Materials and Methods

Study area:

This study was carried out in clusters of villages that represent an endemic focus of visceral leishmaniasis (VL). These villages were located in Gedaref State, eastern Sudan (Figure 1). The disease is well known among people in these villages as kala-azar. The climate of the study area is tropical and could be divided through the year into a hot dry summer (March-June), a warm wet autumn (July-October) and a moderately warm winter (November-February). The average minimum/maximum temperature of the area is 28/44^oC in summer and 18/34^oC in autumn and winter, with annual rainfall of about 600mm.

This study area is generally characterised by reduced vegetation and grass with scattered Acacia trees. The area is flat and composed of cracked alluvial clay soil. People usually live in huts constructed of wood, bamboo and grass (Figure 2). The villages are surrounded by large scale sorghum and sesame fields. Inhabitants mainly work as farmers, woodcutters, and shepherds. Many nomadic tribes roam the area.

Samples collection:

Clinical samples were collected from 100 patients suspected of having kala-azar (patients in whom parasites were not demonstrated microscopically, but who had clinical symptoms and signs suggestive of (VL) or post kala-azar dermal leishmaniasis (PKDL). These patients had at least one of the following features: fever for more than two months, left upper quadrant abdominal pain lymphadenopathy, splenomegaly, wasting or nodules and papules on the skin in case of PKDL. When kala-azar was suspected, inguinal lymph node aspiration was performed and Giemsa-stained thin films were examined for Leishmania parasites; if negative or when no palpable lymph nodes were present, a bone marrow aspiration was performed and the marrow was also microscopically examined. In case of PKDL, skin smear was examined.

Figure 2: A picture of the endemic villages showing the construction of living places and the cracked clay soil.

The clinical samples were collected from Gedaref hospital (the main referral hospital in the area), Gedaref clinic (Gedaref State, eastern Sudan, 411 km from Khartoum) as outpatients and from kala-azar suspected patients in their villages between April 1997 and November 1998. All patients were originating from an endemic area in eastern Sudan. In total 71 cases were found to be microscopically positive. Only the microscopically confirmed kala-azar cases (patients in whom Leishmania amastigotes were demonstrated by microscopy on either lymph node or bone marrow aspirates) were included in this study for molecular characterization purposes. The remaining 29 were included for diagnostic purposes to compare the microscopic method with the polymerase chain reaction (PCR) method for detection of Leishmania parasite.

About 30µl from inguinal lymph node, bone marrow aspirates or skin scraping were collected and spotted on Whatman filter paper #3. Each filter paper sample was stored in a separate polyethylene bag at room temperature for further molecular biological analysis. Five samples were collected from lymph node aspirate from Sudanese patients who had diseases other than VL and were used as negative control.

Forty from the 71 microscopically positive aspirates were simultaneously attempted for culturing. In this case part of the aspirate was also aseptically inoculated into 2 tubes containing NNN (1.4% agar, 0.6% NaCl) and Difco 4N (4% difco 4N blood agar) media, respectively. These media were prepared as described by Evans (1989). 10 ml of defibrinated rabbit blood containing 100 µl gentamicin solution were added to 100 ml of the previous melted medium. 2 ml of either NNN or Difco 4N media were distributed into small sterile tubes, then kept in sloped position until the agar solidified. The tubes were stored in 4^oC until use. Just before inoculation 200 µl of 0.9% NaCl were added to each tube. The cultures were kept at ambient temperature 25^oC and transported to Institüt für Mikrobiologie und Hygiene, Humboldt-Universität zu Berlin, Germany for further sub-culturing in RPMI 1640 medium supplemented with 15% fetal bovine serum and 100 µg/ml penicillin and 100 µg/ml streptomycin. These cultures were sub-cultured weekly (1 ml from the previous culture plus 10 ml from the RPMI medium).

Structural questionnaire filled in by; direct interview for each patient was used for collection of demographic and clinical information. This questionnaire covered the following topics: name of the patient, patient code, sex, age, tribe, residence (usual place of residence and present residence), date of isolation of the parasite, clinical symptoms, microscopy test and stage of treatment.

DNA isolated from further 18 Leishmania donovani strains used in this study were obtained from the collection of the Royal Tropical Institute, Amsterdam. Twelve of them were isolated from the same above-mentioned endemic study area in eastern Sudan (Gedaref State). Six were WHO references namely, MHOM/KE/85/NLB323, MHOM/IN/71/LRC-L51a, MHOM/CN/??/Wangjie1, MHOM/SD/75/LV139, MHOM/SD/68/1S and MHOM/SD/62/LRC-L61.

