDW20.20::cyppr strains
Single worm PCRs were carried out to proof the presence of the GFP gene and the cyp29A3pr and cyp-33E2pr fragments, respectively, in an apparently stable transgenic strain. Fig.
22 shows the image of the gel electrophoresis performed with the single worm PCRs. Worms
from the pha-1(e2123ts) strains were used for a negative control.
Figure 22. Image of the gel electrophoresis with the single worm PCRs. Slots 3 – 8: pDW20.20::cyp33E2pr; slots 10 – 15: pDW20.20::cyp-29A3pr; slots 1,2 and 9: pha-1(e2123ts); slots 2, 5,
8, 10 and 13 display PCR samples for the evidence of the GFP gene; slots 1, 4, 7, 11 and
14 display PCR samples for the evidence of cyp-33E2pr::GFP; slots 3, 6, 9, 12 and 15
display PCR samples for the evidence of cyp-29A3pr::GFP.
- 32 -
Comparison of the expected PCR fragment sizes and the image of the gel electrophoresis
displayed that the correct plasmid was present in the transgenic strain pDW20.20::cyp33E2pr. Deductive, the ballistic transformation was successful. In addition, the image of the
gel electrophoresis from the single worm PCR with the transgenic strain pDW20.20::cyp29A3pr also displayed the correct GFP PCR fragment. However, an incorrect fragment of
cyp-29A3pr::GFP was found in the slots (Fig. 3.11, slots 12 and 15). The PCR fragment only
had a size of about 600 bp, whereas the expected PCR fragment should have had a size of
2915 bp.
To confirm that the incorrect plasmid in the transgenic worms was not due to a mistake in the
pDW20.20::cyp-29A3pr sample, which was used for the ballistic transformation, a control
PCR was run with the pDW20.20::cyp-29A3pr sample
(Fig. 23). Altogether three PCRs were run, with the target
to proof the presence of the GFP gene, the cyp-29A3pr
sequence and the cyp-29A3pr::GFP sequence in the
vector through their amplification. The image of the gel
electrophoresis showed the correct PCR fragments (Fig.
23). The plasmid was intact prior to the ballistic
transformation.
4.7.4
Expression test with xenobiotics
The genome of C. elegans contains 80 cyp genes. Yet, little is known about the biological role
of these enzymes. There have been many investigations regarding the P450 induction
pathways in mammals, revealing that these enzymes are involved as important components in
the biotransformation of drugs and other xenobiotics in mammals as well as in various
endogenous processes. Concerning C. elegans, gene expression studies have demonstrated
that several cyp genes are strongly induced by xenobiotics [13]. Further studies revealed that
especially CYP35 is strongly increased with a concentration-dependent induction [14].
During this study, the influence of the inductors fenofibrate and β-naphthoflavone concerning
cyp-33E2 and cyp-29A3 gene expression was evaluated after an incubation time of 24 and 48
h. Table 15 shows the mean and standard deviation from the medians (median of brightness
originating from all pixels of an image), which were provided by the internet image tool
(http://imagetool.lsmod.de/farbquant.cgi) [cf. app. VII for all results].
- 33 -
Table 15. Results from the quantification of the GFP straining via the internet image tool. Columns
two and three present the means and the standard deviation from the medians of
brightness; n: animals considered in calculation. * p < 0.05 (Kruskal-Wallis One Way
Analysis of Variance on Ranks).
Test series
Control without
inductor
β-naphthoflavone
Mean and standard
deviation of brightness
after 24 h
0.177 +/- 0.03
(n=20)
0.211 +/- 0.044
(n=20) *
Mean and standard
deviation of
brightness after 48 h
0.19 +/- 0.042
(n=19)
0.183 +/- 0.042
(n=20)
0.202 +/- 0.057
(n=20)
0.218 +/- 0.051
(n=20)
Fenofibrate
Regarding the data from the control group permitted by the image tool, a discordant value was
obtained in the group after 48 h. This numeric value was not included in the calculations of
the median and the statistic evaluation.
The cyp-33E2 promoter driven GFP production was slightly induced by fenofibrate but was
not proven as statistically significant in relation to the control group. A statistically significant
difference of GFP straining was proven in animals on β-naphthoflavone plates after 24 h.
However, after the 48 h treatment with β-naphthoflavone there was no obvious difference in
relation to the control group concerning the induction of cyp-33E2 promoter driven GFP
expression.
- 34 -
5
Discussion
The present study was able to localize cyp-33E2 promoter driven GFP expression in the
pharynx and in the anterior part of the intestine. The results suggested cyp-33E2 gene
expression in the muscle, marginal or neuronal cells of the pharynx and in ring 1 of the
intestine. The cyp promoter driven GFP production was slightly induced by the two
xenobiotics, yet a statistically significant induction of cyp-33E2 gene expression in
comparison to the control group was only proven for β-naphthoflavone after the 24 hour
treatment (p < 0.05). However, the transformation of C. elegans with pDW20.20::cyp-29A3pr
failed and the localization of the cyp-29A3 gene expression was not possible.
