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The insert representing the N 1 clone was 2.5 kb in size. When first isolated, a stretch of the N1 clone, long enough to compare against the database, was sequenced.

The N1 clone was in frame with the GAL4 activation domain. Comparison against the GenBank database did not show significant homology to known DNA sequences of the database. The interaction between N1 and GLT-N was not further studied, due to this complete lack of information on this novel clone. Two years later, the N1 sequence was screened again against the GenBank database to look for homology to new database entries. This stretch of the clone N1 cDNA sequence was then found to be highly homologous (84 %) to the Homo sapiens type I imidazoline receptor candidate (I,) cDNA (also called IRAS cDNA), as shown in figure 4.3A (Piletz et al. 1999, 2000). Such high homology between the two sequences suggests that N1 represents a partial sequence of the rat homologue of the human I, gene, and that the slight differences in the sequence are due to species-specific mutations in the genes. The comparison of the protein sequence between the N1 partial sequence and the IjCDNA is shown in figure 4.3B. N1 is likely to represent two thirds of the I, cDNA (as deduced from the start point of the N1 clone and its length).

As described in section 1.6.a in chapter 1, imidazoline receptors were first reported as 'sites' pharmacologically related to a^-adrenoceptors (Eglen et a l 1998). The cDNA candidate, to which the N 1 clone isolated during the yeast two-hybrid screen in this study is homologous, is the only report of a gene coding sequence that could represent the I, receptor. It was identified by screening a human hippocampal lambda gtl 1 cDNA expression library with two antisera directed against candidate imidazoline receptor proteins (isolated biochemically) (Piletz et al. 1999, 2000).

Figure 4.3 Partial sequence comparison of the clone N1 rat cDNA and the Homo sapiens imidazoline receptor candidate (IJ cDNA

A) A partial sequence of the N1 clone cDNA (2.5 kb) isolated during the yeast two-hybrid screen (N l, top sequence, bold) was compared to the Homo sapiens imidazoline receptor candidate (IJ cDNA (I^, bottom sequence) (GenBank accession number 6005787) using the BLAST sequence homology search programme. Vertical lines show nucleotides that are identical in the two sequences. Numbers on the left and right of each stretch of sequence represent the position of the first (left) and last (right) nucleotide with respect to the partial N l sequence used for homology identification (from nucleotides 1 to 421) and to the I, cDNA sequence (from nucleotides 99 to 518).

B) The deduced protein sequence of the partial N l sequence (top) is compared to the II protein sequence (bottom). The middle red lines represent identity (letters), homology (+), or disparity (-) between the two sequences.

A ) Nl ; ^gagctcgtggacacgtccacggtatatgtcatacaggttaccgatggcaaccat^* I l : ^ ^ g a g c t t g t g g a c a c t t a t a c g g t t t a c a t c a t c c a g g t c a c t g a t g g c a g c c a t ^ ^ ^ N 1 : ” gagtggacgatcaaacaccgctatagtgattttcatgacctccatgaaaagctt^°® I l : ^ ^ ^ g a g t g g a c a g t a a a g c a c c g c ta c a g c g a c t t c c a t g a c c t g c a t g a a a a g c t c ^ ° ® Nl : ^°^gttgctgagagaaaaattgacaaaactctacttccacccaaaaagataattgga^“ I l : ^ ° ^ g t t g c a g a g a g a a a g a t t g a t a a a a a c c t g c t t c c g c c c a a a a a g a t a a t t g g g ^ ® ° Nl : "aagaactcgagaagcttggtagaaaagagggagaaagacctggaggtgtacctc^^^ I l : ^“ a a a a a c t c a a g a a g c t t g g t g g a g a a g a g g g a g a a g g a t c t g g a g g t c t a c c t c ^ ^ ^ Nl : ^ cacactctcctgaagaccttccctgatgtggctcccagagttctggcccacttc^^° I l : ^ ^ ^ c a g a a g c t c c t g g c t g c c t t c c c t g g c g t g a c c c c c a g a g t a c t g g c c c a c t t c ^ ® ® Nl : ^’^ctgcattttcacctctatgaaataaatggtgtcactgcagcactggctgaagaa^^^ I l : ^ ^ ^ t t g c a t t t t c a c t t c t a t g a g a t a a a t g g c a t c a c c g c g g c a c t g g c t g a a g a g '* ^ ^ Nl : ^^^ctctttgaaaaaggagaacagcttctaggagccggtgaggtctttgccatcagg^^® I l : ‘‘^ ^ c t c t t t g a g a a a g g a g a a c a g c t c c t g g g g g c c g g c g a g g t c t t t g c c a t t g g a '* ^ ^ Nl ; ^’^cccttgcagctctatgctatcacagaacagctgcagcagggaa'*^^ I l : ‘‘^ ^ c c c c tg c a g c tg ta tg c c g tc a c g g a g c a g c tg c a g c a g g g a a ^ ^ ® B ) Nl 1 ELVDTSTVYVIQ V T D G N H E W T I K H R Y S D F H D L H E K L V A E R K I D K T L L P P K K I I G K N S R S L 60 E LVDT-TVY+IQ V T D G + H E W T + K H R Y S D F H D L H E K L V A E R K I D K - L L P P K K I I G K N S R S L Il 25 ELVDTYTVYIIQ V T D G S H E W T V K H R Y S D F H D L H E K L V A E R K I D K N L L P P K K I I G K N S R S L 84 Nl 61 V E K R E K D L E V Y L H T L L K T F P D V A P R V L A H F L H F H L Y E I N G V T A A L A E E L F E K G E Q L L G A G 120 V E K R E K D L E V Y L - - L L — FP-V-PRVLAHFLHF H - Y E I N G + T A A L A E E L F E K G E Q L L G A G Il 85 V E K R E K D L E V Y L Q K L L A A F P G V T P R V L A H F L H F H F Y E I N G I T A A L A E E L F E K G E Q L L G A G 144 Nl 121 E V F A I R P LQLYAITEQLQQG 140 E V F A I - P LQLYA+TEQLQQG Il 145 E V F A IGPLQLYAVTEQLQQG 164

The interaction of GLT-N and the I, receptor was not further investigated in this study. It will be interesting to further characterise this interaction. If GLT-1 and the I, receptor prove to be true interactors, this research could lead to a better understanding of the function of both proteins in the CNS.