The two murine CD33 cDNA clones, described in chapter 4, differed by an 83 bp insertion in the cytoplasmic region. The site of the 83 bp insertion in m33-B cDNA is flanked by consensus splice exon donor- acceptor sites. The 5' ends of exons are usually G or A and the 3' ends of exons C/AAG (Mount, 1982). This suggests alternative splicing between two exons (Fig. 6.5A). The 83 bp inserted fragment begins with A and ends with GAG, while the first nucleotide, after the insertion (the begining of
Figure 6.4: Im m unohistochem ical analysis o f hum an CD33 antigen in thym us
Frozen thymus sections were stained with CD33 antibody, Anti-Leu-M9 (1:10 dilution), followed by biotinylated rabbit anti-mouse Ig, streptavidin- labelled HRP and developed with DAB. The positive signal appears as brown sta in in g and is localised on cells concentrated a t th e corticomedullary junction, but is also found on cells scattered throughout the cortex and medulla
the next putative exon, position 950) is G. The last three nucleotides of the previous exon, positions 863-865 are AAG.
To clarify whether the 83 bp insertion in m33-B is a cloning artefact or represents a differentially spliced variant, we looked for the presence of this region in cells and tissues by RT-PCR. Primers, allowing the specific detection of both spliced variants were designed. These are shown schematically in Fig. 6.5A. M4 and M l are primers adjacent to the site of the insertion and so would give two bands differing by 83 hp, i.e. products of 344 bp for m33-A isoform and of 427 bp for m33-B. Bands of both expected sizes, corresponding to the two isoforms, are amplified in bone marrow, spleen, thymus, liver, brain and in the multipotential progenitor cell line, A4, the myelomonocytic cell line, WEHI3B and the myeloid cell line. M l (Fig. 6.5B). The m33-A product is the most abundant form, suggesting th at m33-B may be a minor transcript. Alternatively PCR may preferentially amplify the smaller m33-A form. Only one PCR product, corresponding to the m33-A isoform, is detected in the macrophage cell line, P388. No m33-A or m33-B transcripts were detected in EL-4, A-20 or the rem aining cell lines which were also negative in N orthern blot analysis (Fig. 6. IB and Fig. 6.5B).
The absence of m33-B isoform in the P388 cell line is also confirmed by amplification with prim ers M4 and M5 (Fig. 6.5C), M5 being an in te rn a l prim er from the inserted segment (Fig. 6.5A). This pair of prim ers was designed to generate a band of 265 bp only if m33-B is expressed and such a band is detected in all the expected RT-PCR positive samples shown in Fig. 6.5B, namely, bone marrow, spleen, thymus, liver, brain and the cell lines, WEHI 3B, A4 and M l (Fig. 6.5C). The presence of RNA in the samples is confirmed using p-actin oligonucleotide primers in RT-PCR analysis (Fig. 6.5D). The PCR analyses were performed with and w ith o u t reverse tra n sc rip tio n , in order to detect any genomic
A. 83 bp 5' CCTATCaadaGGCAGGÂAQCG- mTGTTCGATACAoa&CTCATCAG... M5 Ml 3" B. M4/M1
ÎI
O CL if) CD 3 > - CO > - X LJJ Z o ÛC LLI > z < X X LLI 1— CD < CO _ l _ CL LU < O OJ bp -1383 -1078 -872 -603 -310 -281 -271 -234 M4/M5 -310 -281 -271 -234 D. ACTIN -603 -310 -281 -271 -234Figure 6.5: RT-PCR analysis o f m urine CD33 sp licin g variants
(A) The 83 bp inserted fragment is boxed and shaded and the consensus exon splice donor/acceptor sites are in lower case and bold. The location of the prim ers, used for detecting alternatively spliced isoforms, is indicated by arrows. RT-PCR analysis w ith prim ers M4/M1 (B) and M4/M5 (C) was performed on RNA from the indicated tissues and cell lines. 20% of the amplification product were separated on 1.5% agarose gel and visualised by etbidium bromide staining. Hae III DNA fragments of 0X174 was used as size m arker. (D) The presence of RNA in th e sam ples was confirmed by amplification of a 428 bp P-actin mRNA fragment after reverse transcription. Control experim ents in the absence of reverse transcriptase produced no definitive bands (data not shown).
contam ination. No definitive signals w ere found w hen reverse transcriptase was omitted (data not shown).
The consensus 375' splice exon boundaries together with RT-PCR resu lts suggest th a t the two cDNA clones represent the products of alternative splicing. The pattern of expression of the murine CD33 splice variants, obtained by RT-PCR does not correlate with the northern blot distribution of the two size transcripts of 3.9 kb and 2.0 kb. Thus, both m33-A and m33-B isoforms are present in thym us as shown by PCR (Fig.6.5B) and only a 3.9 kb size tran scrip t is detected by northern hybridisation (Fig.O.lA). Similarly in spleen, both m urine isoforms, m33- A and m33-B are detected by PCR (Fig.6.5B), while only th e 2.0 kb transcript is observed by northern hybridisation (Fig.O.lA). This suggests th a t both murine CD33 isoforms could be derived from either of the two mRNAs species of 2.0 kb and 3.9 kb. (Furthermore, an 83 bp difference in the size of the transcripts could not be detected by northern analysis). However, final proof of which mRNA transcript represents which splice variant could be obtained by reprobing the northern blot with a specific 83 bp oligonucleotide. A detailed study of the gene structure and, ultimately, analysis of the proteins should resolve this question.