5.1
Ballistic transformation of C. elegans
In each experimental transformation approach performed, about half of the worms died due to
the ballistic bombardment. Yet, no difference was obvious concerning the mortality rate and
the different solution amount applied for each shot. At first, the ballistic transformations with
each plasmid/promoter construct and pBX seemed to be successful. After the worms were
shifted from 15°C to 25°C, worms producing progeny were sight on the NGM worm plates
from every transformation approach. The screening procedure by GFP fluorescence also
indicated that the transformation of C. elegans with pDW20.20::cyp-33E2pr had been
successful, since GFP staining was viewed in many animals. In contrast, only a few animals
from the transformation with pDW20.20::cyp-29A3pr were viewed showing mostly very
weak GFP staining. Single worm PCRs were carried out to prove, whether the correct
plasmid/promoter constructs were present. This revealed that the worms from the
transformation with pDW20.20::cyp-33E2pr possessed the correct plasmid/promoter
construct. However, the single worm PCRs from the transformation approach with
pDW20.20::cyp-29A3pr disclosed that only a small piece of the promoter sequence was
ligated with the plasmid. The possibility that the plasmid could have been incomplete before it
was shot into C. elegans was dismissed after a further PCR with the plasmid solution that had
been used for the ballistic bombardment. A substantial part of the promoter DNA must have
gone lost either during the procedure of the ballistic bombardment or in C. elegans.
Considering the different amount of solution used for the ballistic bombardment, the
transformation result was more sufficient with shots using 20 and 25 µl of the solution.
Moreover, transgenic pDW20.20::cyp-33E2pr C. elegans originating from the shot with 20 µl
fluoresced more intense than the worms from the shot with 25 µl. Thus it seems that the
- 35 -
transformation is more efficient if less amount of the solution is used for the ballistic
bombardment. Since already 5 µl seem to have an influence on the transformation rate, it
would be useful to implement further investigations (using e.g. 18, 19, 20, 21 and 22 µl of the
solution per shot) for a precise adjustment of the solution amount and hence an improvement
of the transformation efficiency. Further on, the GFP expression varied between worms from
the same transformation approach and some worms showed mosaic patterns of GFP
expression. During the transformation shot, the gonads of the hermaphrodites are individually
stricken by a certain amount of gold particles coated with the DNA, leading not only to
unique low-copy-number integrated lines but also extrachrosomal array lines. It is well known
that the expression pattern in extrachromomal lines can vary from animal to animal due to
mosaic loss of the array [50] and that extrachromosomal lines with the same transgenic DNA
can vary in their level of gene expression relative to gene copy number [51]. Hence, this could
be the reason for the mosaic GFP expression in this study. However, studies have shown that
low-copy integrated lines also undergo gene silencing due to a slightly higher number of
copies of the transgene [52]. This suggests that the germ line has a very low threshold for
multicopy sequences and that it is able to discriminate relatively small differences in
transgene copy number [52]. This may be displayed in the decreased level of GFP expression.
5.2
CYP-29A3
As previously mentioned, the ballistic transformation with pDW20.20::cyp29A3pr was not
successful, since a substantial part of the promoter sequence was missing after the
transformation approach. The reason for the missing sequences of cyp-29A3pr in the plasmid
pDW20.20 after the ballistic transformation is unclear. Due to instability in the DNA
sequence of the promoter, the plasmid may have opened up during the shot via helium
pressure. Thereby a piece of the promoter sequence might have gone lost. In addition, recent
work contributing to the characterisation of the two cyp genes is carried out at the MDC in
Berlin and from the Group of Freshwater and Stress Ecology (Department of Biology).
Among other things, this study has the aim of inducing heterologous overexpression of
identified cyp genes, as for example cyp-29A3, in insect cells and to measure their enzyme
activity. Interestingly the transfer of cyp-29A3 was also unsuccessful. These accumulating
problems with this gene in research studies could be caused by instability in the DNA
sequence of the gene. Yet, except a high percentage of adenine and thymine bases in the
sequence which also influences DNA stability, no obvious unusual sequence arrangements as
for example triplet repeats which cause hairpin structures are visible.
- 36 -
5.3
Induction of gene expression by xenobiotics
cyp-33E2 was slightly inducible by fenofibrate, yet it was not possible to prove a statistically
significant difference. An induction of cyp-33E2 was expected, since several cyp genes are
strongly induced by xenobiotics [13]. Moreover, other studies have demonstrated that cyp33E2 is inducible through fenofibrate [15]. This study was not able to prove this.
However, β-naphthoflavone had an inducing effect on the gene expression of cyp-33E2 after
the incubation for 24 h. An inducible effect of this xenobiotic was also expected after 48 h,
since Menzel et al. [13, 14] found out that the gene expression of close related cyp-genes (e.g.
cyp-33E1) was significantly induced by β-naphthoflavone. Yet, the mean of the medians after
the 48 h treatment was similar to the mean of the medians from the control group. Thus, this
study was not able to prove the inducibility of β-naphthoflavone regarding the gene
expression of cyp-33E2 convincingly. This test should be repeated and it would make sense to
test other common cyp inducing xenobiotics, such as benzo[a]pyren and fluoranthene. In case
the transformation of C. elegans with pDW20.20::cyp-29A3pr should be repeated and also
successful, an expression test should occur with clofibrate since it has been proven to induce
the subfamily 29A. It is in addition interesting that both CYP enzymes (CYP-33E2 and CYP29A3) show diverse but overlapping substrate and reaction specifications [15], wherefore the
induction by the same xenobiotics should be analyzed in expression tests.
