The sixty amino acid homeodomain sequences from various proteins are compared to that of the 16-1 clone. Those sequences found to be most closely related to 16-1, as well as three representatives of large homeodomain families (see Chapter 6) -
Antennapedia (Antp), Engrailed (En) and Paired (Prd) - are included in the comparison. Identical conserved residues are marked in bold, and the % conservation of identity is indicated at the end of each sequence.
16-1 10 PRRSRTIFTE 20 LQLMGLEKKF 30 QKQKYLSTPD
Cnox3 CRKPRTVFSD LQLMVLEREF NNRKYLSTPQ
BarH2 QRKARTAFTD HQLQTLEKSF ERQKYLSVQD
NK-1 PRRARTAFTY EQLVSLENKF KTTRYLSVCE
ChkHox3 PRRARTAFTY EQLVALENKF RATRYLSVCE
EgHbxl RRRARTAFTY EQLVTLENKF QSTRYLSVYE
Antp RKRGRQTYTR YQTLELEKEF HFNRYLTRRR
En EKRPRTAFSS EQLARLKREF NENRYLTERR
Prd QRRCRTTFSA SQLDELERAF ERTQYPDIYT
16-1 40 RLDLAQSLGL 50 TQLQVKTWYQ 60 NRRMKWKKMV 100%
Cnox3 RTNLADRLGL NQTQVKTWYQ NRRMKWKKET 65%
BarH2 RMELANKLEL SDCQVKTWYQ NRRTKWKRQT 58%
NK-1 RLNLALSLSL TETQVKIWFQ NRRTKWKKQN 60%
ChkHox3 RLNLALSLSL TETQVKIWFQ NRRTKWKKQH 60%
EgHbxl RLNLALSLNL TETQVKIWFQ NRRTKWKKQN 60%
Antp RIEIAHALCL TERQIKIWFQ NRRMKWKKEN 45%
En RQQLSSELGL NEAQIKIWFQ NKRAKIKKST 40%
60% identity) can be considered in the same class. Although the 16-1 sequence does show the required relationship to the homeodomains listed, of these homeodomains only NK-1, ChkHox3 and EgH bxl show a consistent relationship with each other, and no single grouping can be made to include the 16-1 homeodomain. Only the BarH2 product, involved in Drosophila eye developm ent (for lens and pigment cell formation and for om m atidial differentiation, (Higashijima et al., 1992)), shows any homology at all with the 16-1 product outside of the homeodomain. This homology consists of 2 co- linear segments upstream of the homeodomain, which retain 33% identity over 27 residues and 25% identity over 35 residues, and additional primary sequence homology in the 3' non-coding region.
Th e close similarities seen between a very large num ber of homeodomains is in part a reflection on the fact that a great number of these have been cloned by homology screening - albeit using d eg en erate sequences - so that the current repertoire of homeodomains has to some extent been pre-selected for its homogeneity of sequences. However, a growing number of 'orphan' homeodomains have been cloned by other means, such as by sequence- specific DNA binding (for instance STF-1 (Leonard et al., 1993)) or by genetic means (for instance B arH I and BarH2 (Higashijima et al., 1992)). It seems likely that many of these 'orphan' factors will in fact turn out to be the founding members of new families of related factors.
An unusual feature of the 16-1 homeodomain is the substitution of the phenylalanine (F8) residue in the recognition helix for a tyrosine (Y8 ).
Although a conserved substitution, this is unique amongst m am m alian homeodomains; for this reason rather than for the overall - and rather poor - homology shared between them, I suggest that 16-1 could be classified together with the Cnox3 and 4 products from Chlorohydra viridissima, which also code for Y8 in helix III, and to the Drosophila BarHI and 2 proteins, for the
same reason. These homeodomains also share the substitution of a threonine residue at position 6 of helix III (T6) for the more usual isoleucine (16) although
this position is not as strongly conserved as position 8. A further unusual
feature of the 16-1 homeodomain may be the proline (P) residue in position 1.
This position is not heavily conserved between homeodomains, but a proline here is highly unusual (amongst all of the published hom eodom ain sequences) and conserved in related homeodomains NK-1 and ChkHox3. Since N-terminal residues of the homeodomain are implicated in DNA minor groove contacts (see figure 6.1) the proline in this position may be of some
significance.
Is 16-1 a full-length clone? The first ATG (methionine) codon of the cloned sequence falls within a perfect Kozak consensus sequence (Kozak, 1986) and gives rise to an open reading fram e which includes the homeodomain, strongly suggesting that this methionine corresponds to the correct start site for translation. Although there is insufficient cloned sequence 5' to this ATG to identify it beyond doubt as the genuine 5' end of the coding region, the complete message size for 16-1 seen by Northern blotting (2kb, see figure 6.5a) implies that our clone must be at least very close to containing the complete coding sequence. This can be concluded since of the apparent 400- 500 bp discrepancy between clone and message size, a large part should consist of 3'UTR and polyadenylated sequences (the clone does not read 3' far enough to include the AATAAA polyadenylation signal).
Clone 16-1 was selected for further investigation, the results of which are presented in Chapter 6. It will be referred to as HIC (for H o m eo d o m ain
protein interacting with CREB).
5.4.iv Clones not further characterized
S e v era l of the clones isolated from the screen rem ain uncharacterized. All of the clones listed in figure 5.6a were subcloned into the TTplink vector and partially sequenced. Several of them were found to be unique sequences with no known homologies. These gave rise to in vitro
translation products not able to interact with CREB in the type of Far Western assay described for the 16-1 clone in the following section, and were not investigated further. However, since it was found that neither the correct frame nor orientation of these sequences in TTplink could be guaranteed, it cannot be concluded that they do not merit further investigation. One such clone (5-1) gives rise to a putative highly charged peptide in the only open reading frame to include the complete clone, but this product cannot be detected in Far W esterns and does not appear to affect CREB-mediated transcription in co transfection assays (data not shown). The sequence of 5-1 and its putative translation product are presented in figure 5.12a.
Clone 14-2 was found, based on sequence comparison, to be the human homologue to the rat cDNA for nuclear pore protein gp210 (fig5.12b). Recent sequence database analyses have revealed possible identities for two more of the clones. Clone 14-1 is the human homologue to the recently cloned rat PKC-regulated chloride channel CIC3 (Kawasaki et al., 1994)(fig5.12c) and clone 16-3 appears to correspond (allowing for sequencing errors) with the recently cloned Human Papillomavirus E6-asso ciated protein E6-AP
(Huibregtse et al., 1993a)(fig5.12d). As already mentioned, these clones were not investigated further because of our inability to reproduce their interaction