METHODS

Identification of recombinant cDNA and genomic clones from phage libraries by plaque hybridisation Preparation of plating bacteria

Host bacteria for bacteriophages (XL 1 blue for lambda zap, and XL 1 blue MRA for lambda dash II) were grown overnight at 30°C, 150 rpm in 100 ml LB containing 0.2% maltose. Cells were pelleted at 5000 rpm for 10 minutes at 4°C and re-suspended in 20 ml 10 mM MgSO₄ to an OD₆₀₀ of 2 (about 1.6x10⁹ cells per ml). The cell suspension was incubated at 37°C, 220 rpm for 1 hr, stored at 4°C and used for a maximum of three days.

Plating of lambda phage

An appropriate volume of phage suspension was mixed with 100 µl (for 88 mm Petri dishes) or 200 µl plating bacteria (for 120 mm petri dishes) and incubated with shaking (100 rpm) for 15 minutes at 37°C. The suspension was then mixed with 4 or 8 ml of molten Top agar (50°C) and carefully spread on agar plates. The plates were incubated for 30 minutes at room temperature to allow the top agar to harden, and were then incubated at 37°C overnight. A uniform bacteria lawn could be observed interspersed with lambda plaques. The plates could be stored at 4°C for several days.

Titration of phage libraries

To determine the concentration of a phage suspension in Plaque Forming Units (pfu) per ml, dilutions of a phage stock were made from 1:10 to 1:10, 000 in SM buffer, and 1 µl of each dilution was plated as described in section 4.1.2. Plates having between 50 to 350 plaque forming units were used to calculate the phage concentration in pfu.

Amplification and cryopreservation of phage stocks

Confluent plates of 3 x 10⁴ pfu/plate were produced and incubated for a maximum of 8 hours at 37°C. SM buffer was then added to the plates and the plates were incubated with constant shaking for 2 hours at 4°C. The lysate was then aspirated and centrifuged for 5 minutes at 10, 000 rpm to pellet residual agarose particles and the titre was determined as in section 4.1.3. DMSO was then added to 1 ml aliquots of bacteriophage stock at a concentration of 7% v/v. The tube was mixed gently, plunged into liquid nitrogen and stored at -80°C until used.

Screening of libraries Screening of libraries with a DIG-labelled A. viteae chitinase probe Screening of libraries by plaque hybridisation

Up to 2 x 10⁴independent plaques were plated on a 120 mm petri dish and cultured for 8 to 12 hours. The plates were incubated at 4°Cfor 30 minutes to allow the top agar to set. Nylon membranes were rinsed in water and then in 5x SSC and allowed to dry at room temperature. The membranes were carefully placed on the surface of the infected plate so that the entire membrane was in contact with the plate. After 30 minutes of incubation at 4°C, the membranes were marked with a hot needle for orientation. Plates were again incubated at 4°C for 30 minutes, after which the membranes were removed. Replica filters were also used to lift plaques from infected plates.

DNA on the filters was denatured by incubation with 0.5 M NaOH/ 1.5 M NaCl for 15 minutes at room temperature, and subsequently neutralised twice by incubation with 0.5 M Tris, 1.5 M NaCl pH 8.0, after which the filters were allowed to dry on a Whatmann filter paper. DNA on the filters was immobilised bycovalently linking the nucleic acids to the membrane by UV cross-linking. Alternatively, the membranes were baked at 80°C for 2 hours. An N-terminal cDNA sequence corresponding to nucleotides 52-1,117 of A. viteae L3 chitinase (Adam et al., 1996; Genbank number U14638) was labelled with digoxigenin (DIG) using the PCR labelling and detection kit from Roche. Hybridisation and detectionwas done as described by the manufacturer (Boehringer, Mannheim). Hybridisation was done with 15 ng/ml of probe in 10 ml of hybridisation buffer at a temperature of 50°C overnight. Stringent washes were done as follows: membranes were washed for 2 x 15 minutes in ample 2x SSC, 0.1% SDS at RT, and then in 0.5x SSC, 0.1% SDS (pre-warmed to wash temperature) for 2x 30 minutes at 65°C under constant agitation. Chemiluminescent detection was done using CSPD (Boehringer Mannheim).

Agarose plugs containing putatively positive plaques were incubated in SM buffer overnight at 4°C to let the phage wander out. The phage suspensions were diluted 1:10 with H₂O and boiled for 5 mins, and 1 μl was used for PCR as in section 4.3.12.1. Primers were vector-specific (T3 or T7) and A. viteae chitinase-specific.

To obtain clonal recombinant phages, phage suspensions identified as positive from the primary and PCR screening rounds were subjected to three further rounds of plaque hybridisation. In order to increase the chances of picking single/ independent plaques, 100-fold less independent plaques were used per new screening round as the proportion of positive plaques increased.