5.4
Gene expression of cyp-33E2 in C. elegans
cyp-33E2 gene expression was sight in cells of the pharynx and always seemed to occur in the
most anterior intestinal ring (ring 1) (cf. Fig. 11). Yet, in most studies which use GFP
fluorescence as a marker for gene expression, this anterior part of the intestine shows GFP
staining. For instance, GFP fluorescence was also sight in this part of the intestine within the
worms originating from the unsuccessful pDW20.20::cyp-29A3pr transformation (cf. Fig. 21).
Therefore, it does not seem clear, whether cyp-33E2 gene expression actually occurs within
these cells.
As mentioned in the introduction, C. elegans mutants with deficiency in the PUFA synthesis
show next to developmental defects also osmosensory, machanosensory and olfactory deficits
[21, 23, 41]. Hence, it is not surprising that cyp-33E2 expression occurs in the pharynx, a
neuromuscular organ responsible for nutrition, including detection of bacteria, pumping
activity and digestive functions [4]. This indicates that the CYP-dependent eicosanoids may
contribute in some kind of way to the feeding behaviour of C. elegans. In addition, hints
indicate many similarities between the pharynx and the vertebrate heart, whose
- 37 -
cardiomyocytes are places with high activity of CYP-dependent eicosanoids. For example,
several results from studies suggest that the C. elegans pharynx and the vertebrate heart could
have evolved from an organ of a common ancestor and further on that an evolutionary
conserved mechanism underlies heart and pharynx development [53]. This would explain why
similar signal molecules can be found in both organs. However, the resolution capacity of the
microscope [Nikon Eclipse E200] was not good enough for distinguishing the different cells
sufficiently, thus it was difficult to tell exactly from which cells the GFP fluorescence actually
originated and hence gene expression occurred within the pharynx. Therefore, it would make
sense to examine the GFP staining a second time with methods that allow identifying and
viewing single cells. A possible method would be laser capture microdissection (LCM).
Nevertheless, cyp-33E2 gene expression probably occurred in the muscle cells, the neuronal
cells M1, M2 and I4 and/or in the marginal cells (cf. 4.7.2).
As mentioned previously, a deficit in PUFAs can lead to neurological defects which could
reinforce the assumption that cyp-33E2 gene expression occurs in the neuronal cells of the
pharynx. Yet, the functions of the neurons within the pharynx are relatively unknown since it
has been demonstrated that the nervous system is not essential for pumping [54]. Some
neurons (MC, M3, M4 and M5) are required for efficient pumping [1, 55], but these do not
include the neurons which could have been showing GFP fluorescence. Avery and Horvitz
[54] were able to show that M1 neuron plays a minor role in effective efficient trapping of
bacteria in the procorpus. This could suggest that CYP-33E2 contributes with its eicosanoids
in addition to the pm cells via M1 neuron to pharyngeal pumping. Yet, M2 and I4 (Fig. 20)
have no effect towards pharyngeal functions and feeding behaviour [55], wherefore the reason
for gene expression of cyp-33E2 in these cells is questionable. However, the marginal cells
(mc) reinforce strength to the muscle organ. Two theories presume that mc could either have
motor functions within the pharynx or they may enable the synchronous contraction and
relaxation of all pharyngeal muscles within a segment by acting as relay stations, which
synchronously transmit signals from motor neurons to surrounding pharyngeal muscles. CYP33E2 derived eicosanoids could have some kind of signalling function within these two
theories.
In contrast to the possibility of neuronal localisation, plenty of evidence including the effect
of PUFA deficits, present studies concerning the regulation of ion channels in pharyngeal
muscle cells and also parallels to mammals imply that the cyp-33E2 gene expression occurs
rather within the muscle (pm) cells of the pharynx than in neuronal or marginal cells. A closer
look at the functionality of the pharynx regarding feeding reinforces this hypothesis. Normal
- 38 -
feeding is performed by two motions in the pharynx, namely pumping and isthmus peristalsis
[56]. Whereby the pumps for opening the lumen in a triangle way are performed by a nearsimultaneous contraction of the corpus, the anterior isthmus and the terminal bulb, the isthmus
peristalsis carries bacteria which is trapped in the anterior isthmus to the grinder by a
peristaltic wave of contraction in the posterior isthmus [56]. Hence, all muscle cells are
essential for the contractility of the pharynx during feeding, which explains why GFP
fluorescence was sight in all muscle cells (Fig. 18 most likely represents the threefold radial
symmetry of pm5 muscle cells and Fig. 16 the threefold radial symmetry of the three pm3
cells) and CYP-33E2, since it may contribute to feeding behaviour through its eicosanoids, is
present in all of these muscle cells.
In addition, there are many analogies between the pharyngeal muscle cells and the smooth
muscle cells and cardiomyocytes, respectively, both places of high CYP-derived eicosanoid
activity, which support the hypothesis that cyp-33E2 gene expression occurs within the
muscle cells of the pharynx. First of all, there is evidence that like the heart, the pharynx is a
rhythmically contracting neuromuscular pump and that the muscle cells of the pharynx have
an independent contractile activity reminiscent of cardiac myocytes [54, 56]. Secondly,
regarding similarities between smooth muscles cells and pharyngeal muscle cells, both types
of muscles are nonstriated muscles and are to be found in similar places within the animals, as
for example in the male and female reproduction tracts. Further on, both muscle types
contract with recurrent intracellular Ca2+ transients, whereby smooth muscle cells can also
derive their calcium from extracellular fluid. These results indicate that the pharyngeal
muscles possess similar features to both types of cells. Therefore, they could resemble an
intermediate between both muscle types, which is regulated by homologous enzymes and
signal molecules, hence solidifying the theory that cyp-33E2 gene expression also occurs
here.