DNAextraction: DNA extraction from clinical samples spotted on filter papers:

DNA was isolated as described previously by Meredith et al. (1993). Briefly, filter papers with spotted biological material (Lymph node, bone marrow aspirates and skin smear) were punched out with a paper puncher. After each sample was obtained a clean sheet of paper sprayed with 90% alcohol was punched 10 times in order to prevent DNA contamination from one sample to the next. Two punched out discs (approximately 15 µl of aspirate) were placed in 250 µl lysis buffer (50 mM NaCl, 50 mM Tris- HCl; 10 mM EDTA pH 7.4; 1% {vol/vol} Triton X-100; 200 µg of Proteinase K per ml) and incubated in water bath for 3 hours or overnight at 60^oC. Thereafter the mixture was subjected to phenol -chloroform extraction. An equal volume of bufferd phenol was added, shaked gently for 10 minutes and centrifuged at 13000 rpm. The upper water phase was then transferred to a new 1.5 ml eppendorf tube containing an equal volume of chloroform-isoamyl alcohol (24:1), shaked and centrifuged as mentioned above. Again the upper water phase (±250 µl) was extracted and for precipitation 1/10 volume of 3M NaAc and 2 volumes of absolute ethanol were added. The tubes were left overnight at - 20^oC. Thereafter, samples were centrifuged for 15 minutes at 12000 rpm. DNA pellets were washed with 70% ethanol then dried using speed vacuum dryer (Speed Vac, Savant, Hicksville, NY, USA) for 5 minutes. The dry pellets were then resuspended in 50 µl TE buffer (10mM Tris; 1mM EDTA pH 7.5) and stored at 4^oC until use.

DNA extraction from cultured Leishmania:

DNA was isolated from 8 successful cultures as described previously by Schönian et al. (1996) with slight modifications, 2 ml cultured cells were suspended in 5 ml Net buffer (50 mM NaCl; 10 mM EDTA; 50 mM Tris- HCl pH 7.4). Sodium dodecyl sulphate (SDS) was added to a final concentration of 0.5% and the mixture was shaked until the solution was viscous. RNase was added to a concentration of 100 µg/ml and the mixture was incubated in a water bath for 30 min at 60^oC. Proteinase K was added to a final concentration of 100µg/ml and the samples were incubated again at 60^oC in a water bath for 3 hours or overnight. Then samples were subjected to phenol/chloroform extraction, precipitation with NaAc and ethanol and redissolution in 200 µl TE buffer as described above for extraction of DNA from filter papers. The DNA concentration was measured spectrophotometrically using UV/ Visible Spectrophotometer (Pharmacia LKB. Ultrospec 111).

PCR amplification: Internal transcribed spacer (ITS):

The entire ITS region was amplified with the following leishmania specific primers: LITSR (5^’-CTGGATCATTTTCCGATG-3^’) and LITSV (5^’-ACACTCAGGTCTGTAAAC-3^’). No amplification products were observed when human, Trypanosoma cruzi, Escherichia coli, Candida albicans, Trychophyton terrestre and Microsporum audouinii DNA was used as template in this PCR approach. The ITS1 and ITS2 regions (see Figure 3) were separately amplified with the primerpairs L5.8S (5^’- TGATACCACTTATCGCACTT-3^’)/LITSR and L5.8SR/ (5^’-AAGTGCGATAAGTGGTA-3^’)/LITSV; respectively. Primers were synthesized commercially (TIB-MOLBIOL, Berlin, Germany).

Figure 3: The position of the internal transcribed spacer (ITS) in the ribosomal operon amplified with leishmania specific primers. Primer sequences are given in the text.

For SSCP and sequence analysis the two parts of the ITS2, A and B (Figure 3) were amplified separately using the primer combinations LIS2MR (5^’- AGAGTGCATGTGTGTAT-3^’)/ L5.8SR and LIS2MV (5’-ATACACACATGCACTCTC-3^’)/ LITSV, respectively.