In vivo excision

The cDNA libraries used in this study were constructed in the vector Lambda UniZap XR (Stratagene). This vector is designed to allow in vivo excision and recircularization to form a pBluescript phagemid containing the cloned insert. An ExAssist helper phage contains a mutation that prevents replication of the phage genome in the non suppressing E. coli host (SOLR) and thus allows only the excised phagemid to replicate (Short et al., 1988). In vivo excision was done according to the manufacturer's instructions. Briefly, 5-ml cultures of XL 1 Blue MRF´ and SOLR cells were grown at 30°C overnight in LB broth. The cells were pelleted and resuspended in 2.5 ml 10 mM MgSO₄to an OD₆₀₀ of 1.0 (8x10⁸ cells/ml). An excision mix was made of 200 μl of XL 1 bLue MRF´ cells, 250 μl phage stock (1x10⁵ phage particles) and 1 μl ExAssist helper phage (titer: 1 x10⁷ pfu/ml). The mixture was incubated for 3 hours at 37°C with shaking. During this period the excision reaction takes place leading to the formation of pBluescript phagemid packaged as filamentous particles. The tube was heated at 70°C for 20 minutes to lyse the lambda phage particles and cells. Following centrifugation (1000xg for 15 minutes), the supernatant containing the excised phage particles was then stored. To plate the cells, 100 ul of phage supernatant was added to 200 μl SOLR cells (OD₆₀₀=1) and incubated at 37°C for 15 minutes. The cells were then plated on LB-ampicillin plates and incubated overnight at 37°C. Bacteria colonies were isolated containing the pBluescript double-stranded phagemid with the cloned cDNA insert.

Purification and manipulation of DNA Isolation of lambda DNA and sub-cloning of genomic inserts

DNA was typically purified from clonal plaques and used for restriction digestion analysis, PCR, Southern and subcloning. The genomic libraries used in this study were constructed in lambda dash II (Stratagene). This vector, unlike lambda UniZap, can not be in vivo excised. To circumvent this short-coming, the inserts were released by digestion and cloned into plasmids.

Preparation of crude phage lysates

Confluent plates of 3 x 10⁴ pfu/plate were produced and incubated for a maximum of 8 hours at 37°C. SM buffer (10 ml) was then added to the plates and the plates were incubated with constant shaking for 2 hours at 4°C. The lysate was aspirated and centrifuged for 5 minutes at 10, 000 rpm to pellet agarose particles.

Isolation of phage DNA

DEAE cellulose columns were prepared and used for isolation of DNA. To prepare these columns, hydrochloric acid (0.05N) was slowly added to DE52 powder (Whartmann DE52 number 4057-050) with continuous stirring until a pH < 4.5 was attained. Concentrated NaOH was then added until the pH was 6.8. The resin was let to settle, the supernatant was aspirated and the ion exchanger was then washed 4 to 5 times with several volumes of LB-Medium. The slurry was adjusted to 60% resin, NaN₃ was added to a final concentration of 0.02% and the slurry was stored at 4°C for up to three months. Columns were prepared by pouring 8 ml DEAE slurry in a 10-ml disposable syringe resulting in a bed height of 6 cm. After the DEAE-cellulose had settled, the column was washed with 5 ml LB and subsequently equilibrated in LB.

The supernatant from two plates was loaded onto a 6-8 ml DEAE column. During this chromatographic step, bacterial DNA and RNA are separated through ion-exchange chromatography; the ion-exchange resin preferentially binds contaminants in the lysate such as E. coli DNA, RNA and proteins. The eluate containing the phage particles were then collected and pooled. The column was washed again with 2 ml LB- Medium and the eluate added to the first. The NaCl concentration of the eluate was adjusted to 70 mM and phages were precipitated with 2 volumes of ethanol (20 minutes at –20°C, and then centrifuged for 15 minutes at 12,000 rpm). The pellet was then washed with 2 volumes of 70% ethanol and subsequently dissolved in 2 ml TE with 0.2% SDS to release the phage DNA. The mixture was extracted twice with 2 volumes of Phenol (TE saturated). DNA was precipitated from the aqueous phase with 2 volumes of ethanol without any salt. The phage DNA pellet was then washed twice with 70% ethanol and dissolved in 200 µl TlowE. The yield was about 75 µg per 10 ml phage lysate.

Release and sub-cloning of genomic inserts into pBluescript II SK

Purified recombinant lambda DNA and pBluescript II SK (-) were separately digested with Not I and dephosphorylated as described in sections 4.3.8 and 4.3.9. Genomic inserts ranging in size between 10-14 kb were then ligated into pBluescript vector (3kb) using a 1:1 molar ratio of insert and vector (see section 4.3.10.2).

Small scale Plasmid isolation from E. coli

Plasmid DNA was isolated as described by Del Sal et al., 1989, with some slight modifications. This technique relies on the property of the detergent cetyl-trimethyl ammonium bromide (CTAB) of preferentially precipitating nucleic acids at low NaCl concentrations (< 0.6 M), while leaving proteins and polysaccharides in solution.

Essentially, a single bacterial colony harbouring a plasmid of interest was used to inoculate 6 ml of LB medium (with an appropriate antibiotic for selection) and grown over night at 37°C. Bacterial cells were recovered by centrifugation at 4,000 rpm for 10 minutes. The pellet was re-suspended in 200µl of STET buffer, 4µl of a 50-mg/ml lysozyme solution was added and the samples were incubated at room temperature for 5 minutes. Samples were boiled for 45 seconds and then centrifuged at 13,000 rpm for 10 minutes in an Eppendorf centrifuge. Pellets were recovered, 5 µl of RNase A (10 mg/ml) were added to the supernatants and the samples were incubated for 10 minutes at 68°C. Nucleic acids were precipitated by the addition of 10 µl of CTAB (5% w/v), followed by centrifugation at 14,000 rpm for 5 minutes. Pellets were re-suspended in 300 µl of 1.2 M NaCl and the plasmids were re-precipitated by the addition of 750 µl ethanol and incubation at - 20°C for 1 hour. Samples were then centrifuged at 14,000 rpm for 15 minutes and the pellets were washed twice in 70% ethanol and allowed to air dry. Plasmids were re-suspended in 40 µl H2O. The yield was between 20 to 40 µg of plasmid DNA.

Electrophoresis and detection of DNA on agarose gels

Agarose gel electrophoresis was used for the routine analysis of DNA. Agarose was cast in 1x TAE buffer containing 0.5 µg/ml EtBr. The DNA samples (0.1 to 5 µg) were dissolved in DNA loading buffer and separated in agarose gels at 10 volts per cm. The concentration of the agarose gel was relative to the size of DNA fragments to be separated, and was typically between 0.7 and 1.2%.

Isolation of DNA from agarose gels

Following electrophoresis, a DNA fragment was excised from agarose gels using a clean scalpel, and the DNA was purified using the NucleoSpin® Extraction Kit (Clontech) according to the manufacturer's instruction.

Isolation and concentration of DNA from aqueous solutions Extraction with Nucleospin kit

The NucleoSpin DNA Extraction Kit (Clontech) was used according to the manufacturer’s instruction for the isolation of DNA from PCR reactions, gel fragments and other aqueous DNA solutions.

Phenol chloroform extraction

To free DNA from protein contaminants, the volume was adjusted to at least 300 µl with TE buffer, and extracted with an equal volume of Tris-equilibrated phenol:chloroform:isoamyl alcohol (25:24:1) (Sambrook et al., 1989). The aqueous phase was extracted twice with chloroform to remove traces of phenol, and the DNA was concentrated by ethanol precipitation.

Precipitation of DNA

Ice cold sodium acetate, pH 4.5 was added to a DNA sample to a final concentration of 0.3 M, followed by the addition of 2.5 volumes of ice cold absolute ethanol. The sample was incubated at -70°C for 15 minutes and centrifuged at 12, 000 rpm for 15 minutes at 4°C. The pellet was washed in 70% ethanol, air-dried, and re-suspended in an appropriate buffer.

Isolation of high molecular weight genomic DNA from A. viteae

About 200 mg to 1 g of adult A. viteae were snap-frozen in liquid nitrogen and ground to powder using a mortar and a pestle. Up to 100 mg of powdered worm material was suspended in 1.2 ml of digestion buffer containing Proteinase K (100 µg/ml), and incubated with shaking at 50°C for 12 to 18 hours until the sample became viscous with a visible sludge. The samples were extracted three times with an equal volume of phenol/chloroform/isoamyl alcohol. The viscous aqueous phase was transferred each time with a wide-bore pipette (0.3-cm-dimeter orifice) into in to a new tube. RNA was digested by the addition of 20 µg/ml RNase A and incubating for 1 hour at 37^o C. Genomic DNA was then precipitated by adding ½ volume of 7.5 M ammonium acetate and 2 (original) volumes of 100% ice-cold ethanol. Stringy precipitates of DNA were carefully transferred in to new tubes using a blunt spatula, and washed twice with ample amounts of 70% ethanol (2 times the original volume). The DNA pellet was air dried and re-suspended in TE buffer until dissolved. The concentration of the gDNA was determined by photometry and the DNA was analysed on a gel.

Determination of DNA Concentration

The concentration of DNA in solution was determined using a spectrophotometer at OD₂₆₀, and subsequently confirmed by comparing the intensity and size of DNA from a sample to those of standard size markers with known concentrations.

