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Use of various programs (Methods Section 2.23.1) to search available databases identified two identical rat sequences (Accession numbers D 14437; X71070). Homologues of rat transgelin were also found in mouse (96% aa identity), human (99% amino acid identity, Accession M95787) and chicken (84% amino acid identity. Accession M83105). Rat transgelin was also found to display 59% amino acid identity with human C4i (M Smith unpublished). Transgelin displayed 64% amino acid identity with a rat neuronal protein designated NP25 (Accession M84725). Transgelin contained 38-43% identity with various calponin isoforms (Accession M63559, D14437, X71071, Z19542, Z19543), and 39% identity with a Drosophila protein designated mp20 (Accession Y00795). Transgelin was found to have 17% overall amino acid identity with C elegans unc87 proteins (but contained short stretches displaying up to 74% conservation) (Accession U04711 and U04712).

A multiple alignment of the rat and sheep transgelin amino acid sequences with chick SM22a, C 4\ rat NP25, mp20, chick calponin beta and one of the repeat regions in unc-87 is shown in Figure 15. The alignment clearly demonstrates the homology between the proteins, illustrated by the occurrence of 43 positions of amino acids that are identical in all of the aligned proteins (Fig. 15*) together with 44 positions identical in all but one sequence (including conservative changes)(Fig. 15*) and 20 positions at which only conservative changes occur in more than one protein across all aligned proteins, namely 53% of the residues in transgelin have been very highly conserved within all proteins in the family (including proteins from humans and Drosophila ). These highly conserved residues are distributed throughout the protein (25/50 aal-50; 25/50 aa51-100; 31/50 aalOl-150; 27/50 aal51-200) with a slightly higher representation at the N- and C-termini respectively (20/30 in residues 1-30 and 21/30 in residues 171-200).

* * * * • • • * * • • • • # * * * _{« * *}

1 MANKGPSYGM S R E V Q S K IE K K Y D EELE ER L VEWIVMQCGP DVGRPDRGRL 5 0

2 MANKGPSYGM S R E V Q S K IE K K Y D EELE ER L V E W IW Q C G P DVGRPDRGRL 5 0

3 MANKGPAYGM S R D V Q S K IE K K YDDELEDRL V EW IV A Q C G S SV GR PD RG RL 5 0

4 MANRGPAYGL SR E V Q Q K IE K Q Y D A D L E Q IL IQ W IT T Q C R K DVGRPQPGRE 5 0

5 M ANRGPSYGL S R E V Q E K IE Q KYDADLENKL V D W IIL Q C A E D IE H P P P G R T 5 0

6 M SL E R A V R A K IA S KRNPEMDKEA Q E W I E A I I A E K FPA G Q --- 3 9

7 M SNA N FN R G PA Y G L SAEVKNKLAQ KYDPQTERQL RVW IEGATGR R I G D --- 4 8

» * * ★ * * * * • • . * * * • • * 1 GFQVWLKNGV IL S K L V N S L Y P E G SK PV K V P EN PPSM V FK Q MEQVAQFLKA 1 0 0 2 GFQVWLKNGV IL S K L V N S L Y PD G SK PV K V P EN PPSM V FK Q MEQVAQFLKA 1 0 0 3 GFQVW LKNGI V L S Q L V N S L Y P D G S K P V K IP D SPPT M V FK Q M EQ IA Q FLK A 1 0 0 4 NFQNWLKDGT V L C E L IN A L Y PEG Q A PV K K I -Q A ST M A FK Q M E Q ISQ F L Q A 9 9 5 HFQKWLMDGT V L C K L IN S L Y P P G Q E P I P K I -S E S K M A F K Q M E Q ISQ F L K A 9 9 6 SY EDVLK DG Q V L C K L IN V L S PN A V PK V N — — SSG G Q FK F M EN IN N FQ K A 8 5 7 NFMDGLKDGV I L C E L IN T L Q P G S V Q K V N -- --D P V Q N W H K L E N IG N F L R A 9 4 * * , * * • • • * * * • • * • * • * • • •