Amplification reactions were performed in volumes of 50 µl. 3 µl DNA or 1 µl DNA (for nested PCR) were added to a PCR mix containing 200 µM of each dNTP (Pharmacia Biotech, USA); 1x PCR buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1.5 mM MgCl₂); 2 U Taq polymerase (Perkin Elmer, Roche Molecualr System, USA) and 25 pmol of each primer. Samples were overlaid with sterile, light mineral oil (Sigma, Deisenhofen, Germany) and amplified as follows: initial denaturation at 95^oC for 2 min followed by 34 cycles consisting of denaturation at 95^oC for 20 sec, annealing at 53^oC for primer pairs LITSR/LITSV; LISTR/L5.8S and LITSV/L5.8SR or at 57^oC for primer pairs LITSV/LIS2MV and L5.8SR/LIS2MR for 30 sec and extension at 72^oC for 1min. This was followed by a final extension cycle at 72^oC for 6 min.

PCR was run in Robocycler Gradient 40, Stratagene. Amplification products were subjected to electrophoresis in 1% agarose NA (Pharmacia Biotech AB, Uppsala, Sweden) at 100 Volts in 0.5x TBE buffer (0.023 M Tris-borate, 0.5 mM EDTA) and visualized under UV light after staining for 15 min in ethidium bromide (0.5 µg/ml). One kilo base pairs (1kbp) DNA ladder (Life Tech, USA), was used as a molecular size marker. Amplified PCR products were documented by photography (on Polaroid films, USA) or by a camera (Gene Eagle eye 11, Stratagene, Heidelberg).

Nested PCR was performed to obtain sufficient PCR products for subsequent single-stranded conformation polymorphisms (SSCP) analysis or sequencing. ITS1 and ITS2 were amplified by nested PCR from previous ITS PCR products and, ITS2 A and B from ITS2 products.

Major surface protease msp (gp63) gene:

To design primers for the amplification of gp63 sequences, sequences of L. donovani, L. infantum, L. chagasi and L. major as well as of different classes of gp63 genes (S, L & C) that were submitted to Gene Bank were aligned. Then primer sequences were designed from the highly conserved regions (with close annealing temperature, according to the equation {4(G+C) +2(A+T)}-3 to 5^oC across the above mentioned species. Part of the coding region of the gp63 gene (from position 445-461 to position 1802-1817) was amplified with the following primers: gp63-1 (5^’-TCCACCGAGGACCTCACCGA-3^’) and gp63-6 (5^’-CTGGCACACCTCCACGTACG-3^’). For SSCP and sequence analysis (Figure 4), this part was divided into 3 parts X (from position 445-461 to position 869-887), Y (from position 850-868 to position 1209-1224) and Z (from position 1189-1204 to position 1802-1817), which were amplified separately using the primer combinations gp63-1/gp63-2 (5^’-GTCGTACCGCGACGCAATGT-3^’); gp63-3(5-ACATTGCGTCGCGGTACGAC-3^\)/gp63-4 (5^’-GTAGAAGCCGAGGTCCTGGA-3^’) and gp63-5 (5^’-TCCAGGACCTCGGCTTCTAC-3^’)/GP63-6, respectively. These primers were synthesised commercially (TIB-MOLBOL, Berlin, Germany).

Figure 4: Schematic presentation showing the division of gp63 gene into 3 parts. Primer sequences are given in the text.

Amplification reactions were performed in volumes of 50 µl. 3 µl DNA were added to a PCR mix containing 200 µM of each dNTP (Pharmacia Biotech, USA); 1x PCR buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1.5 mM MgCl₂); 2.5 U Taq polymerase (Perkin Elmer, Roche Molecular System, USA) and 10 pmol of each primer. Samples were overlaid with sterile, light mineral oil (Sigma, Deisenhofen, Germany) and amplified as follows; initial denaturation at 94^oC for 3 min followed by 30 cycles consisting of denaturation at 94^oC for 30 sec, annealing at 63^oC for primer pairs gp63-1/gp63-6 and gp63-1/gp63-2; at 64^oC for primer pairs gp63-3/gp63-4 and at 65^oC for the primer pairs gp63-5/gp63-6 for 30 sec and extension at 72^oC for 1 min. This was followed by a final extension cycle at 72^oC for 6 min. PCR was run in Robocycler Gradient 40, Stratagene. Visualization and documentation of PCR products were performed as mentioned above for ITS region.

To obtain enough PCR products for subsequent SSCP analysis or sequencing, X, Y and Z regions were amplified by nested PCR from previous XYZ region.