Further on, recent work by the group from Wolf-Hagen Schunck at the MDC demonstrated
that CYP-dependent omega-3 epoxidation results in novel metabolites regulating the
contractility of cardiomyocytes and smooth muscle cells in mammals. Providing an example,
it was proven that EPA treatment protects against angiotensin II-induced end-organ damage in
rats, implying that n-3 PUFAs possess antiarrhytmic properties [57]. In addition, further
studies have demonstrated that 17,18-EETeTr also stimulates vascular calcium-dependent
potassium (BK) channels in the vascular smooth cells of mammals. Hence, concerning
vasodilatation, CYP/EPA-dependent eicosanoids function as efficient vasodilators [58] [59].
Considering the similarities of pharyngeal muscle cells to cardiomyocytes and smooth muscle
- 39 -
cells, it is quite likely that CYP-33E2-derived eicosanoids also contribute to muscle
contraction, for example by converting EPA similar as in the cardiac of mammals to
antiarrhytmic metabolites (e.g. 17,18-DHETeTR, 19/20-OH-EPA and 17,18-EETeTr [15]),
and thus play a role within the contraction of the pharynx. Besides, C. elegans contains many
ion channels, ranging from TRP [60], calcium [61], potassium [62] and sodium channels [63],
some of which are proven to be involved in osmotransduction, machanotransduction and
olfaction alike PUFAs. For instance, studies have shown that mutations in genes encoding ion
channels in pharyngeal muscle cells change the feeding behaviour [64]. Since CYP-dependent
eicosanoids alter the activity of various ion channels in mammals [16, 17, 65], it is likely that
CYP-33E2-derived eicosanoids contribute to feeding behaviour, as for example pharyngeal
pumping, via the regulation of ion channels. In addition, C. elegans ion channels possess
pronounced homologies to their mammalian counter parts. For instance, the C. elegans gene
egl-19 encodes the α1-subunit of a homologue of voltage-activated Ca2+-channels from the
vertebrate L-type [66]. These homologies regarding ion channels and the analogies with
mammals concerning cell composition and effect of PUFAs within the organs confirm the
theory that CYP-dependent eicosanoids may modulate the activity of various ion channels in
the pharyngeal muscles of C. elegans and thus may contribute to feeding behaviour.
5.5
Perspective
Recent work at the MDC and the Group Freshwater and Stress Ecology at the HumboldtUniversität is carried out for determining the enzymatic activity of CYP-33E2 and CYP-29A3
subsequent to overexpression in insect cells. A further characterisation would be possible by
testing the effect of their metabolites concerning feeding behaviour after cyp gene silencing.
Thereby, to analyse the effect on the different ions, the ion activity within the pharynx cells
should be tested. Further investigations concerning other CYP enzymes contributing to the
metabolism of PUFAs, such as AA und EPA, could possibly detect other biological functions
of these signalling molecules within C. elegans and may elucidate evolutionary conserved
mechanisms, for example the protection of pumping mechanisms via the regulation of ion
channels. In addition, investigations for revealing the specific ion channels where defined
eicosanoids operate would be from great interest. C. elegans could then be used for analysing
the mechanisms of action of eicosanoids at their target location. This may also open up new
pharmaceutical strategies for the prevention and therapeutically approach for the variety of
diseases, including hypertension, diabetes, metabolic syndrome and also acute renal failure, in
which these eicosanoids play a crucial role.
- 40 -
Appendix
I
Acknowledgment
First of all, I would like to thank Prof. Dr. Christian E. W. Steinberg for admitting me in the
Group of Freshwater and Stress Ecology and for providing me with this interesting Bachelor
thesis. In addition, I would like to express my gratitude to my supervisor Dr. Ralph Menzel. I
especially thank him for his encouragement and his support during the course of my work.
I would also like to thank the whole group of Freshwater and Stress Ecology for the pleasant
working atmosphere at the laboratory. In particular, my special thanks to Kerstin Pietsch for
her helpful advice and especially for her patience when answering my (numerous) questions.
Further on, very special thanks go to my friends and above all to my family for their support
and in particular for their tolerance during the writing of this report.
- 41 -
II
List of Abbreviation
AA
A. dest.
app.
bp
C. elegans
CPR
CTAB
CYP, P450
E. coli
EPA
h
IPTG
LB
NGM
PCR
PUFA
PVP
RPM
RT
UV
III
Arachidonic acid
Aqua destillatum
Appendix
Base pair
Caenorhabditis elegans
NADPH-cytochrome P-450
reductase
Cetyltrimethylammonium bromide
Cytochrome P450
Escherichia coli
Eicosapentaenoic acid
Hour
Isopropyl-beta-Dthiogalactopyranoside
Luria-Bertani
Nematode Growth Medium
Polymerase Chain Reaction
Poly Unsaturated Fatty Acid
Polyvinylpyrrolidone
Revolutions per minute
Room Temperature
Ultraviolet
C. elegans life cycle
- 42 -
IV
Vectors
IV.I
pGEM-T
Figure II. pGEM®-T Vector circle map and sequence reference points
[source: www.promega.com /tbs/tm042/tm042.pdf ].
