Supplementary material for Eichmüller et al. (January 9, 2001) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.021386498

Material and Methods

Immunoscreening.

 The immunological screen was performed essentially as described (1). All sera were diluted in Tris-buffered saline (TBS; pH7.5, containing 0.2% dry milk powder) and preabsorbed with Escherichia coli proteins (mechanically disrupted or lysed by phages without insert).

E. coli

 transduced with recombinant l-ZAP phages were plated onto NZY-agar at a concentration of 2000 plaques/plate and expression of recombinant protein was induced with isopropyl b-D-thiogalactoside. Plates were incubated at 37°C over night and protein was blotted to nitrocellulose membranes for 4 hours at 37°C. The membranes were washed with TBS containing Tween-20 (0.05%), blocked with 5% dry milk powder in TBS, and incubated with serum from either patients or healthy controls at a final concentration of 1/100. Reactive proteins were detected with an alkaline phosphatase-coupled secondary antibody (goat-anti-human IgG, Fc fragment; Dianova, Hamburg, Germany) and visualized by 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. Positive phagemids were further analyzed using sera of Mycosis fungoides (n = 15) and Sézary Syndrome (n = 3) patients, as well as healthy controls (n = 10). Positive phagemids were subcloned to monoclonality and submitted to in vivo excision of the pBluescript plasmid (protocol as described by the manufacturer of the library: Stratagene). DNA was isolated with QIAprep spin miniprep following the manufacturer’s protocol (Qiagen). The size of the insert was analyzed by SmaI/KpnI digestion and gel electrophoresis. Sequences were determined using an automatic fluorescent sequencer (Model 377; Perkin–Elmer/Applied Biosystems, Forster System) and the dye terminator method according to the manufacturer's protocol (ABI PRISM Big Dye Ready Reaction Terminator Cycle Sequencing Kit, Perkin-Elmer). Primers were chemically synthesized. The sequences of the clones were completely determined on both complementary strands.

RNA Isolation.

 All cell lines were tested to be mycoplasm-free before use (Mycoplasm Detection Kit, Roche Diagnostics). Total RNA was isolated from tissues and cell lines using a variant of the guanidinium thiocyanate method (RNA-Clean, Angewandte Gentechnologie Systeme GmbH, Heidelberg, Germany) as recommended by the manufacturer. Total RNA was treated with DNAse. Briefly, RNA was diluted in 0.1 M sodiumacetate (pH 5), 5 mM MgSO4 and incubated with DNAse (Roche Diagnostics; diluted in 25 mM Tris·HCl, pH 7.6, 1% glycerol; final concentration: 1/500) for 10 min at 25°C. RNA was isolated by phenol/chloroform extraction, precipitated, and finally checked for RNA integrity by an RNA gel . cDNA was obtained using a first strand cDNA synthesis kit (Roche Diagnostics) and random primers following the manufacturer’s instructions.

Reverse Transcription (RT)-PCR Conditions.

Primers for RT-PCR were selected using either the program primer (Lincoln et al., MIT, Cambridge, MA) or the program Prime (written by Irv Edelman). The following primers (for, forward; rev, reverse primer) were selected (length of PCR product and annealing temperatures are given in parentheses):
 

GAPDH

for: gtt tac atg ttc caa tat gat tcc ac, 


rev: tca tat ttg gca ggt ttt tct aga c (638 bp, 57°C);

se1-1

for: gca aaa gca att aga cgc tac c, 

rev: cac agc cct gtt ctt ctt tag c

(596 bp, 57°C);

se2-1

(SCP-1)

for: gta cag cag aaa gca agc aac tga atg, 

rev: gga aat tgg att cta aag cag ttc ctt c

(564 bp, 55°C; as described by Türeci et al., 1998);

se2-2

for: cta tga atc caa gac caa agg c, 

rev: ctc cac ttt ggt cct tgt tag c

(539 bp, 59°C);

se2-5

for: acc cac gca gat ttg gaa tc, 

rev: agg ctg atc act ggc tgt g

(911 bp, 59°C);

se14-2

(CTAGE-1)

for: cct tat tgt aca ctg ggg ctt c, 

rev: cag aca caa gga act gaa gta acg

(620 bp, 60°C);

se14-3

for: cac tgc caa gat aga caa gca g, 

rev: gct ctt atc cag gaa gtc cat g

(256 bp, 59°C);

se20-4

for: tac agg atc tca gac ata tct cca tg, 

rev: aaa tgt ctt ccc act gca taa tag tc

(424 bp, 59°C);

se20-7

for: taa gga aac aat tca gtc aca taa gg, 

rev: ctg tag ctt agc aat ttg ttct tct g

(553 bp, 59°C);

se20-9

for: tta tga ggc tta gaa ttt caa cca c, 

rev: aaa ggc ttt caa aac att ttt caa c

(554 bp, 59°C);

se20-10

for: gta gag atc aga gag ttg tga cat ctg, 

rev: tat tac ttt tca ctg tta cac tgc tgg

(537 bp, 59°C);

se33-1

for: gcc aca gag aat gaa cca ctt aac, 

rev: gag gga cta tca gtt gct gtt tg

(692 bp, 60°C);

se37-2

for: gca tct aat aga acg cta cta cca cc, 

rev: ctg tga gct atc acc tat cct tga g

(568 bp, 60°C);

se57-1

for: gtg aca gtg acc aca gaa att ccc cc, 

rev: cac gtt tct cag agc tgc tgc tcc

(381 bp, 63°C);

se70-2

for: gct gca cag aaa acc tta ctt gtt tcc acc,

rev: ctc gta aat gca gaa atc tcc aat gcc c

(585 bp, 56°C);

se89-1

RAP140for: tcc aca gcc tat tgg ctc act tgg ac,

RAP140rev: gca cac act gct cct cca cct gac 

(RAP140: 688 bp, se89-1: 628 bp, 57°C);

se89-1rev: gcc ctt tag tgt gtc tgt aat tgg aat cag 

(in combination with RAP140for: 561 bp, 60°C).

RT-PCR was done using an MJ Research PCT-200 (Biozyme, Oldendorf, Germany) with the following settings: Denaturation for 5 min at 95°C, annealing for 1 min at variable temperature (see above), elongation for 2 min at 72°C, denaturation for 1 min at 95°C, 35 cycles, and final elongation for 10 min at 72°C. The quality of the cDNA was tested by RT-PCR using primer for GAPDH. All RT-PCRs were done in at least two independent experiments.

Northern Blot Analysis.

 RNA from tumor and normal tissues as well as cell lines were used for Northern blotting. RNA was separated on formaline/MOPS gels, checked for RNA integrity, and blotted onto Nylon membranes. Products obtained by RT-PCR as described above were labeled using the random primed DNA labeling kit (Roche Diagnostics) and 32P-dCTP. Membranes were prehybridized (2 hr at 63°C), incubated with specific probes overnight at 60°C, and subsequently washed in progressively higher stringency with the final wash in 0.2 SSC/0.1 SDS at 65°C. Autoradiography was conducted at –80°C for up to 2 weeks using Kodak XR-Omat film and intensifying screen. Filters were stripped and rehybridized with GAPDH-probe to prove RNA integrity and comparison of RNA loading amount.

Sequence Analysis.

Computational analyses was done using the husar package from the Biocomputing Service Group at the German Cancer Research Center. All sequences were analyzed with respect to possible ORFs. Database searches were performed on EMBL and GenBank databases with BLAST programs for nucleic acids (BLASTN) as well as for protein sequences (BLASTP) using the deduced putative amino acid sequences as queries. Computational prolongation of incomplete sequences was attempted using EST-CLUSTERING (developed by Ebeling and Glatting, German Cancer Research Center), a program that aligns available EST sequences and produces a elongated contig.