1 AED Y G V TK TD M FQTVDLFEG KDMAAVQRTV M ALGSLAVTK NDGHYRGDPN 1 5 0

2 AED Y G V TK TD M FQTVDLFEG KDLAAVQRTL M ALGSLAVTK NDGTYRGDPN 1 5 0

3 A E D Y G W K T D M FQ TVD LFEA KDMAAVQRTL V A L G SL A V T K NDGHYRGDPN 1 5 0

4 A E R Y G IN T T D IFQTVDLW EG KNMACVQRTL MNLGGLAVAR D D G L F SG D P N 1 4 9

5 A E V Y G V R T T D IFQ TVDLW EG KDMAAVQRTL M ALGSVAVTK D D GC YRG EPS 1 4 9

6 L K E Y G V P D ID VFQ TV D LY EK K D IA N V T N T I FALGRATYKH A D F K G P F --- 1 3 2

7 IK H Y G V K PH D IF E A N D L F E N TNH TQ VQ STL lA L A S Q A K T K G N --- NVG 1 3 9

, • * * , * * , * * * , * * • •

1 WFMKKAQEHK R E F T D SQ L Q E GKHVIGLQMG SNRGASQAGM T G Y G R P R Q II 200

2 WFMKKAQEHK R E F T E S Q L Q E GKHVIGLQMG SNRGASQAGM T G Y G R P R Q II 200

3 WFMKKAQEHK R E F S E S Q L K E G K N IIG LQ M G TNKGASQAGM S - Y G R P R Q I I 1 9 9

4 W FPK K SK EN P R N FSD N Q LQ E GKNVIGLQMG TNRGASQAGM TGYG M PRQ IL 1 9 9

5 WFHRKAQQNR R G FSEEQ LR Q GQNVIGLQMG SNKGASQAGM TGYGMPGRSC 1 9 9

6 LG PK PA D E C K RD FTEEQ LK A G Q TIVG LQA G SN K G A T Q A G - -N L G A G R K IL 1 8 1

7 LGVKYAEKQQ R R FQ PEK LR E G R N IIG L Q M G TNKFASQQGM TAYGTRRHLY 1 8 9

8 unc-87-repeat 4 IP L Q S G TNKFASQKGM T G FG T A R R E T 1 S 2 0 1 2 S 2 0 1 3 S 2 0 0 5 K M L Y SE C PL V ER TD VPHH SL 2 1 9 6 LGK 1 8 4

7 D P K L G T D Q P L D Q ATISLQ M G TNKGASQGMT VY GLPRQVY D PKY CDA PG LL 2 3 9

7 G E D G L N H SF Y N S Q 2 5 2

Figure 15. Alignment of homologous sequences. The deduced rat transgelin sequence (1) is shown aligned with (2) sheep transgelin (this study), (3) chicken SM22a (Nishida et al 1992), (4) human C4^ (M Smith unpublished), (5) rat NP25 (unpublished, EMBL Accession M84725), (6)

Drosophila mp20 (Ayme-Southgate et al 1989), (7) chicken calponin p (Takahashi & Nadal- Ginard 1991) and (8) repeat 4 from unc-87 (Goetinck & Waterston 1994b). Hyphens represent

gaps introduced to optimise alignments. The location of residues conserved in all sequences is marked with an *, those identical in all but one with an •.

Alignment of a region in transgelin (residues 175-197) with seven of the repeat motifs present in C elegans unc-87 illustrates the degree of conservation in this peptide (ranging from 60-74% conservation over 23 amino acids)(Fig. 16). The protein kinase C motif in transgelin (Section 3.14(a)) is conserved in four of the repeats, while the calcium- calmodulin dependent protein kinase motif (Section 3.14(a)) is conserved in six of the seven repeats in unc87.

Rat transgelin (175-197) IGLQM GSNRG ASQAG MTGYG RPR

unc-87 repeat 1 (250-272) IPSQA GWNKG DSQKL MTNFG TPR (60%) unc-87 repeat 2 (298- 320) VRLQS GTNKY CSQRG MTGFG SGR (74%) unc-87 repeat 3 (351-373) VRLQA GTNKY DSQKG MTGFG TGR (69%) unc-87 repeat 4 (397-419) IPLQS GTNKF ASQKG MTGFG TAR (74%) unc-87 repeat 5 (444-466) IPSQM GSNQY ASQKG MTGTG QPR (69%) unc-87 repeat 6 (485-507) VRLQS GTNRF ASQAG MIGFG TGR (74%) unc-87 repeat 7 (531-553) IPSQA GWNKG DSQKK MTSFG APR (65%)

Figure 16. Alignment of the unc-87 repeat motif. The rat transgelin sequence between amino acids 175-197 is shown aligned with the seven repeats found in unc-87 (Goetinck & Waterston 1994b). The numbers in brackets represent the amino acid conservation between transgelin and that repeat. The potential phosphorylation target residues in transgelin are underlined.