Genes coding for mucins identified in other species include those for; at least two rat intestinal mucins which are the homologues of MUC2 and possibly MUC3 and a subm andibular mucin (Gum et al. 1991; Xu e/ al. 1992), the mouse M u d and M uc5ac genes (Spicer et al. 1991), frog integumentary mucins (FIM) (Hoffmann et al. 1993), porcine submaxillary mucin and gastric mucin (Timpte et al. 1988; Turner
et al. 1995), bovine subm axillary m ucin (B hargava et al. 1990) and canine tracheobronchial mucin (Shankar et al. 1992). These will be briefly described in the following section.
1.5.1. Rat mucins
Rat M uc2 clones from the am ino term inal, central region and carboxyl terminal have been isolated by a number of groups.
The clones 1-1, 8-1 and 21-1 were isolated from a XZAPII rat intestinal library using a 5' non tandem repeat probe from MUC2. The com bined sequence o f these
clones encode a 1513 residue peptide in which the first 1391 residues are rich in cysteine whilst the remaining 122 amino acids are com prised o f irregular tandem repeats rich in serine, threonine and proline (Ohmori et al. 1994). The non-repetitive region show s 80% identity with the amino term inal region o f M UC2 and the repetitive region approximately 38%. This evidence suggests that this is the amino terminal o f the rat Muc2.
V R IA was isolated from a XZAPII rat jejunum library using antiserum raised against deglycosylated rat intestinal mucin (Hansson et al. 1994). The sequence of this clone encodes a peptide which has 7 cysteine residues in the first 53 amino acids and the following 182 residues are rich in serine, threonine and proline. There is no tandem repeat structure although certain motifs such a TTT are present which are repeated 13 times. The cysteine rich region shows 59% similarity with the region between the degenerate tandem repeats and tandem repeat region o f MUC2.
The clone MLP 2677 which corresponds to the carboxyl terminal o f M uc2 was isolated using a 0.5kb H in d lll fragm ent from a PCR product obtained w ith prim ers designed from the amino acid sequence of an 118kDa glycopeptide from rat intestine (Xu, Huan et al. 1992). This clone encodes an 837 residue peptide which contains 4.5 tandem repeats at the N terminal of 11 to 12 amino acids rich in serine, threonine and proline, while the remaining 767 amino acids are rich in cysteine residues (Huan et al. 1992). Probes from MLP and MUC2 recognise the same 9.0kb fragm ent when hybridised to Northern blots of rat and human RNA (Xu et al. 1992). A probe from M LP also m apped to rat chrom osom e 1 which contains a region syntenic with the region o f human chromosome 11 which contains M UC2 (Klinga- Levan et al. 1996). These results all suggest that this clone is the carboxyl terminal of a rat M UC2 homologue Muc2.
Muc2 appears to be expressed in the intestine and colon o f the rat in a pattern sim ilar to that o f M UC2 (Xu et al. 1992). It is also interesting to note that there are sim ilarities in the biosynthesis o f rat Muc2 and human MUC2. Tytgat and colleagues show ed that the rat colonic mucin (RCM) could be im m unoprecipitated using a
specific anti human MUC2 indicating this is the rat homologue (Tytgat; et al. 1994). The mucins o f both species have sim ilar characteristics in SDS PAGE experim ents and in respect o f their relative mobility, com position and buoyant density. The precursor also appears to dimerise before glycosylation like M UC2 (Tytgat; et al. 1994). A protein polymorphism o f the precursor (Tytgaf
et al. 1994) was also observed which may indicate genetic polymorphism as in MUC2 (Griffiths et al. 1990).