IV.II pDW20.20
HindIII
SphI
Sse8387I
PstI
BspMI
XbaI
BamHI
SmaI
XmaI
BalI
NheI
Asp718I
KpnI
AscI
BssHII
SapI
BspLU11I
XhoI
BsaAI
Bst1107I
4000
MunI
PDW2020.TXT
BsaBI
1000
3000
4292 bps
FseI
NaeI
NgoMIV
Ecl136II
SacI
AhdI
2000
FspI
PvuI
ScaI
SpeI
ApaI
Bsp120I
SspI
AflII
Figure III. pDW20.20 circle map and sequence reference points
[source: Group Freshwater and Stress Ecology].
- 43 -
V
Overview of promoter sequence
The primer sequences are marked in red.
V.I
cyp-33E2
aagatctcattattcgttcgttactttgttgaacgaatgatttttttaatttgacaaatgactagaaattgtgaaatcattgtgcaaattgcgggtt
ctactgttaaaacttccggtattttcctctctcttttaataaattttatataatcaataaataaatcaatcaataaaatgttgtttcaatgatccatgg
aagagatacaatatcgattttctgaagaagtgaagactgattatcagctgttttttcggtggaagctggtgacaaagggggtggacttcta
gctggcatggatgacgacttagtctaaaaacagtaattatagaagaatgcaaatagaataaatagtaaatgttatcagaaagcttacatcg
atatcagctttcaactttgcaataaacagatcaatataatcgtcggtaagttcgttgagagtagtcatttttaacaaatattttgcagaaataaa
acttttccgaaaaatgggaaacgtggaaaaccaattttgaatggattttttgaatatttaagttagtttctaaaacagattaatcgaatttcacg
gcaaaaaagttttctgtttcacgcttacctgcctacattcttgttatccttttgagatgccgaagtgtagtctgccacatttttcaaccaattactt
ttaacataagctaatgacagtgtatcttatttcaatatttcggttattttttgtaagaacattcaaccattttcatcattttatctgtttatcattccga
acattactaattctaaattttaaaagtaataaataatatttcttatcattttcctccaacttttcctcccattttcatggcctctgcattcattttgtggt
cggtaaacctagaaaaatcatttctacaggaaactttagagatagtagagaaaagtttgtgactgtttatgtcttcacactctaaccaaaca
cacagttttgtgttcatatgtctaaatctcttattcttgcattaaatactctgttaggtgagcaaacttcagttgttttcattataaatactgtttgca
gaagtacgttaaagcagggcttactttattgaatattagttttcactctcctttgaaatttttttttaattttctcttttcagaatgatattatttattgtt
ctatctattgtttcaatctacctcttcgacttattctactgg
V.II
cyp-29A3
atttcttgtaaaaacgactgaattttgtttcattttatgatacaaaaaaattgtaacacaataatataaaatttcagattactcgtttttttacaactt
ctagggcttcaatgggaggcaggcgcggttttagggcttgacgcctgcctccaaccagggctgtgcgacatccggatttttcgacaaac
cctaattttttcggcgatcggcatttgccggttgccgaaaaaacctgtttttcggtacttttcggcattcttaaaatattctcaaaatatttgatgt
tttgttgtttttcttgtattttctaaatctaattagttcagttcgaaaatgataaggagtgcctttgtttaaattttcaagcaaagatcaattttatatg
accagactgttgggatagtcaaaaaaggatcccagaggcaagaacgattcaaaataattgtacaagtacacagccatagacagcatac
ttgatggttttatgatggggtgacgatgttatcaactaaaagaatatctcctctatacggctaataattcgaatattttcatttttgggaagttttg
ggagtacaggtggctggagtgttcggatacaatagacacagagatgccccttgatttcaacttaatcccggacttcttaatgaaacctatg
tcatttgtcgctctgaacatttttcagtacatttcttcaaaactttcgaggtccgtaacatggctccaaatgttaatttttgcagatttttgatgatt
tctaggatgttaagaggtcagaatttgttttgaaattttggtaaaatttcagatttttttcaaaaaattttaaaaatttccaaaaaaaattctgaat
gtttgcctgccactgaacatcctagaaatcatcaaaaatctgcaaaaattaacatttggagccattttacggaccttgaaagttgtgaaaaa
atgcactgaaaaattttcagagcgacttttttccagttcgaaatgctcctctgccgctaaattttttgtaggggcctatttagaagatgctcttt
aacatagggttcattcagaagaccgggattaagttgaaatacttcccttgtaagctcgttaggattaagtttgattgggttccttaagattaac
gctttagacatatttattttgatgaacttgttatttaaaaatcgcccgaactcgtgagatcagaagatctcggaagtaaaaaaggattactgta
ggaatgacgtggcaggacattttctagacaaagctttgcagaaattatttgataactttttttttcagaagttatgctcgtaactttttacaatct
atacataataactattcaagttcacataaattttcaaattttcaaagaaaaaaaattgaaaaatattttttaggtaaccgcaaatataagcttcct
tgattaaatgaagcgaaacctcttggcattactaatgagatcattttcttgggcttggtaatactacaaacttcagaaatgagttttcttcaga
gatcactgatgttacacaactacaaactacgttaaattttttaggacaattgttttttatgtttgcaaaattagcaaacagggtttcaaatcaagt
atcaaagaaaaaatgagaaaaccccctttttttaaacagctttattcggtaaattgaggctgttttttgtcaggtaggtcatcaaaacagctc
agatgctactctaccaaaaatggaaattcggagagaaaacttcagaatatcgacaatttgaaaatgtttacttaaattttttaaaaagtagaa
tttttttggaaagtttcgaaaatttgtttttcttgaaaacttaaactttcaaaatctatccacaaaattatttttccatttctaaaacccactgtaagt
gtgcagttttctaatttttgctgacctaaacttttctagccaagaccaaaaagaactaattcgctagaagaacaggttctcgtacccgccac
attgttttcaagtctacaaactccaatgtgttgggtgataagaaattgataaagtataaaagatggaaatcaaccaaaaaatctagtttgata
aagttagatcatcaaaatgtcattgattctgccctgtttattgatcattttacttttatttattgt
- 44 -
VI
Restriction analysis from pGEM-T::cyppr – possible orientations from inserts in
pGEM-T
Tth111I
HindIII
SpeI
NotI
SbfI
PstI
AccI
HincII
SalI
EcoICRI
SacI
MluI
BfrBI
NsiI
SapI
PciI
BseYI
4000
AlwNI
pGEM-T::cyp-33E2pr a
SacII
SfiI
SphI
ZraI
AatII
PspOMI
ApaI
3000
1000
4062 bps
2000
AhdI
BsaI
BpmI
NgoMIV
NaeI
BsaAI
DraIII
BtgZI
ScaI
TatI
XmnI
Figure IV. Possible Orientations a of cyp-33E2pr sequence in pGEM-T.