Anonymous DNA markers:

Six anonymous DNA regions were amplified, using primers developed at the Institüt für Mikrobiologie und Hygiene, Humboldt Universität zu Berlin as described by (Lewin, 2000) for population genetic studies in Leishmania. These primers were synthesized commercially (TIB-MOLBIOL, Berlin, Germany). The primer sequences and the PCR conditions were shown in table (1).

Prime pair

Sequence

Primer each pmol

DNTPs

μM

DNA

ng

Annealing temp.^oC.

cycles

L413H

L413R

5^’-CTCACGCTTTGTGCTTGTGT-3^’

5^’-CAACAAGGCGTATTTCCACG-3^’

15

50

6

54

30

L0114H

L0114R

5^’-CTACCAAGAAGGGTGGCAAG-3^’

5^’-GGTGCAGTACTCGTACCTAC-3^’

60

200

8

51

34

LK413H

LK413R

5^’-ACAGACGTACACACGGGC-3^’

5^’-TCGTCTTCTTGTTGCTTGCC-3^’

15

50

6

50

31

L510H

L510R

5^’-ATAGGTTAACGGCAACGCAC-3^’

5^’-TGACAGAGACACACAACGAC-3^’

30

100

10

52

34

L0308H

L0308R

5^’-ACACCATTCACGGCAGGCAA-3^’

5^’-CCCTCAATCTCTACCTCCAC-3^’

60

200

8

52

34

L0110H

L0110R

5^’-GGCAAAGAAAAAGAGCAGCG-3^’

5^’-CTTGTCGTGCGTTGAATATC-3^’

15

100

6

50

34

Table 1: Specific primer pairs and PCR conditions (per 50 μl) for the amplification of anonymous DNA markers. One U Taq polymerase and 1x PCR buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1.5 mM MgCl2) were added into all reactions.

All amplification reactions were performed in volumes of 50 µl and amplified as follows; initial denaturation at 95^oC for 3 min followed by number of cycles depending on the primer (Table 1). These cycles consist of denaturation at 94^oC for 1 min, annealing temperature (Table 1) for 30 sec; extension at 72^oC for 1 min and a final extension at 72^oC for 5 min. Amplification reactions were performed in Perkin Elmer Thermocycler 9600. The PCR products were visualized and documented as described previously for ITS region.

Optimization of PCR protocols:

PCR protocols, applied with different primers were optimized mainly with regard to MgCl₂and primer concentration, amount of template DNA and cycling program for both direct and nested PCR. To detect DNA contamination from other samples a negative control (PCR mix plus water instead of DNA) were done. For diagnostic purposes further additional negative control (PCR mix plus DNA from patients who had diseases other than VL) was performed for diagnostic purposes. These two negative controls were performed with each set of PCR amplification reaction.

Single stranded conformation polymorphism (SSCP):

SSCP is performed by denaturing the double -stranded DNA products as follows: 10 µl of each PCR product were added to 3 µl denaturing buffer (0.01 M EDTA, 1% SDS) and 2 µl loading buffer (80% Glycerine, 0.1 M EDTA pH 8, 10 mM Tris-HCl pH 8, 0.1% Bromophenol blue). The mixture was heated to 98^oC for 15 min and immediately chilled on ice and kept at 4^oC for 15 min. These samples were loaded on acrylamide gel 350x450x0.8 mm [37.5 ml MDE-gels solution (FMC Bioproducts, Rockland, USA), 9 ml 10x TBE (10.8%Tris, 5.5% Boric, 0.02 M EDTA pH 8), 103.5 ml distilled water, 60 µl NNNN-Tetramethylene diamine (TEMED), 600 µl 10% APS (Ammonium peroxide sulphate)]. The samples were subjected to electrophoresis in 0.5xTBE (0.023 M Tris-borate, 0.5 mM EDTA) which was run in a cold room (7-8^oC) at 3 kV, 300 mV and amount of Watt and running time depending on the region under investigation (Table 2). Following electrophoresis, the gel was fixed in 1% Nitric acid for 15 min, subsequently the gel was washed in distilled water for 20 sec, then stained in 2% AgNo₃ for 25 min. After washing for 10 min in distilled water, the gel was placed in freshly prepared developing solution (3% Sodium carbonate with addition of formaldehyde up to 0.05%).