The partial cDNA clone RMUC176 was isolated by screening a rat jejunum library using antiserum raised against deglycosylated intestinal mucin (Gum et al. 1991). This clone consisted of 18bp tandem repeats which code for a 6 amino acid repeat with the consensus sequence TTTPDV. A 9kb fragment is detected with this cDNA clone on Northern blots of RNA from small intestine and colon, w hich is consistent with this being an intestinal mucin. The partial cDNA clone M 2-798 was isolated from a rat intestinal XZAPII library and detects the same fragm ent as RM U C176 but detects something different to M LP on Northern blots o f rat RNA (Khatri et al. 1993). The sequence o f M2-798 encodes a peptide which consists of tandem repeats with the same consensus sequence as that encoded by RM UC176 follow ed by a unique sequence of 82 amino acids. These results indicate that RM UC176 and M2-798 are from the same rat intestinal mucin gene but are not rat M uc2. RM UC176 has been mapped to rat chromosome 12 which appears to have a region syntenic with human chrom osom e 7q22 indicating that these two clones correspond to Muc3 the rat homologue o f human MUC3 (Klinga-Levan; it al.
1996).
At least one other rat m ucin gene has also been cloned by T suda and colleagues from a rat airway cDNA library screened with SMUC41 (Tsuda et al.
1993). Although this mucin was isolated using a human MUC2 probe the sequence o f the tandem repeats, TTTTIITI, and overall lack o f homology indicates that this is not part of the rat Muc2 gene. This gene is expressed in rat trachea (after exposure to S02/Sendai virus or endotoxin) and intestine.
A lthough the gene for rat M u d has not yet been cloned a probe from the repetitive region of mouse Muc 1 was used to localise a homologous sequence on rat
chromosom e 2. This suggests the presence of a rat homologue to both human M U C l and mouse M u d as rat chromosome 2 contains a region syntenic with human chromosome lq21 (Klinga-Levan, et al. 1996).
1.5.2. Mouse mucins
At least two mouse m ucin genes have been cloned w hich are probable homologues o f human M U C l and MUC5AC. The M u d gene has been cloned by two groups. Spicer and colleagues obtained the complete cDNA for mouse M u d (Spicer et al. 1991), whilst Vos and colleagues isolated a number of genomic clones by screening a ^gtlO library with a cDNA clone containing the majority of the non repetitive region of M U C l (Vos et al. 1991). The genetic structure o f the M u d gene has been determined and it is comprised of 7 exons, of which exon 2 contains 16 tandem repeats (Vos et al. 1991). Interestingly the tandem repeat o f M u d does not appear to be polym orphic, and there appears to much greater m ore variation betw een individual M u d repeats than in human M U C l. The peptide sequence predicted from the full length cDNA has a relatively high percentage o f threonine, serine and proline which is characteristic o f mucins. A num ber of regions show a high level o f conservation between the deduced peptide sequences of human M U C l and mouse M u d . The transmembrane region (90%) and the cytoplasmic tail (87%) are both highly conserved. Two regions which are not well conserved are the extra cellular region and the tandem repeats. The lack o f conservation o f the tandem repeats has been observed in the rat and other species and may indicate that the precise sequence is not functionally important (Spicer et al. 1991). H owever one aspect that does appear to be conserved is the high proportion o f hydroxyl residues able to form 0-glycosidic linkages.
At the nucleotide level the promoter sequence is also generally well conserved (Vos, de et al. 1991). Studies o f the expression o f M u d show that, like M U C l, it is expressed on the epithelial surface of a wide variety of organs e.g. stomach, pancreas, lung, trachea, kidney and salivary glands (Braga et al. 1992). It is interesting to note that these immunohistological results were obtained using an antibody specific to an epitope in the cytoplasmic region o f M U C l which together with the high level o f conservation im plies that the peptide sequence o f this region is im portant in m aintaining function. Indeed this region of M U C l appears to be well conserved throughout mammals (Pemberton et al. 1992).
Partial clones for a mouse gastric mucin were isolated by screening a stomach cDNA library with chicken antibodies raised against deglycosylated mouse gastric mucin (MGM) (Shekels et al 1995). The tandem repeat region is comprised of 48bp re p e a ts w h ich code fo r a 16 am ino acid re p e at w ith the seq u en ce QTSSPNTGKTSTISTT. This repeat sequence shares no significant similarity with any other mucin so far identified however the non repeat region shows 75 to 80 % identity with MUC5AC. There is also a lower level o f similarity with M UC2 and one o f the rat intestinal mucins. W hen this gene was mapped it was localised to the region o f mouse chromosome 7 homologous with human chromosome 1 Ip 15. These results indicate that this gene may be the mouse homologue o f human MUC5AC.