1 unit of absorbance of dsDNA at OD₂₆₀ = 50 µg/ml dsDNA

1 unit of absorbance of ssDNA at OD₂₆₀ = 33 µg/ml ssDNA or RNA

The OD₂₈₀ was also measured and the ratio between the two ODs indicated the purity of the DNA solution. For pure DNA,

1.8 ≤ absorbance at OD₂₆₀ / absorbance at OD₂₈₀ ≤ 2.0

A value less than 1.8 indicates contamination with proteins or with aromatic substances like phenol, while a value greater than 2.0 indicates possible contamination with RNA.

Restriction digestion of DNA

DNA was digested at the optimal temperature of restriction enzymes according to the pipetting scheme below.

Reagent

Volume (µl)

DNA (0.1 – 5 μg)

5

10 x restriction enzyme buffer

2

10 x Bovine Serum Albumin (BSA)

2

Restriction enzyme (3-20 units)

1

HPLC water

10

Volume

20

Tab. 12: Pipetting scheme for restriction digestion of DNA

Following digestion, the mixture was either heated at 70°C for 15 minutes to inactivate the enzyme, or extracted with phenol chloroform.

5' Dephosphorylation of digested DNA

The removal of 5’ phosphate terminals from digested dDNAs ends prevents vector self-ligation and reduces background in ligations, especially when only one restriction site is used. Shrimp alkaline phosphatase (SAP) (0.5 units) was added to the restriction digests and incubated for 15 minutes at 37°C. Enzyme inactivation was achieved by heating the mixture at 65°C for 15 minutes.

Ligation of DNA fragments Ligation of PCR fragments into T-overhang vectors

PCR products were ligated into the pGEM-T vector (Promega, Madison, WI) accord- ing to the following protocol.

The reaction mixture was incubated overnight at 16°C. The ligation reaction was used to transform competent bacteria and single clones were picked for the isolation of recombinant plasmid DNA that was sequenced and its insert subcloned into an appropriate vector where necessary.

Reagent

Volume (µl)

Vector (pGEM-T) DNA (50ng)

1

2X ligation buffer (Promega)

5

Insert DNA

3 (in three molar excess of the vector)

T4 DNA ligase

0.5

HPLC water

0.5

Reaction volume

10

Tab. 13: Pipetting scheme for ligation of PCR fragments into T-overhang vectors

Ligation of DNA fragments with sticky ends

Insert DNA was digested out of a vector using appropriate restriction enzymes, while the target vector was also digested with the same enzymes. Both were then ligated following the scheme below.

Reagent

Volume (µl)

Vector DNA (50ng)

1

10 x ligation buffer (New England Biolabs)

2

Insert DNA

3 (in three molar excess of the vector)

T4 DNA ligase

1

HPLC water

13

Reaction volume

20

Tab. 14: Pipetting scheme for ligation of DNA fragments with sticky ends

mRNA isolation, reverse transcription and RT-PCR

Worms (1g) were homogenised with a plastic pistil in 1000 µl of RNA isolation reagent (Peqlab) according to the manufacturer’s instruction, and total RNA was isolated by phenol chloroform extraction followed by ethanol washes. Total RNA was reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Promega) in the presence of random hexamer primers.

Polymerase chain reaction (PCR)

Thermostable DNA polymerases were used for the amplification of DNA as described (Saiki et al., 1988, Bej et al., 1991). Two types of PCR amplifications were used in this study: standard PCR for the amplification of DNA fragments up to 3 kb and Mid-range PCR for the amplification of fragments up to 9 Kb in size.

Standard PCR

These were done in 50 µl reaction volumes as shown in Table 15. Optimal annealing temperatures for primer pairs relative to a target template were obtained using the Oligo 4.0 software. PCR was performed on an MJ Research PT-200 DNA machine according to the thermal profile on Table 16.

Reagent

Volume (µl)

Forward primer (10 pmol/ul)

1

Reverse primer (10 pmol/ul)

1

dNTP mix(40 mM)

1

DNA template

1 – 5 (between 100 to 500 ng)

10 X reaction buffer

5

HPLC water

ad 50

Taq polymerase (5 U/ul)

0.5

Tab. 15: Pipetting scheme for standard PCR

Phase

Temperature (°C)

Duration

Cycles

Denaturation

94

2 min

1

Annealing

53-60

40 s

Elongation

72

1 min per 1 kb

Denaturation

94

20 s

30

Annealing

53-60

40 s

Elongation

72

1 min per 1 kb

Denaturation

94

20 s

1

Annealing

53-60

40 s

Elongation

72

15 min

4

Tab. 16: Thermal profile for standard PCR

Mid-range PCR

This PCR technique was used for the amplification of genomic DNA fragments between 3 and 9 kb (Barnes et al., 1994; Cheng et al., 1994). These were done using the Peq Lab mid-range PCR system as follows:

Reagent

Volume (µl)

Forward primer (10 pmol/ul)

2

Reverse primer (10 pmol/ul)

2

dNTP mix(40 mM)

1.75

DNA template

1 – 5 (between 100 to 500 ng)

10 X reaction buffer

5

HPLC water

ad 50

Mid-range polymerase (5 U/ul)

0.5

Tab. 17: Pipetting scheme for Mid range PCR

PCR was performed on an MJ Research PT-200 DNA machine as follows:

Phase

Temperature (°C)

Duration

Cycles

Denaturation

94

2 min

1

Annealing

53-60

40 s

Elongation

68

1 min per 1 kb

Denaturation

94

20 s

30

Annealing

53-60

40 s

Elongation

68

1.5 min per 1 kb

Denaturation

94

20 s

1

Annealing

53-60

40 s

Elongation

68

15 min

4

Tab. 18: Thermal profile for mid range PCR

Southern blotting Digestion, electrophoresis, and blotting of genomic DNA

Genomic DNA (20 μg) were digested with different restriction enzymes in 100 μl reaction volumes as described in section 4.2.7, and electrophoresed in 0.7% agarose gels (section 4.2.2). The DNA was capillary-blotted onto a nylon membrane at room temperature in 20x SSC overnight (Sambrook et al., 1989). The membrane was then baked at 80°C for 2 hours to fix the DNA onto the membrane.

Radioactive labeling of A. viteae chitinase probe

A. viteae chitinase PCR fragments (25 ng) were labelled with 50 μCi α-³²P dCTP using the RadPrime DNA labelling system (Life Technologies). The pipetting scheme was as follows:

Item

Volume (μl)

A. viteae chitinase PCR fragment (25ng)

5

dATP (500 μM)

1

dGTP (500μM)

1

dTTP (500μM)

1

Random primers solution

20

α-³²P dCTP (3000 Ci/mmol, 10mCi/ml)

5

Distilled water

16

Klenow fragment

1

Tab. 19: Pipetting scheme for radioactive labeling of probes

The mixture was incubated at 37°C for 10 minutes and the reaction stopped. The radioactive probes were purified using Sephadex^TM MicroSpin G50 columns (Amersham Biosciences Europe GmbH). The labelling efficiency was about 30%.

Hybridisation and detection

Membranes were washed in 6x SSC at room temperature and prehybridised at 65°C in 10 ml hybridisation buffer (6x SSC, 5x Denhardt’s reagent, 0.5% SDS, 100μg/ml salmon-sperm DNA) for 1 hour. The probe was denatured by boiling for 2 min in a water bath and added to the hybridisation buffer. Membranes were hybridised overnight at 65°C in a roller bottle. Stringent washes were done in 2x SSC, 0.5% SDS for 5 minutes at room temperature, 2x SSC, 0.1% SDS for 15 minutes at room temperature, and 0.1% SSC, 0.1% SDS for 2 hours at 65°C. The membranes were rinsed in 0.1x SSC, covered in Saran Wrap, and exposed to a phosphorimager plate (Fuji Film) for 3 hours, and the bands were scanned using a phosphoimager.

Microbiological methods Preparation of competent E. coli

Competent E. coli cells were prepared essentially as described by Inoue et al. (1990). E. coli were streaked on LB agar plates with antibiotics and cultured overnight at 37°C. Twelve large colonies were picked with a sterile toothpick and used to inoculate 125 ml SOB in a 1-liter Erlenmeyer flask. Flasks were incubated at 18°C with vigorous shaking (220 rpm) and the bacteria grown to an OD of 0.6 (mid-log phase). Bacteria cultures were poured into Falcon tubes and incubated on ice for 10 minutes and then centrifuged at 2500x g (3000 rpm) in an Eppendorf 5403 bench top centrifuge. The pellet was resuspended in 40 ml ice-cold TB buffer, incubated on ice for 10 minutes and centrifuged as above. The pellet was then carefully resuspended in 10 ml of TB, and DMSO added drop-wise to a final concentration of 7%. The bacterial suspension was incubated for ten minutes on ice, after which 1 ml aliquots were snap-frozen in liquid nitrogen and stored at – 80°C for up to one month.

Transformation of competent E. coli

Eppendorf tubes (1.5 ml) were pre-chilled on ice and 10 ng of purified plasmid DNA in a total volume of 20 μl were pippeted into the tubes. Competent cells were thawed, and 200 μl were dispensed into the tubes on ice. The tubes were flicked gently to mix and incubated on ice for 30 minutes. The cells were then heat-shocked by heating for exactly 45 seconds in a 42°C water bath, and then incubated on ice for 2 minutes. Room temperature SOC medium (800 μl) was added to each tube on ice. The tubes were then incubated at 37°C while shaking vigorously (220 rpm) for one hour. Antibiotic selective agar plates were plated with 50 to 150 μl of the transformation mixture and incubated overnight at 37°C. Positive controls were competent cells transformed with 10 ng pBluescript SK (-) plasmid, while negative controls were competent cells transformed without DNA.

Screening of bacterial colonies for plasmids/ recombinant plasmids

Two methods were routinely employed to identify bacteria colonies that contain the recombinant plasmids or plasmids of interest.

Blue-white screening

Most bacteria strains (e.g. JM109,DH5 alpha and XL 1 Blue), used as hosts for initial cloning of a target gene, code for an inactive carboxy-terminal portion of ß-galactosidase due to a mutation in the lac Z gene (lacZdeltaM15). On the other hand, many vectors contain the regulatory sequences and information that could be induced (e.g. by IPTG) to code for the missing amino terminal (alpha region) of the ß-galactosidase gene. Both host and plasmid encoded fragments are inactive. However, if a bacterial host is transformed with a plasmid expressing the missing amino terminal of the enzyme, both fragments associate to form an active enzyme in a process called alpha complementation. Such bacteria can be recognised because they metabolise the chromogenic substrate 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) in the presence of isopropyl-ß-D galactopyranoside (IPTG) to form blue colonies. Insertion of a target gene in the alpha peptide region coding for the ß-galactosidase enzyme results to insertional inactivation of the alpha peptide. Bacteria carrying plasmids with the target gene will form white colonies since they lack active ß-galactosidase. Thus, transformed bacteria were plated on LB-agar plates supplemented with 0.5 mM IPTG, 80 μg/ml X-gal and antibiotics.

Restriction analysis of isolated plasmids

In order to verify constructs of plasmids/ inserts resulting from cloning, plasmid DNA was isolated and analysed. Independently transformed bacterial colonies were picked and grown in 5 ml LB/ antibiotic overnight. Plasmid DNA was isolated (section 4.3.2) and digested (section 4.3.8) at restriction sites used for cloning of the insert DNA. The digested DNA was then analysed by agarose gel electrophoresis (Section 4.3.3).

Bacteria cultures and long-term storage of bacterial stocks

Bacteria host strains used in this study included XL 1 Blue, BL 21 (DE3), and Origami B (DE3). Bacteria were streaked on LB agar plates and grown overnight at 37°C. For long-term storage of bacteria, a single colony was grown to an OD₆₀₀ of 0.6 and 1 part was mixed with 9 parts of Hogness freezing medium (36 mM K2HPO4, 13 mM KH2PO4, 20 mM sodium citrate, 10 mM MgSO4 and 40 % glycerol). Tubes with bacteria stocks were snap-frozen in liquid nitrogen and stored at -80°C. Bacteria recovery involved scraping the surface of a bacteria stock with a sterile loop and streaking an LB plate with the appropriate antibiotics.

Expression and purification of recombinant proteins from E. coli

The pET expression system was used for prokaryotic expression of recombinant N-terminal A. viteae chitinase. In this system, target genes are cloned under the control of the T7 promoter which is not recognised by E. coli RNA polymerase. Some E. coli host strains (like BL21 (DE3) and Origami B (DE3)) have a chromosomal copy of the T7 RNA polymerase gene under control of lacUV5. Transfer of recombinant pET plasmids into such hosts results in an IPTG-inducible gene expression system. The cDNA encoding the N-terminal domain of chitinase was cloned into the pET 22 b (+) vector and transformed into Origami B (DE3 cells). The pET 22 b (+) vector has a pelB signal sequence that leads to the localisation of an expressed protein into the periplasmatic space of the E. coli host.

A single bacterial colony was picked and used to inoculate LB (100 μg/ml Ampicillin, 12.5 μg/ml Tetracyclin, 15 μg/ml Kanamycin and 1% glucose) and the culture was grown overnight at 37°C, 150 rpm. The overnight culture was diluted 1:50 in fresh LB medium and further grown at 37°C till an OD₆₀₀of 0.4. The culture was shifted to room temperature and further grown to an OD₆₀₀ of 0.6. Protein expression was induced with 1mM IPTG for 4 hours. Bacteria were pelleted by centrifugation at 6000 rpm for 15 minutes at 4°C. The pellet was then used for the extraction and purification of periplasmatic proteins and proteins in inclusion bodies.

For extraction of periplasmatic fluid, bacteria pellet from a 500 ml culture was resuspended in 30 ml 30mM Tris-HCl pH 8, 20% sucrose. EDTA was added to a final concentration of 1 mM and the suspension stirred for 10 minutes at room temperature. Cells were collected by centrifugation at 10,000xg at 4°C for 10 minutes. The pellet was then re-suspended in 30 ml of ice-cold 5 mM MgSO₄ and stirred slowly on ice for 10 minutes to release the periplasmatic proteins. The suspension was centrifuged at 10,000xg to pellet the cells and the supernatant was used for purification of periplasmatically expressed A. viteae chitinase. The insoluble fraction in inclusion bodies was purified as described below.

The pellet was resuspended in lysis buffer A, 100 ug/ml lysozyme and incubated for 30 minutes at 4°C. The suspension was then sonicated for 1.5 min, 30 cycles. After centrifugation (13,000 rpm, 20 min, 4°C), the supernatant was transferred into a falcon tube, incubated with 1 ml Nickel chelate matrix (Qiagen, Hilden) and incubated for 1hr at room temperature with constant gentle shaking. The matrix with bound protein was then loaded onto a column and washes were done with 20 ml of buffers B and C, respectively. Elution was done in 20 ml of buffer E, and collected in 1ml fractions. The fractions were analysed by SDS-PAGE for protein and the positive fractions were pooled. This pool was then dialysed in phosphate buffers with reducing amounts of urea and the protein concentration was determined. Aliquots of the protein were stored at -20°C until used.

Protein analytical methods Determination of protein concentration

Protein concentration was determined by the BCA test using the BCA kit (Pierce, Rockford, USA). The basis of this reaction is the biuret reaction: reduction of Cu²⁺ to Cu¹⁺ by a peptide bond under alkaline conditions. Chelation of two molecules of bicinchoninic acid (BCA) by the cuprous ion (Cu¹⁺) produces a water soluble complex, whose solution has a deep purple colour and an absorbance at 562 nm. A linear standard curve was made with 0.05 to 2mg BSA.

Sodium dodecly sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Analytical polyacrylamide gel electrophoresis in sodium dodecyl sulfate was performed as described by Laemmli (1970). Samples were heated for 5 minutes at 100°C in sample buffer containing 125 mM Tris-HCL, pH 8.3, 1% SDS, 2.5% ß-mercaptoethanol and 10% glycerol. Samples were applied at volumes of 10-30 μl to a 12.5% slab gel cast in a mini-gel apparatus (Hoefer). The electrophoresis was performed at 20mA with electrode buffer (25mM Tris-HCL, pH 8.3, 192mM Glycine and 0.1% SDS). The run was stopped when the bromophenol blue dye front reached the end of the gel. Protein bands were then revealed by staining with Coomassie Brilliant Blue R250, and the gels dried for a permanent record.

Chitinase activity assays

Chitinase assays were performed using 4-methylumbelliferyl ß-N, N', N’’-triacetylchitotrioside (GlcNA)₃UMB. (GlcNA)₃UMB was dissolved in reaction buffer (20mM NaPO₄, pH 6.0, 200mM NaCl, 1mM EDTA) and 95μl of 20 μM substrate solution were mixed with 5μl of enzyme and incubated for 2 to 10 minutes at 25°C. The reactions were stopped by adding 1.9 ml of 0.5 M glycine, pH 10.5. Release of free methylumbelliferone was measured by a fluorescence spectrophotometer at 360 nm excitation and 450 nM emission wavelengths. The fluorometer was scaled at the beginning of the experiments such that 20 nM substrate had a fluorescence reading of 10 units.

Immunochemical and immunological methods Western blot Transfer of proteins onto nitrocellulose membranes

Proteins separated by SDS-PAGE were immobilised onto nitrocellulose membranes by electrophoretic transfers (Towbin et al., 1979). A transfer cassette was assembled in transfer buffer using a sponge followed progressively by a 3 MM- Whatmann paper, nitrocellulose membrane (pore size 0.2 uM), SDS-PAGE gel, another Whatmann paper and finally a second sponge. The cassette was introduced into a transfer tank between electrodes so that the gel with the protein was towards the negatively charged cathode and the nitrocellulose membrane towards the positively charged anode. A constant current of 70 mA was then applied across the cassette at room temperature overnight. To determine the transfer efficiency, the membrane was reversibly stained in Ponceau S dye (0.1% Ponceau S, 7% trichloroacetic acid in distilled water). After complete washing of the Ponceau stain, unbound sites of the membrane were blocked by incubation for 45 minutes in 5% skimmed milk powder in PBS.

Immunodetection of immobilised proteins

Nitrocellulose membranes with immobilised proteins were incubated with a primary protein specific antibody for one hour at room temperature, followed by 3 x 5 minutes washes in wash buffer (PBS, 0.02% Tween 20). The membrane was then incubated in a secondary conjugate antibody for one hour followed by washes as above. Detection was done by using a substrate for the alkaline phosphatase enzyme conjugate, 5-Brom-4-chlor-3-indolylphosphate (BCIP) and tetrazolium chloride (NBT) in alkaline phosphatase buffer. All antibodies were diluted in blocking solution (5% skimmed milk powder in PBS).

Coupling of peptides to KLH

Synthetic peptides (20 amino acids long) were synthesised with two extra N-terminal cysteins and coupled to KLH (Keyhole limpet haemocyanin) to make them immunogenic. Coupling was done using the Imject maleimide activated mcKLH kit (Pierce USA) and the coupled proteins were used at a concentration of 100µg in vaccinations.

Immunisation studies

The protective potential of A. viteae N-terminal chitinase was evaluated in the Meriones/A. viteae natural host-parasite system. Eight to ten-week-old Meriones were anaesthesized with ketamin:xylazin:normal saline (1:1:8) and immunised with 25 μg recombinant A. viteae chitinase or 100 μg KLH-coupled synthetic A. viteae chitinase peptide in adjuvant. The animals were immunised three times in a two-weekly interval, after which they were challenged with 70 freshly isolated L3s by injecting in the neck region. Microfilaria load was verified at weeks 4, 8 and 12 post infection (pi), and the animals were dissected at week 12 pi for isolation of adult worms and determination of protective potential.

Two adjuvants were used for immunisation, depending on the immune reaction desired: Alum was used for humoral-mediated immune reactions, while STP was used for cell-mediated immune responses.

Bleeding of animals for production of sera

Mice and jirds were bled from the retro-orbital sinus using a microhaematocrit capillary, and the blood was stored at 4°C overnight. Blood samples were then centrifuged (10,000 rpm, 20 minutes, 25°C) to separate the sera from blot clots. Sera were stored in 100 ul aliquots at -20°C.

Parasitological methods Maintenance of the life cycle of A. viteae

The life cycle of A. viteae was maintained in Ornithodoros moubata essentially as described by Lucius and Textor, 1995.

Quantification of microfilarial load in blood of jirds

Infected Meriones were anaesthesized with ketamin:xylazin:normal saline (1:1:8) and bled from the retro-orbital sinus using a heparinised glass capillary tube. 20 μl blood were mixed with 100μl Teepol (10% in H₂O), and the microfilaria load was counted using a Fuchs-Rosenthal counting chamber.

Isolation of filariae Isolation of adult A. viteae

Meriones were anaesthetized and fully bled from the retro-orbital sinus. Following dissection of the jirds, adult A. viteae were isolated from the subcutaneous and intramuscular tissues, the iunguinal and subscapular regions and some times in the thoracic chamber. Animal carcasses were then incubated in normal saline (0.9% NaCl) overnight to allow the rest of the worms to wander out.

Isolation of L3 from the vector Ornithodoros moubata

L3s were isolated using a Baermann apparatus comprising of a funnel with a tap. The inner part of the funnel was covered with bandage and filled with Ringer's solution. Ticks were cut medially and briefly rinsed in a Petri dish with Ringer’s solution to remove rests of blood meal and loose tissue. The ticks were incubated in warm Ringer’s solution in the funnels for 1 hr for the L3s to migrate into the solution. The tap was then opened to collect the L3s in 50 ml falcon tubes. The larvae were amply washed in Ringer's solution and rinsed in RPMI if they were to be used for culture.

Isolation of uterine microfilariae from gravid female worms

Adult female A. viteae worms were isolated on day 90 post-infection (average length, 6 cm; embryogenesis starts on day 28 post-infection) and cut into 3mm segments in Ringer's solution. Developmental stages were separated from the worm segments by filtration. The stages obtained included: oocytes attached to the rachis of the ovary, fertilized eggs, multicellular stages, invaginated stages, pretzel and ring stages, and elongated microfilariae.

Computer analysis and statistical methods Analysis of DNA sequences

Genomic DNA sequences obtained from primer walking were assembled into contigs by using the program MacVector 7.2 (Accelrys, USA). Assembled sequences were fed into Artemis^® (www.sanger.ac.uk/Software/Artemis/) for visualisation of sequence features, its six-frame translations and exon-intron prediction. Exons thus predicted were verified by comparison to a cDNA sequence and by further analysis using Netgene (www.cbs.dtu.dk/services/NetGene2/). Brugia malayi genomic chitinase DNA sequences were retrieved from the server for the B. malayi genome project at while EST sequence analysis was done at the server of the Filaria genome project (http://nema.cap.ed.ac.uk/fgp.html).

Statistical analysis

The worms recovered from immunised and non-immunised Meriones in immunisation experiments were compared using the Mann-Whitney U-test at the 95% significance level (p<0.05).