The cDNA for human GDNF was cloned by RT-PCR from the fetal brain RNA and ligated to TA cloning vector (Invitrogen) according to manufacturer’s protocol. Several clones were grown, isolated and sequenced in both directions.
Cloning of human and rat GFRa -2 cDNA
Several human clones similar to GFRa -1 were found in the EST database search. The rat cDNA was cloned from adult rat hippocampus library and the 5’-end of the human cDNA was amplified by PCR from human fetal cDNA. The cloning procedures are described more in detail in (II).
The probes for in situ and Northern hybridizations of human samples were synthesized from cDNA inserts of human GDNF (636 bp), GFRa -1 (2537 bp, a kind gift from Dr. G.Fox, Amgen), GFRa -2 (1490 bp) and mouse ret (714 bp) clones. The probes for FISH were the human 1490 bp GFRa -2 cDNA and a mouse 10 kb genomic fragment containing the initiation ATG-codon.
Fluorescent in situ hybridization
The fluorescent in situ hybridization of metaphase chromosome lymphocytes was performed using the 1490bp human cDNA and 10kb mouse genomic probes as described in (II).
The detection of the GFRa -2 mRNAs from different human tissues was done by hybridizing radiolabeled GFRa -2 probe to Human and Human Fetal Multiple Tissue Northern Blot filters as described in (II).
Human recombinant GDNF was purchased from Promega and PeproTech Inc. Baculovirus -produced rat GDNF was a gift from Dr. Carlos Ibáñez. The antibodies to Ret and phosphotyrosine were from Santa Cruz and Transduction Laboratories, respectively.
Receptor tyrosine phosphorylation assay
The immunoprecipitation and Western blotting of the Ret protein with anti-Ret and anti-phosphotyrosine antibodies are described in detail in (I) and (II).
The tissues of patients were taken from autopsy or blood samples with the full ethical permission of the patents or the hospital in question (Hospital for Children and Adolescents in Helsinki, Karolinska Institutet in Stockholm and Turku University hospital). The tissues were treated as described in (III-V).
In the animal experiments tissues from Sprague-Dawley and Wistar rat and mouse embryos were used. In these experiments, the animals were mated overnight and the next day was defined as embryonic day 0 (E0).
Mouse antibodies to human p75NTR (Boehringer) were used at 1:10 dilution to stain acetone-fixed cryosections of placenta, kidney and spleen of 3 Meckel syndrome patients and 3 age-matched controls (V). The staining was visualized by FITC-conjugated antibodies to mouse IgG.
In situ hybridization was performed as described (Wilkinson and Green, 1998), with slight modifications (Kallunki et al., 1992) that are described in (III and IV).
The extraction of polyA mRNA from the fetal and newborn colon, and the RT-PCR protocol are described in more detail in (III).
The tissues for organ cultures were microdissected and cultured in Trowell-type dishes as described in (IV).
SSCP analysis with the radiolabeled PCR fragments was performed as described (Orita et al., 1989) with some modifications (Suomalainen et al., 1992).
The sequencing of direct PCR fragments and their cloned inserts of patient samples were done in the DNA synthesis and sequencing core-facility of the Institute of Biotechnology. Different samples were sequenced either in ALF or ABI sequencers according to the current protocol for either solid-phase or cycle sequencing.
The mutation frequency analysis by minisequencing was performed as described (Syvanen et al., 1993).
Radiation hybrid mapping panel provided by Research Genetics was utilized to order the genes of NGFR and HOXB6 as well as the polymorphic microsatellite markers D17S806, D17S1607, D17S957, and D17S807 as described in (V). Radiation hybrid mapping data was analyzed using multipoint maximum likelihood approach and the FORTRAN programs RH2PT and RHMAXLIK from the package RHMAP version 2.01 (Boehnke et al., 1991). In multipoint analysis we carried out branch and bound strategy assuming equal fragment retention as well as allowing centromeric effect. Breakage probabilities theta were converted to additive distances d (cR) according to the formula d = -ln(1-theta ) (Cox et al., 1990).
CFLP mutation analysis of the GDNF gene PCR products of the Parkinson’s disease patients was done according to manufacturer’s (Boehringer Mannheim) protocol.