Materials

Commercial Kits and Enzymes

Collagenase A

Biochrom, Berlin

RNase A

Boehringer Mannheim, Mannheim

T4 DNA ligase

New England Biolabs, USA

DNA restriction enzymes

New England Biolabs, USA

Mid-range Polymerase

PeqLab, Erlangen

Sawady Taq Polymerase

PeqLab, Erlangen

Lysosyme

Sigma, Deisenhofen

Qiagen protein purification kit

Qiagen, Hilden

Nucleospin DNA extraction kit

Clontech, Heidelberg

Nucleospin plasmid prep kit

Clontech, Heidelberg

BCA protein assay Kit

Pierce, USA

KLH protein conjugation kit

Pierce, USA

PGEM-T^TM Cloning kit

Promega, Madison, USA

Random Primers DNA labeling System

Life Technologies, Karlsruhe

Nick Translation System

Life Technologies, Karlsruhe

Laboratory Equipment and consumables

Chromatographic columns

Pharmacia, Freiburg

Agarose gel electrophoresis apparatus

AGS, Heidelberg

SDS-PAGE apparatus

Pharmacia, Biotech

Video documentation apparatus E.A.S.Y.RH

Herolab, Wiesloch

Peltier Thermal Cycler, PTC-200

MJ-Research, Massachusetts

Bandelin Sonoplus HD 200 sonicator

Berlin

Spectrophotometer

Eppendorf

DE52 powder ( DE52 number 4057-050)

Whatman, USA

Chromatography paper (3MM)

Whatman, USA

Dialysis membrane

Serva, Heidelberg

Nitrocellulose membrane (0.2 µm)

Schleicher & Schuell

Cell culture plates (Flat & round bottom)

Costar, Bodenheim

HPLC water

Roth, Karlsruhe

CSPD

Boehringer; Mannheim

Synthetic oligonuceotides

Oligogonucleotides were delivered as lyophilised powders and dissolved to final concentrations of 100 pmol/μl in water as per manufacturer’s instruction. This stock solution was then dissolved 1:10 in water to obtain working solutions with concentrations of 10 pmol/ μl. A total of 10 pmol primer was used in standard PCRs, while 20 pmol were used in long range PCR.