DNA region

Watt

Running time (hr)

ITS1

12

17

ITS2

27

17

ITS2 A

10

15

ITS2 B

15

20

Gp63-X

18

18

Gp63-y

14

17

Gp63-Z

25

17

413

10

10

114

10

10

K413

10

10

510

10

12

308

10

10

110

20

18

Table 2: Conditions for SSCP analysis of different PCR products

PCR- fingerprinting:

The PCR- fingerprinting technique was performed as described by Schönian et al. (1996). The following oligonucleotides which anneal to mini- and microsatellite DNA sequences were used as single primers in the PCR experiments: The core sequence of phage M13 (Huey and Hall, 1989) 5^’- GAGGGTGGCGGTTCT-3^’; the simple repeat sequence (GTG)₅ ^{5^’-GTGGTGGTGGTGGTG-3^’; and (GACA)₄ 5^’-GACAGACAGACAGACA-3^’(Ali et al., 1986) as well as the T3B oligonucleotide 5^’-AGGTCGCGGGTTCGAATCC-3^’ which was derived from an intergenic spacer of tRNa genes (McClelland et al., 1992).}

Primer

PCR-program

T3B

(GTG)₅

M13 core

(GACA)4

*ID

95^oC 2min

95^oC 2min

95^oC 2min

95°C 2min

Denaturation

95^oC 20sec

95^oC 20sec

95^oC 20sec

95°C 20sec

Annealing

52^oC 30sec

50^oC 30sec

50^oC 60sec

50°C 60sec

Extension

72^oC 80sec

72^oC 80sec

72^oC 20sec

72°C 20sec

No. of cycles

32

32

27

35

**F. extension

72^oC 6min

72^oC 6min

72^oC 6min

72°C 6min

Table 3: PCR program for PCR-fingerprinting using the primers T3B, (GTG)5, M13 core and (GACA)4. *ID: Initial denaturation. ** F: Final

Amplification reactions were performed in volumes of 50 µl containing 10 ng template DNA for the primers (GACA)₄ and T3B, 50 ng for the primer (GTG)₅ ^{and 25 ng for the M13 core primer; 1x PCR buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1.5 mM MgCl₂); 3 mM Mg-acetate; 200 µM each of dNTPs (Pharmacia Biotech, USA) and 2.5 U Taq polymerase (Perkin Elmer, Roche Molecular System, USA). The primers (GTG)₅
^{and (GACA)₄ were added to a final concentration of 5 pmol per 50 µl assay where as T3B and M13 core were added at final concentrations of 10 pmol and 25 pmol, respectively. Samples were overlaid with sterile, light mineral oil (Stigma, Deisenhofen, Germany) and amplified in a Perkin-Elmer Thermocycler 9600 as shown in table (3).}}

Samples were concentrated to a volume of approximately 20 µl using speed vacuum dryer (Speed Vac. Savant-Hicksville, NY, USA) and subjected to electrophoresis in 1.2% agarose NA gel (Pharmacia Biotech AB, Uppsala, Sweden) for 5 hours at 100 Volts in 0.5x TBE buffer (0.023 M Tris-borate, 0.5 mM EDTA). Amplification products were detected under UV light after staining the gel with Ethidium bromide (0.5 µg/ml) for 15 minutes. The gel was run once again for band sharpening at 125 Volts for 15 min. Finger printing profiles were documented by photography (on Polaroid films, USA) or by a camera (Eagle Eye 11, Stratagene, Heidelberg).

Restriction fragment length analysis (RFLA):

The entire amplified ITS region, was digested using 10 different enzymes (Alu1, Bst U1, Cfo1, Dde1, EcoR1, Fsp1, Mse1, Msp1, Nde1 and Taq1) according to the conditions recommended by the supplier. Briefly, 17 µl of the DNA were restricted by addition of 10 units of each enzyme and 2 µl of the corresponding buffer and incubated at 37^oC (with exception of Taq1 which is incubated at 65^oC) for 2 hours. Restriction products plus 3 µl loading buffer (80% glycerine, 0.1 M EDTA pH 8.0, 10 mM Tris -HCl pH 8.0) were subjected to electrophoresis in 1.3 % metaphore agarose (for fine analytical separation of small nucleic acids and PCR products, FMC Bio Products Rockland, ME, USA) for 2 hours at 80 Volts in 0.5 TBE buffer (0.023 M. Tris-borate, 0.5 mM EDTA). DNA fragments were visualized under UV light after staining for 15 min in ethidium bromide (0.5µg/ml). The gel was run again for 15 min at 100 Volts for increased resolution. Restriction products were documented by photography (on Polaroid films, USA) or by a camera (Gene Eagle Eye 11,Stratagene, Heidelberg).