SpeI
NotI
SbfI
PstI
AccI
HincII
SalI
EcoICRI
SacI
MluI
BfrBI
NsiI
SapI
PciI
HindIII
BseYI
4000
Tth111I
AlwNI
pGEM-T::cyp-33E2pr b
SacII
SfiI
SphI
ZraI
AatII
PspOMI
ApaI
3000
1000
4062 bps
2000
AhdI
BsaI
BpmI
NgoMIV
NaeI
BsaAI
DraIII
BtgZI
ScaI
TatI
XmnI
Figure V. Possible Orientations b of cyp-33E2pr sequence in pGEM-T.
- 45 -
SpeI
NotI
Sse8387I
PstI
BspMI
AccI
HincII
SalI
NdeI
Ecl136II
SacI
BstXI
MluI
NsiI
BstXI
AccI
XmnI
XmnI
SapI
BspLU11I
AlwNI
cyp-29A3 Promotor
XmnI
4000
1000
pGEM-T::cyp-29A3pr a
4950 bps
AhdI
3000
BsaI
GsuI
2000
GsuI
XmnI
BstXI
SacII
StyI
NcoI
SfiI
SphI
AatII
Bsp120I
ApaI
ScaI
XmnI
NgoMIV
NaeI
BsaAI
DraIII
Figure VI. Possible Orientations a of cyp-29A3pr sequence in pGEM-T.
SpeI
NotI
Sse8387I
PstI
BspMI
AccI
HincII
SalI
NdeI
Ecl136II
SacI
BstXI
MluI
NsiI
BstXI
SapI
XmnI
GsuI
BspLU11I
AlwNI
XmnI
cyp-29A3 Promotor
pGEM-T::cyp-29A3pr
b
4000
1000
4950 bps
AhdI
3000
SacII
StyI
NcoI
SfiI
SphI
AatII
Bsp120I
ApaI
BsaI
GsuI
2000
XmnI
XmnI
AccI
BstXI
ScaI
XmnI
NgoMIV
NaeI
BsaAI
DraIII
Figure VII. Possible Orientation b of cyp-29A3pr sequence in pGEM-T.
- 46 -
VII
Data from image internet tool
Table I.
Control group; bold value: discordant value.
After 24 hours
Median
Standard
deviation
Average
Mean
value
Median
Mean
value
0.2078
0.1922
0.1647
0.2275
0.2078
0.1882
0.1686
0.1882
0.1961
0.2745
0.1843
0.2118
0.1294
0.1458
0.1569
0.2784
0.149
0.1216
0.1765
0.3529
0.1785
0.1754
0.1771
0.1614
0.1334
0.1983
0.1754
0.1621
0.2577
0.1706
0.1736
0.1844
0.2373
0.211
0.1869
0.221
0.204
0.2103
0.1752
0.1529
0.2319
0.18075
0.1882
0.189
0.02969
0.03106
0.04205
0.05511
0.17661
0.18999
0.19611
0.20342
0.1765
0.1686
0.1529
0.1059
0.1843
0.1765
0.1529
0.2196
0.1569
0.1569
0.1804
0.2157
0.2157
0.1804
0.1961
0.1922
0.1974
0.1686
0.1255
0.2092
Median
After 48 hours
- 47 -
0.3054
0.2016
0.1761
0.2293
0.2151
0.1836
0.1749
0.1923
0.1951
0.2675
0.191
0.2085
0.1418
0.1458
0.172
0.1765
0.1574
0.187
0.3711
0.1764
Table II. Fenofibrate
After 24 hours
Median
Standard
deviation
Average
After 48 hours
Median
Mean value
Median
Mean value
0.2235
0.1529
0.1882
0.2157
0.2
0.1216
0.1608
0.234
0.2078
0.1216
0.1608
0.2118
0.2588
0.349
0.2235
0.1542
0.149
0.3059
0.1804
0.2157
0.2039
0.2314
0.1921
0.187
0.2138
0.1934
0.1391
0.183
0.2258
0.2087
0.1324
0.183
0.2011
0.2584
0.3707
0.2216
0.154
0.1623
0.3462
0.1774
0.2088
0.19725
0.1882
0.2627
0.2261
0.2157
0.2824
0.2235
0.2941
0.1843
0.2667
0.1843
0.1569
0.2275
0.1608
0.1765
0.2941
0.2392
0.1529
0.149
0.2941
0.1804
0.2196
0.1941
0.2971
0.2398
0.2275
0.2935
0.2407
0.3095
0.1894
0.2763
0.1954
0.1671
0.2331
0.1717
0.187
0.3035
0.2464
0.1722
0.1617
0.2998
0.1919
0.2303
0.0573868
0.20176
0.0597174
0.20951
0.050695729
0.21797
0.0515874
0.229885
- 48 -
Table III. β-naphthoflavone
After 24 hours
Median
0.1529
0.1765
0.1569
0.2196
0.1529
0.149
0.2118
0.251
0.2471
0.2654
0.1804
0.149
0.2301
0.2261
0.251
0.2588
0.2235
0.251
0.2784
0.1882
Median
After 48 hours
Mean value
0.1563
0.1772
0.1709
0.2261
0.1614
0.151
0.2102
0.2614
0.2474
0.2666
0.1896
0.1513
