Expression o f W I-17997 EST

A pre-made Northern blot containing Ipg of polyA+ RNA lane from different human tissues was used for Northern blot analysis of W-17997 EST (Clonetech, USA). The 1.7kb insert of IMAGE clone 155338 (AC R69484) was used as a probe. Northern blot analysis confirmed the presence of one transcript o f approximately 2.2kb in all of the tissues analysed (figure 3.3), in addition that the full-length transcript is similar in size to the largest cDNA clone sequenced.

2.4kb 1.35kb

Figure 3.3 Northern blot analysis of WI-17997. One transcript of approximately 2.2kb is shown in all of the tissues analysed (as indicated by the red arrow). The Northern blot was probed with the (P^^) radiolabelled 1.7kb insert of IMAGE clone 155338 (AC R69484).

3 .2 .3 P rotein id e n titie s and p o ssib le fu nction

Hypothetical protein BC013995 (322 amino acids) represented by EST WI-17997 shows homology o f 81%, over aligned region o f 244 amino acids to a mouse protein encoded by a gene called Myadm

(Myeloid-associated differentiation marker) (Pettersson et al., 2000). This predicted 32-kDa protein contains multiple putative hydrophobic transmembrane segments and has several potential consensus sites for phosphorylation (figure 3.4).

Unlike the human gene, Myadm exhibits a restricted expression pattern within the hematopoietic system. In view o f this expression pattern, Myadm has been suggested to serve as a new marker for hematopoietic differentiation. The ubiquitous expression pattern o f the human gene is however suggestive o f it having additional functions besides a possible involvement in myeloid differentiation.

Moreover, protein similarities were also identified with H.sapiens

mucin protein DKFZp434B0635.1 (pir: T345949) (26%/ 341aa),

R.norvegicus plasmolipin (pir: A55046) (25%/ 159aa), A.thaliana

glycine-rich protein (pir: T49100) (29%/ ISSaa) and C.elegans

hypothetical protein T05C12.10 (pir: T23754) (24%/ 277aa) for each o f the homologies percent identities and length o f the aligned region are shown (figure 3.4).

3 .2 .4 G en om ic o r g a n isa tio n o f h y p o th e tic a l gene L O C 9 1 6 6 3

The genomic organisation o f this novel gene, now identified as a hypothetical gene LOC91663, was established by aligning the collated cDNA sequence (see section 3.2.1) with the genomic sequence o f the BAC clone 331H23 using the BLAST program to identify exon/intron boundries (Pearson and Limpman, 1988; Altschul et al., 1990).

C h a p t e r 3, C h a r a c t e r i s a t i o n o f WI - 1 7 9 9 7 E S T

LOC91663 was found to consist o f 2 exons, 86bp and o f 924bp in size respectively for exon 1 and 2. Translation was initiated and terminated within the second exon. The two exons follow the GT/AG rule for exon/intron boundaries. The gene spans 8.6kb o f genomic DNA sequence (figure 3.5). Based on the genomic sequence, the stop codon o f LOC91663 was found to be approximately 9kb far from the start codon o f

PRKCG (Al-Maghtheh et al., 1998). The genomic organisation o f the

hypothetical LOC91663 gene was further aided by the analysis o f the genomic sequence o f 3 3 1H23 by NIX program.

Mouse m y e .upprtn M P --- V T V T R T T I T T T T S S S T T V G S AR AL TQ P LG L LR LL QL I ST CV A FS LV AS V GA W 54 Human BC013995 61 T G S M G N W S M F T W C F C F S V T LI IL I VE L CG LQ AR F PL SW R NF PI TF A CY AA LF C LS AS I IY TG MGNW+MFTWC FC F +V TL II L IV EL GLQA F P L S W R N F P I T F A C Y A A L F C L S + S I I Y 120 Mouse m y e .upprtn 55 T G P M G N W A M F T W CF C FA VT LI I LI VE L GG L OA HF PL S WR NF PI T FA CY AA L FC LS S SI IY 114 Human BC013995 121 PT TY V QF LS H GR SR DH A IA AT FF S CI A CV AY A TE VA WT R AR PG EI T GY MA TV P GL LK V LE PTTYVQFL+HGR+RDHAIAAT F S C + A C + A Y AT EV AW T RA RP G EI TG YM A TV P GL LK V E 180 Mouse m y e .upprtn 115 P T T Y V O F L A H G R T R DH AI A AT TF S CV A CL AY AT E VA WT RA R PG EI T GY MA TV P GL LK VF E 174 Human BC013995 181 T F V A C I I F A F I S D P N LY QH QP A LE WC V AV Y AI CF IL A AI AI LL N LG EC T NV LP IP F PS FL

TFVACIIFAFI +P LY + P ALEWCVAVYAICFILA + ILLNLG +C T NV LP IP F P+ FL

240

Mouse m y e .upprtn 175 T F V A C I I F A F I G E P L L Y N Q K P A L E W C V AV YA I CF IL AG V TI LL N LG DC TN V LP IP FP T FL 234

Human BC013995 241 SGLA

SGLA

244

Mouse m y e .upprtn 235 SGLA 238

Figure 3.4 Alignment of human hypothetical protein BC013995 against the mouse Myeloid up-Regulated protein. Amino acids highlighted in red represents the mouse transmembrane regions.