Radioactive cycle sequencing: Template purification:

The quality of the sequence data depends directly on the purity of DNA products. Hence prior to sequencing, 100 µl of the PCR products were purified by loading them on 1% agarose NA gel (Pharmacia Biotech AB, Uppsala, Sweden). Electrophoresis was performed in 0.5x TBE buffer (0.023 M Tris-borate, o.5 mM EDTA). Agarose blocks containing amplified DNA fragments were cut out from the gel under UV light using sterile scalpel and purified using QIA quick gel extraction kit (QIAGEN, Hilden, Germany) according to the instructions given by the manufacturer.

Sequencing cycles:

Sequencing was performed using ThermoSequenase radiolabeled terminator cycle sequencing kit (Amersham Life Science, Inc.26111 Miles Rd., Cleveland, OH44128 USA) according to the instructions given by the supplier. Briefly, purified PCR products were amplified twice using the forward primer on the first amplification and the reverse primer on the second amplification. 7 µl purified DNA, 8 µl distilled water, 2 µl Thermo Sequenase^TM reaction buffer, 1 µl of 3 pmol solution single primer and 2 µl Thermo Sequenase^TM _{DNA polymerase were mixed together. The mixture was divided into four 0.5 ml microcentrifuge tubes, labelled A, C, G and T each one containing 4.5 µl reaction mix. To each tube the corresponding 2.5 µl radiolabeled termination reaction mix (0.5 µl of [α-³³P] ddNTP, 2 µl dGTP) was added. Then each tube was overlaid with sterile light mineral oil (Sigma, Deisenhofen, Germany). Thereafter amplification reactions were performed in Biometra- Trio-Thermoblock as follows: initial denaturation at 95^oC for 2 min, followed by 32 cycles consisting of denaturation at 95^oC for 30 sec, annealing according to the primers (the same annealing temperature used for PCR amplification of the specific region), and extension at 72^oC for 80 sec. After PCR cycle sequencing is completed 4µl from the stop buffer were added to each tube. The tubes were stored at -4^oC until use for electrophoresis.}

Preparation of the sequencing plates and electrophoresis:

Long run ranging sequencing gel glass plates (450x350mm) were cleaned twice with distilled water, and once with 70% ethanol. One of the glasses (earless glass) was wiped with acrylease (Stratagene, Heidelberg). Spacers (0.4 mm) were placed between the plates of both sides and the bottom side. The two glasses were fixed together using clips.

Amplification products were subjected to electrophoresis. Polyacrylamide gel 450x350x0.4mm (48% urea, 6% rotiphorese gel 40 [Fa. Roth, Karlsruhe], 15ml 20x Glycerol tolerant buffer, added to this 20 µl TEMED and 1 ml 10% APS) was poured into the prepared plates. Then a comb was inserted and the gel was allowed to polymerize at room temperature for 2 hours. Samples were preheated at 70^oC for 10 min before loading onto the gel to prevent formation of secondary structure. Three µl of each sample (labelled A, C, G and T respectively) were loaded on the gel. The samples were run in 1x Glycerol- tolerant gel buffer (21.6% Tris-hydroxymethyl-aminomethane GR, 7.2% Taurine, 0.4% EDTA) at 1500 Volts for 3 hours (short run), for 6 hours (middle run) and at 1000 Volts for 13 hours (long run). Then the gel was fixed in 10% glacial acetic acid for 20 min, transferred to GB002 Gel blotting paper 460x570mm, wrapped with Saran Wrap and dried in a Slab gel dryer (Savant-Hicksville, N.Y., USA) at 80^oC for 1.5 hour. After complete drying Saran Wrap was peeled off and the radiation was measured in impulses per second using a LB122 Berthold β- δ detector. The dried gel was attached to X-ray hyperfilm^TM MP (Amersham Buchler GmbH, Braunschweig, Germany) and kept in the dark room on carton file. After a time of exposure dependant on the amount of measured radioactivity (if less than 200 IPS, 10 days; greater than 200 IPS, 3 days; more than 800 IPS one day), the autoradiograph was developed using (Hyperprocessor, Amersham). The sequence was read and interpreted in both directions using the computer program for sequencing (Esee, the eye ball sequence editor, version 1.09e).