0.2332
0.2262
0.251
0.2613
0.222
0.2509
0.2736
0.1907
Median
0.2196
0.2078
0.2039
0.1216
0.1686
0.2261
0.2078
0.1647
0.1294
0.1569
0.149
0.1412
0.149
0.1569
0.1569
0.1451
0.251
0.1882
0.302
0.2196
Mean value
0.2125
0.2098
0.2152
0.1305
0.1734
0.2327
0.2057
0.1785
0.1536
0.1719
0.1641
0.1512
0.1602
0.1652
0.1574
0.1598
0.2555
0.1924
0.3088
0.2184
0.2039
0.19725
0.2196
0.2303
0.05739
0.059717
0.05069
0.051587
0.20176
0.20951
0.21797
0.22989
Standard
deviation
Average
- 49 -
VIII
Chemicals
Substances
Acetic acid
Acetone
Agar
Ampicillin
Bacto Pepton
Bactotrypton
Calcium chloride (Ca2+ *2H20)
Chloroform (CHCl3)
Cholesterol
Dichloromethane (CH2Cl2)
Dimethyl sulfoxide (DMSO)
EDTA (Ethylendinitritetraessigsäure)
Ethanol
Ethidium bromide (10ml/mg)
Fenofibrate
Glycerine
Hydrochloride acid (HCl)
HotStar Taq Polymerase 8250 units) + 10x
buffer
IllustraTM GFXTM PCR DNA and Gel Band
Purification Kit
IPTG (Isopropyl-β—dthiogalactopyranoside)
Lysozym
Magnesium chloride (MgCl2*6H2O)
Magnesium sulphate (MgSO4*7H2O)
Methanol
Potassium chloride (KCl)
Potassium dihydrogen phosphate (KH2PO4)
Potassium hydrogen phosphate
(KHPO4*3H2O)
PureYield Midiprep System
Saccharose
Sodium chloride (NaCl)
Sodium dihydrogen phosphate
(NaH2PO4*H2O)
Sodium hydroxide
Spermidin
Tetracycline
TRIS (Tris(hydroxymethyl)-aminomethan)
Triton X-100
Yeast extract
Company
Carl Roth GmbH + Co. KG
J. T. Baker
Serva GmbH
Carl Roth GmbH + Co. KG
OTTO NORDWALD
Carl Roth GmbH + Co. KG
Fluka Chemie AG
Carl Roth GmbH + Co. KG
Sigma-Aldrich Chemie GmbH
Carl Roth GmbH + Co. KG
Carl Roth GmbH + Co. KG
MERCK
MERCK
Sigma-Aldrich Chemie GmbH
Sigma-Aldrich Chemie GmbH
Carl Roth GmbH + Co. KG
MERCK
QIAGEN
GE Healthcare
Appli Chemie GmbH
Carl Roth GmbH + Co. KG
MERCK
MERCK
Carl Roth GmbH + Co. KG
Carl Roth GmbH + Co. KG
Carl Roth GmbH + Co. KG
MERCK
Promega
Carl Roth GmbH + Co. KG
Carl Roth GmbH + Co. KG
MERCK
MERCK
Sigma-Aldrich Chemie GmbH
Sigma-Aldrich Chemie GmbH
Serva Feinbiochemica GmbH
Serva Feinbiochemica GmbH
Carl Roth GmbH + Co. KG
- 50 -
IX
Laboratory equipment
Apparatus
Autoclave
Autoclave
Agarose gel apparatus
Centrifuge
Digital scale
Electrophoresis power supply
Heating block
Heating block
Heating block
Incubator
Incubator
Laboratory-Grade Water
Systems
Magnet stirrer
Magnet stirrer
Microcentrifuge
Microcentrifuge
Micro scales
Microscope
Microscope
Microscope
PCR apparatus
pH meter
Photometer
Pipettes
Quartz cuvettes
Shaker
Sterile working bench
Steam sterilizer
UV apparatus
Vortex
Vortex magnet stirrer
Vortex minishaker
X
Model
Varioklav 25T
5050 ELV
Agagel Mini, Biometra
Megafuge 1.0 R
scale 510-23
EV243, Consort
Thermomixer comfort
Thermomixer 5436
Thermostat 5320
INB 500
UNB 500
RiOs
Company
H+P
Systek
Blomed Analytik GmbH
Heraeus Instruments
Kern
Fisher Bioblock Scientific
Eppendorf
Eppendorf
Eppendorf
Memmert
Memmert
Millipore
MR 200
Variomag Monotherm
1-14
Variomag Monotherm
Explorer Pro 214C
Eclipse E200
SMZ 1000
Axioplan
FPROGO50
pH 526
Specgene
Research® pro
QS 10.00 mm
GFL 3005
Safeflow 1.2.
Varioklav
TFL-20M
Vortex Genie
REAX 2000
MS1
Heidolph
H+P
Sigma
H+P
Ohaus
Nikon
Nikon
Zeiss
Techne
WTW MultiCal®
Techne
Eppendorf
Helm
Hilab
BIOAIR Instruments
H+P
FLUO_LINK
WINN B.V.
Heidolph
Roth [IKA®]
Software
-
Adobe Photoshop
-
Clone Manager Professional Version 8 Software
-
Image internet tool (http://imagetool.lsmod.de/farbquant.cgi)
-
Office (Microsoft)
-
Quantity One 4.2 (image and analyzing gels)
-
SigmaStat 3.5 [SPSS]
- 51 -
XI
Reagents and stock solutions
XI.I
buffers
Table IV. Elution buffer, pH 8.0
Components
Concentration
Tris-HCl
10 mM
Table V. Freezing buffer
Components
NaCl
KH2PO4
Glycerol
1 N NaOH
0.1 M MgSo4
Concentration for 100 ml
0.585 g
0.68 g
30 g
0.56 ml
Autoclave
0.3 ml
Table VI. M9 buffer
Components
Concentration for 1 L
KH2PO4
Na2HPO4 (2xH2O)
NaCl
Aqua dest.
3g
7.5 g
5g
500 ml
Autoclave
1 M MgSo4
1 ml
Table VII. STET-Puffer Lyse
Components
Concentration for 1 L
Tris/HCl
EDTA
Saccharose
Triton X-100
50 mM
50 mM
8%
0.1%
Autoclave
1 M MgSo4
1 ml
- 52 -
Table VIII.
TAE-buffer
Components
Concentration for 1 L
Tris-Acetate
EDTA
40 mM
1 mM
pH=8.5
Table IX. TE-Puffer
Components
Concentration for 1 L
Tris/HCl
10 mM
pH=8.0
EDTA
1 mM
Worm lysis buffer
Table X.
Components
Concentration for 1 L
Tris/HCl
10 mM
KCl
50 mM
MgCl2
2.5 mM
NP40
0.45%
Tween 20
0.45%
Gelatine
0.01%
pH=8.3; autoclaved and saved as aliquots at -20°C
XI.II Stock solutions
Solutions
Agarose
Ampicillin
CTAB
Components
0.7-2.0%, dissolved in 1xTAE buffer
100 µl/20ml
cationic detergent 5% w / v. in Aqua dest.; filtrate sterile
Lysozym-stock solution
Polyvinylpyrrolidon solution
20 mg Lysozym / ml Aqua dest.; filtrate sterile
10 mg + 490 µl A. dest, 99.5 ml 99% Ethanol
Protein kinase K
RNase A-stock solution
10 mg/ml in sterile A. dest
10 mg RNaseA(Sigma)/ml TE-Puffer
Warm up to 95°C for 15 minutes; cool down slowly to RT
and store in small portions at -20°C
100 µl worm lysis buffer
1 µl proteinase K
72.6 mg/ 10 ml; filtrate sterile
Single worm lysis mix
Spermidin solution
- 53 -
XII
Media
XII.I Preparation of C. elegans growth media
Table XI. NGM-Agar
Components
NaCl
Agar
Bactopepton
5 mg/ ml Cholesterol
1 M KH2PO4
Aqua dest
For 500 ml
1.5 g
8.5 g
1.25 g
800 µl
12.5 ml
486 ml
Autoclave
1 M MgSo4
1 M CaCl2
0.5 ml
0.5 ml
XII.II Preparation of E. coli OP50 growth media
Table XII. LB (Luria-Bertani), pH 7.5
Components
Bactotrypton
Yeast extract
NaCl
Aqua dest
Concentrations for 1 L
10 g
5g
10 g
1L
Autoclave
Table XIII. LB-Agar, pH 7.5
Components
Bactotrypton
Yeast extract
NaCl
Agar
Aqua dest
Concentrations for 1 L
10 g
5g
10 g
20 g
Fill up to 1 L
Autoclave
Table XIV.
SOC-medium, pH 7.5
Components
Bactotrypton
Yeast extract
NaCl
KCl
Aqua dest
Concentration for 1 L
20 g
5g
0.5 g
2.5 mM
975 ml
Autoclave
2 M MgCl2
1 M Glucose
5 ml
20 ml
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XIII
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XIV
Affidavit
I hereby declare that the Bachelor Thesis has been written only by the undersigned and
without any assistance from third parties.
Furthermore, I confirm that no sources have been used in the preparation of this thesis other
than those indicated in the thesis itself, and that quotations used were identified.
Berlin, May 2009
Place, Date
Signature
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