PCR Primers Primers used in long range PCR

Name

Sense

Clone

Sequence

T7- long

all

GTA ATA CGA CTC ACT ATA GGG C

T3-long

all

AAT TAA CCC TCA CTA AAG GGA AC

F3

+

all

CTACGTTCGCGGATGTTAC

F4

+

all

TGGGCTTGAAAGTGAGGTAAG

R4

-

all

TGTTTGCTCACTTTCAAGCCC

R7

-

12

TCCCAACTGCCGTGTAAATCA

R8

-

12

CATTTCCGACAGTAATACGAT

R5

-

1

CCGGCAGAAATACAATGCTTG

Primers used for standard PCR

Sequencing primers Universal sequencing primers Primers on pGEM-T vector

Name

Sense

Sequence

SP6

-

ATTTAG GTG ACA CTA TAG

T7

+

TAA TAC GAC TCA CTA TAG GG

Primers on pBluescript SK (-) vector

Name

Sense

Position

Sequence

T3

-

772-791

ATTAACCCTCACTAAAGGGA

T7

+

626-645

TAATACGACTCACTATAGGG

M13 forward

+

603-620

GTA AAACGACGGCCAGT

M13 reverse

-

811-828

CAGGAAACAGCTATGAC

Primers used for sequencing A. viteae chitinase genomic clone 1

Name

Sense

Position

Sequence

F3

+

5679-5698

CTACGTTCGCGGATGTTAC

F3walk

+

6107-6127

ACACCTATGCACTCACATTC

F4

+

8698-8719

TGGGCTTGAAAGTGAGGTAAG

F31

+

8075-8094

TCGCCATCGACAACGAATC

F5

+

8653-8674

AAGTTGTGGCAAAGGTCCATA

F6

+

6521-6542

CATCAGTTTTTGCTGTTCGTT

F7

+

8852-8873

GGACCATACGGAAAAATGATT

F7walk

+

9208-9229

CAGATAATACTGAAATGCCAG

R4

-

8698-8709

TGTTTGCTCACTTTCAAGCCC

R5

-

6025-6046

CCGGCAGAAATACAATGCTTG

R5walk

-

5473-5493

CAATAGATGCTACTCGACAG

R6

-

4837-4858

GGAATTCAATGAGTTATGCTG

R6walk

-

4280-4301

GACGTATAACACAAAGTACG

R7

-

7772-7792

TCCCAACTGCCGTGTAAATCA

M13 forward

-

vector

GTA AAA CGA CGG CCA GT

R3n1

-

12355-12376

TCAGGCGCTAAGAAAAGAGAG

R3n1walk

-

11550-11573

TAAGGCTCATTTAACTTCGATAT

R3n2

-

10722-10743

TGGACCATTTCGGGTTTTCTG

Rur6

-

3706-3727

ATTCTTTGCACCGCNTGTTTC

Rur6walk

-

2948-2968

TAGGTAAAGCGAAAGAGCTG

Rur6.1

-

2217-2239

GTGAAAATGCGGAAACGGAATA

Rur6.1walk

-

1392-1412

ACTTCCTGAGGCTCTCCCTG

Primers used for sequencing A. viteae chitinase genomic clone 9

Name

Sense

Position

Sequence

F4

+

6645-6666

TGGGCTTGAAAGTGAGGTAAG

F>e10_9

+

8187-8208

GGAGAGCCTCAGGAAGTAGTG

K9_4pups

-

9414-9435

ACTAGGAAATTGGGAAAATACAAG

Rb4_4pupstr

+

10265-10286

TGCACTGTCCCATTTATTGTC

K9_3pup.

-

10516-10537

CTTTGTACCGCTTGTTTCTTGTT

M13 forward

+

vector

GTA AAACGACGGCCA GT

M13 reverse

-

vector

CAGGAA ACAGCT ATG AC

K9-FV

+

780-801

GGAAATGCAAATACAAACGGC

K9-RV

-

12925-12946

AGGATGTGAGTGCATAGGTG

K9-V1

-

11359-11380

GTACTTTATCAAACGTACCGT

K9-V2

-

9973-9993

GAAATAAAATCAGGAAGTTGC

K9-V3

-

8632-8650

TCAATTCATTTGACTGCCAC

K9-V4

+

2228-2249

CGAATATTCGTTTATCCATTG

K9-F1

+

3773-3795

GTAGGAAATGTCACATAACATC

K9-F2

+

4408-4427

CACTGATCGATGTAATATGC

K9-F3

+

5227-5240

CTGTGACGATCACTACCAGC

K9-F4

+

6117-6138

GTGAATAGCAACTTCTTTAGC

Primers used for sequencing A. viteae chitinase genomic clone 12

Name

Sense

Position

Sequence

F3

+

1374-1372

CTACGTTCGCGGATGTTAC

F3.walk12

+

2108-2129

GACGAAGATACCGAATGGTC

F4

+

4587-4607

TGGGCTTGAAAGTGAGGTAAG

F5

+

4542-4562

AAGTTGTGGCAAAGGTCCATA

F5.1

+

5288-5309

TACGTGCATCAAACACAGTC

F8

+

4130-4151

TGGCAAGGAAACGGTGGATAA

R4

-

4588-4608

TGTTTGCTCACTTTCAAGCCC

R7

-

3662-3683

TCCCAACTGCCGTGTAAATCA

R8

-

1709-1729

CATTTCCGACAGTAATACGAT

R13

-

543-564

AACCACGCTGAAGCCAAAATA

R13.1

-

950-971

GTAAGACTTGTACACTTCTG

F13

+

5836-5857

GTTTATTTCCGCATCACAGTG

Plasmids Cloning plasmids Cloning plasmid for genomic inserts: pBluescript (Stratagen)

TA- overhang vector for cloning PCR products

Source: Promega

Plasmids harbouring genomic and cDNA inserts: λ Dash II and λ Zap, respectively

Lambda dash II replacement vector map (source: Stratagene)

Lambda Uni-Zap XR insertion vector

Expression vector:Vector maps of pET 22 b(+) expression vectors (Novagen)

Buffers and solutions Molecular biology Buffers for separation, purification and storage of DNA

Buffers for purification of plasmids: CTAB miniprep

Buffers for purification of lambda DNA

Buffers and reagents used for screening of libraries

Buffers and reagents used for Southern blotting

Media and buffers for E. coli

Protein and immuno- chemistry SDS-PAGE

Western blot

Buffers for native purification of N-terminal chitinase with Ni²⁺-NTA

Buffers for denaturing purification of N-terminal chitinase with Ni²⁺-NTA

Synthetic peptides

Buffers and solutions for chitinase enzyme activity assay

Stock solutions

Antibiotic stock solutions

E. coli host strains and plasmids

Databanks, softwares and online services

Blast server for ESTs:

Sequence analysis:

Brugia malayi genome:

Filaria genome Project:

Softwares

Genomic DNA analysis, Artemis: www.sanger.ac.uk/Software/Artemis/

Clustalw: http://www.ebi.ac.uk/clustalw/

Netgene: www.cbs.dtu.dk/services/NetGene2/

MacVector 7.2: Accelrys, USA

Oligo 4.0: