MATERIALS AND SPECIFIC METHODS 1 Normal controls

This work was performed on RNA from the haemopoietic cells of 11 haematologicaUy normal individuals. The RNA was extracted by a standard technique as described in section 3.3 and stored at -80°C.

4.2.2 Reverse transcription-polym erase chain reaction

Initial RT-PCRs were perfomed exactly as described in section 3.5.2 using primer pairs G lU/G lD, G2U/G2D and G3U/G3D. Further investigation of the PCR product of the G lU/GlD primers utilised a two-round semi-nested PCR technique. The first round was again performed as described using a G1U/G2D primer combination. The product was loaded onto a 1% low-melting point agarose minigel prepared with Ix tris borate EDTA (TBE) buffer and run at a constant 45mA until adequate separation of the bands had occurred. The cDNA fragments were then excised under ultra-violet light visualisation in as small a volume of agarose as possible and placed in a 7Q0C waterbath to liquify. 2jal of the melted agarose containing the target DNA was used unmodified as the template for a IOOjjI second round PCR containing Ix Taq polymerase buffer, 2.25mM magnesium, 0.2mM dNTP, 200ng each of GIU and G ID primers and 2.5U Taq polymerase (Promega, Madison, Wisconsin, USA). 35 amplification cycles were performed as before (950C for 30s, 6Q0C for 30s, 720C for 60s) after an initial dénaturation stage of 95^C for 5 mins and the program completed with a 5 minute extension stage at 720C. These products were again run out on a 1 % low melting point agarose minigel and the individual bands excised.

4.2.3 PCR of genomic DNA

One ug genomic DNA derived from cells of the erythroleukaemic ceU line TF-1 was amplified in a lOOjil GlU/GlD PCR reaction. The conditions were as described above except for an increased extension time of 2 minutes at 72<>C per cycle to allow for the increased fragment length due to the encompassed intronic sequence.

4.2.4 Cloning and sequencing of PCR products

Cloning of PCR products was performed as described in section 3.6. Sequencing was approached in three different ways: (i) direct sequencing of the purified DNA product from a two-rounded semi-nested PCR using the fmoF^ DNA sequencing system (see section 3.7.1), (ii) direct sequencing of the purified product of a PCR amplification of a cloned

DNA sequence using the fnioF^ DNA sequencing system and (iii) sequencing of alkali denatured plasmid DNA containing the inserted target sequence using the 70770 sequenase^'^ version 2.0 sequencing system (see section 3.7,2).

4.3 RESULTS

4.3.1 G-CSFR isoforms in normal haemopoietic cells

RT-PCR of RNA from normal haemopoietic cells using the primer pairs G2U/G2D and G3U/G3D gave a single band on agarose gel electrophoresis. Each corresponded to the expected size predicted from the published wild-type G-CSFR cDNA sequence (Fukunaga etal, 1990b), i.e. 321 and 279bp respectively. Using primer pair GlU/GlD however two bands were obtained, a major fragment (A) of 345bp, the size predicted for the wild-type receptor, and an additional larger fragment (B) of approximately 420bp in size and comprising about 10% of the total [Fig 7]. These two products were investigated further by performing two rounds of a semi-nested PCR to increase the amplification and maintain specificity. 35 cycles using primers G1U/G2D were followed by 35 cycles with primers GIU/GID. This revealed four bands, the original two bands (A) and (B) in approximately the same proportions as before, and two additional minor bands, (C) and (D) [Fig 7]. All four fragments were sequenced, band A directly off the purified PCR products and bands B, C and D off clones prepared from these products. As expected sequence of the major band (A) was consistent with nucleotides 2(X)6-2251 of the wild-type G-CSFR (Fukunaga et d , 1990b). The largest band (B) also corresponded to a published isoform, the 81 bp insertion of exon 17’ (Fukunaga et al, 1990b) [Fig 6]. The smallest band (C) had a deletion of 82bp from nucleotide 2128 to 2209. This was at a different site to the deletion giving rise to the previously reported soluble G-CSFR and comparison with the genomic sequence showed that it corresponded to complete deletion of exon 16 (Seto et d , 1992). Sequencing of the final band, band (D) revealed a 60bp insertion between nucleotides 2033 and 2034, the boundary between exons 14 and 15.

1018— 506-517— 396— 344— 298— 220-

Fig 7 Agarose gel electrophoresis of the product o f two rounds o f PCR (G1U/G2D amplification, followed by G IU /G ID ) on cDNA from haematologicaUy normal neutrophils. Lane 1 = size marker.

The origin o f this insert w as further investigated by PCR amplification o f genomic DNA using primers G IU /G ID . This encompassed the 3 ’ 28bp o f exon 14, exons 15 and 16 in their entirety and the 5 ’ 42bp of exon 17 in addition to the three associated introns and gave a fragment o f approximately 1200bp in size. Sequencing from this fragment showed that the 60bp insertion was immediately upstream of, and in continuity with, exon 15 and resulted from the use o f an alternative donor splice site in the intron (Seto et al, 1992). We have called this 60bp sequence exon 15’ by analogy to the exon 17’ o f the previously published isoform. The relationship of these four isoforms to the genomic sequence is depicted in Fig 8. 6 0 bp insertion 82 bp deletion

I i

m

Fig 8 The intron-exon organisation of the G-CSFR showing the sites o f alternative splicing resulting in the two novel mRNA transcripts described.

4.3.2 G-CSFR isoform expression in acute myeloid leukaemia

The relative mRNA expression of the four different G-CSFR isoforms, (A)-(D), was evaluated in RNA from blast cells of 40 patients with AML. Both single round RT-PCR using the primers GIU and G ID and the semi-nested PCR approach described above (using primers G1U/G2D followed by primers GIU/GID) were performed but with the reduced number of 25 cycles per round in order to maintain semi-quantitative amplification. In all the patients the level of expression of the two major isoforms (A and B) was the same as the TF-1 and haematologicaUy normal RNA samples without any gross increase in the level of the two minor isoforms (C and D) being evident.

4.3.3 G-CSFR isoform expression in Kostm ann’s syndrome.

The same experiment was performed on mRNA extracted from the PB or BM of nine patients with KS. The relative levels of expression of the receptor isoforms in each patient was again the same as in the normal controls.