SISTEMA DE CONTROL DISTRIBUIDO

VASE, a 30 bp exon, is alternatively-spliced at the splice junction of exons 7 and 8 (see Figure 4.1). The inclusion of VASE in NCAM mRNA has b ee n dem onstrated to dow n-regulate neurite outgrow th (D o h erty et a l,

1992b). It must therefore influence the interaction of NCAM w ith either itself or other m olecules. Several characteristics of the VASE-encoded polypeptide recom m end it as a m odulator of binding strength or specificity. The VASE peptide sequence is hydrophilic in character, suggesting that it protrudes from the surface of the Ig-fold into the aqueous environm ent. Secondly w h en NCAM and im m unoglobulin sequences are aligned, the p osition of the exon 7/8 splice site in NCAM partly coincides w ith a hypervariable region in antibodies (Small et a l, 1988). As the latter is thought to be involved in specific antigen binding, it m ay be that the VASE- encoded polypeptide, by virtue of its analogous position within NCAM, has the potential to influence intermolecular contact.

VASE was originally characterised in a cDNA clone from rat brain (Small et a l, 1987) and later dem onstrated to lie within 10.5 kb o f sequence w hich separates exons 7 and 8 in NCAM DNA (Small et al., 1988). Its presence has also been dem onstrated in m ouse brain RNA (Santoni et a l,

1989b), and its sequence found to coincide with that found in rat NCAM. Initially, its inclusion was thought to be restricted to the nervous system, and as such, subject to rigid tissue-specific control. Since its discovery, a variety o f neural and muscle cell lines and tissues have b een analysed to

Figure 4.1

a ) R o d e n t s e q u e n c e o f V A S E w i t h f l a n k i n g s e q u e n c e s a n d c o r r e s p o n d i n g a m i n o a c i d s

The underlined sequence is that of VASE (Small et al., 1987; Santoni et a i, 1989) .

b ) S c h e m a t i c r e p r e s e n t a t i o n o f a l 4 0 k D a p o l y p e p t i d e e n c o d e d b y R N A c o n t a i n i n g V A S E

The five immunoglobulin-like domains are in the amino- terminal half of the molecule, as illustrated. Two fibronectin

type Ill-like repeats are found in the extracellular portion, proximal to the plasma mem brane. VASE is situated in the fourth

dom ain. K e y :

= plasma m em brane

= constitutively-spliced NCAM polypeptide = VASE-encoded polypeptide

= fibronectin type III like repeat

Figure 4.1

a)

e x o n 7 e x o n 8

5* GAA AAG GCA TCG TGG ACT CGA CCA GAG AAG CAA GAG ACT CTA 3'

Glu Lys Ala Ser Trp Thr Arg Pro Glu Lys Gin Glu Thr Leu

b )

test this hypothesis (see Table 4.1), and VASE is now know n to be present in NCAM isoforms found in rat heart tissue (Reyes et al., 1991). H ow ever, little if any was detected in myoblast and neural cell lines or tissues o f the p erip h eral nervous system (Small and Akeson, 1990). Furtherm ore, its varying levels of inclusion in different neural tissues and at different stages o f developm ent, suggest spatial and temporal control of alternative splicing. H ow ever, w ith respect to the major size classes o f neuronal NCAM, (i.e. NCAM-120, -140, an d -180) alternative splicing of VASE is th o ught to be stochastic (Andersson et a l, 1990b, Reyes et a l, 1993).

It w as therefore o f interest to examine the splicing patterns at the ex o n 7 /8 splice junction in neuroblastom a and rhabdom yosarcom a cell lines. Differences in splicing patterns betw een the tw o types o f cell line might assist in their differential diagnosis.

4 .2 S ou th ern B lottin g o f PCR-am plifled cDNA S p an n in g th e E x o n 7 /8 Splice Ju n ction

Alternative splicing, by virtue of its tissue-specific regulation, m ay be a m ean s by w hich to distinguish tum ours of different lineages. The altern ativ e splicing p attern at the exon 7/8 splice junction o f eig h t n e u ro b la sto m a cell lines an d one rhabdom yosarcom a cell line w as investigated by PCR amplification of DNA com plem entary to RNA spanning this region.

(a ) M ethods

RNA was extracted from cell lines using guanidium isothiocyanate. Two extractions w ere m ade of each cell line growing exponentially. Each line w as resuscitated from two different stock cultures.

Complem entary DNA was generated by reverse transcription o f RNA tem plate, using an oligonucleotide prim er com plem entary to a region in ex o n 8, dow nstream from the exon 7/8 splice junction (Figure 4.2.1). The prim er w as designed according to recom m endations (Innis an d G elfand,

Table 4.1

VASE e x p r e s sio n in h u m an c e ll lin e s and d istin ct rat b rain r e g io n s

The data presented summarises results obtained by Small and Akeson, 1990. Extent of VASE inclusion was assessed according to the intensity o f signal and is a comparative measure of VASE- containing PCR products, m easured against VASE-lacking PCR products.

Table 4.1

C ell Line T isue T ype VASE In c lu s io n

- Basal Ganglia + - Brain Stem + - Cerebellum ++ - Cortex ++ - Mid-brain ++ - H ippocam pus ++ - Thalamus +++ - Olfactory epithelium +/- - Spinal cord +

- Dorsal root ganglia +

- Adrenal +/- - Embryonic heart +/- - Adult Heart +++ B35 Neuronal +/- B104 Neuronal +/- RN22 Schwanoma +/- C6 Glial +/- B12 Glial +/- L6 Muscle +/- PC12 Phaeochrom ocytom a +/-

Figure 4.2.1

E x o n s 7 and 8 and th e p o sitio n s o f p rim ers an d th e non-VASE