A C T A G T T C T C T C T C T C T C T C T C C G T C T T T G C T T C A G C C G C A G T C G C C A C T G G C T G C C T G A G N G T G A G G T T G TG G G G A C C C C A G A G G G C A T A A G C A A C A G T C T A G G G T C T T TA T G G C C A G A G A G A G G A A TG G G C T G G G T C T C A G A A T G T C T G G G T C C C T A G A A A G A C T G G G G C T G G G C C A G G A T T C T T G G G G T T C T G C A G A G G A G A G G G C T G A G G A C C TG G A C T G T G G A G G T C A T A C G T C T G G A A GG G G G A T C C T C T A G A G T C G A C C A A C A G C A T G C A G C T G C C T G C T G / / A T G A G G T C A C T C T A G A G G T A T C C C C A G C C A A A C A G T T A A C TG A A C G G G G A GGGGAGGGGG C G G G G C T T A C C C G G G G T C A C C G C C A G G C C C T G C T G G G T T C C A C G C C A C G T T C C T G T C T C C G C A G G G C G T T C G A T C C C T A G A G G G A G G A G C C T G T C C A A A C G G A C G C T A A C G G C C T A C C T C C C C C T C A G 6 T G C T C T T A C A G C C T G T T C C A A G T G T G G C T T A A T C C G T C T C C A C C A C C A G A T E x o n I C T T T C T C C G T G G A T T C C T C T G C T A A G A C C G C T tS A G T G A G T A G G C A G C G G G C C T G G A C T C C G G G G T C C G A G GGAGGAGGGG C T G G G G G C C T G G A C T C C G G G G T C C G A G G G A G G A G GG G CTG G G G G C C T G G G C T C C T C G G T C C G A G G G A G G A G G G G CT G G G G G C C T G G G G G C C T G G G C T C C T G G G T C C G A G G G AGGAGGGGC T G G G G G C C T G G G C T C C T G G G T C C G A G G G A G G A G G G G C TG G G G G T C T G G A C T C C G G G G T C C G A G G G A G G A G G G G C T G G G G T C T G G A C T C C T G G G TC CG A G G A A G G A G G G G C T G G G G T C T G G A C T C C T G G G T CCG A G G A A G G A G G G G C T G G G G G C C T G G G C T G C T G G G T C C G A G G G AGGAGG G G C T G G / / G G G T G A C T A G A G G T TC C A A A C G A A A G C C C A C A T C T G G G A A C T C C C T ^ m T T A G A G G T C A T A T T A A T T T G T C C C T G G G G TA G G CA A T G G C T A A G G G A C C T G G A T T C T T ^ G T T T G T G A C T G T A T A A G G A C A C T G T C T T T C C C C T T T T T G C A G T C C A T G C C A G T G A C G G T A A C C C G C A C C A C C A T C A C A A C C A C C A C G A C G T C A T C T T C G G G C C T G G G G T C C C C C A T G A T C G T G G G G T C C C C T C G G G C C C T G A C A C A G C C C C T G G G T C T C C T T C G C C T G C T G C A G C T G G T G T C T A C C T G C G T G G C C T T C T C G C T G G T G G C T A G C G T G G G C G C C T G G A C G G G G T C C A T G G G C A A C T G G T C C A T G T T C A C C T G G T G C T T C T G C T T C T C C G T G A C C C T G A T C ^ ffC C T C A T C G T G G A G C T G T G C G G G C T C C A G G C C C G C T T C C C C C T G T C T T G G C G C A A C T T C O C C A T C A C C T T C G C C T G C T A T G C G G C C C T C T T C T G C C T C T C G G C C T C C A T C A T C T A C C C C A C C A C C T A T G T C C A G T T C C T G T C C C A C G G C C G '^ C G C G G G A C C A C G C C A T C G C C G C C A C C T T C T T C T C C T G C A T C G C G T G T G T G G C T T A C G C C A C C G A A G T G G C C T G G E xon 2 A C C C G G G C C C G G C C C G G C G A G A T C A C T G G C T A T A T G G C C A C C G T A C C C G G G C T G C T G A A G G T G C T G G A G A C C T T C G T T G C C T G C A ÏC A T C T T C G C G T T C A T C A G C G A C C C C A A C C T G T A C C A G C A C C A G C C G G C C C T G G A G T G G T G C G T G G C G G T G T A C G C C A T C T G C T T C A T C C T A G C G G C C A T C G C C A T C C T G C T G A A C C T G G G G G A G T G CjACCAACG T G C T A C C C A T C C C C T T C C C C A G C T T C C T G T C G G G G C TG G C C T T G C T G T C T G T C C T C C T C T A T G C C A C C G C C C T T G T T C T C T G G C C C C T C T A C C A G T T C G A T G A G A A G T A T G G C G G C C A G C C T C G G C G C T C G A G A G A T G TA A G C T G C A G C C C C A G C C A T G C C T A C T A C G T G T G T G C C T G G G A C C G C C G A C T G G C T G T G G C C A T C C T G A C G G C C A T C A A C C T A C T G G C G T ^ T G T G G C T G A C C T G G T G C A C T C T G C C C A C C T G G T T T T T G T C A A G G T C T A A G A C T C T C C C M G A G G C T C C C G T T C C C T C T C C A A C C T C T T T G T T C T T C T T G C C C G A G T T T T C T T T A T G G A G T A C T T C T T T C C T C C G C C T T T C C T C T G T T T T C C T C T T C C T G T C T C C C C T C C C T C C C A C C T T T T T C T T T C Ç T T C C C A A T T C C T T G C A C T C T A A C