Segunda hipótesis especifica

The single bands obtained for the above genomic PCRs were NA-45 paper-purified (section 2.6.3.1) and cloned into the plasmid vector pCR2 (Invitrogen, see sections 2.6.4.1, 2.6.5 and 2.6.6). Desired recombinants were selected by colony PCR (section 2.6.8) and three clones were sequenced for each product in order to map the intron/exon boundary and to obtain intronic sequence information.

The size of the introns is given by the difference between the size of the amplified genomic PCR fragment and that calculated from the cDNA sequence. This information, shown in table 3.3 shows that, whilst particular intron sizes are very roughly conserved between the mouse, dog and man, there is no consistent relative sizing for each of the different species. Figure 3.24 shows the positions of the introns relative to the cDNA sequence whilst figure 3.25 extends this information to include intronic sequence adjacent to these exons.

The positions of the introns map to exactly the same nucleotide positions as those in

the human sequence (Weber et at, 1991) indicating that the genomic structure, as well as the

nucleotide and amino acid sequence conservation is extremely high. All intron/exon boundary sequences conform to the 'GT' ('GU' in RNA) consensus at the extreme 5’ end of the intron and a pyrimidine (somewhat interrupted) stretch followed by an 'AG' dinucleotide at the 3' end of the intron. In the vicinity of the 3' intronic region, there is at least one A residue which is situauted between 18 and 37 bases upstream of the 'AG' which would theoretically be candidates for the 2' hydroxyl bond with the 5' phosphate group of the G nucleotide which is thought to stabilise the lariat structure formed during RNA splicing (Cech,

1983, 1986).

The sequence information generated for the end of each exon is sufficient (except for the 5' end of intron 16, which requires further sequencing) to design oligonucleotide primers which can be used to screen the entire coding sequence plus splice donor and acceptor sites for exons 17 to 22 from genomic DNA. This characterisation will be useful when attempting to screen dogs with GPRA from breeds in which this candidate gene is being investigated.

Characterisation o f pdeb in normal dogs

1 93 0 1940 1950 1960 1970 1980 _________ F 6 - > _________________________ C A A G

GAG GAG ACC CTG AAC ATC TAC CAG AAC CTG AAC CGG CGG CAG CAC GAG CAC GTG ATC CAC

E E T L N I Y Q N L N R R Q H E H V I H

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

1 9 9 0 _ , . J 2 0 0 0 2 0 1 0 2 0 2 0 E x o n i 6 / E x o n 1 7 2 04 0 CTC ATG GAC AT.A GCC ATC ATC GCC ACC GAC CTG GCA CTC TAC TTC AAG AAG AGG ACA ATG

L M D I A I I A T D L A L Y F K K R T M

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * H O M O L O G O U S d o m a i n* * * * * * * * * * * * * * * * * * * * * * * * * * 2 0 5 0 2 0 6 0 2 0 7 0 2 0 8 0 2 0 9 0 2 1 0 0

* * * x 17F -> __________________* TTC CAG AAG ATC GTG GAT GAG TCT AAG AAC TAC GAG GAC AGG AAG AGC TGG GTG GAG TAC

F Q K I V D E S K N Y E D R K S W V E Y 2 1 1 0 2 1 2 0 E x o n i 7 /E x o n 1 8 2 1 5 0 2 1 6 0

* <-X17R ____________ W 1 1 1->_______________ .CTG TCC CTG GAG ACG ACG CGG AAG GAG ATA GTC ATG GCC ATG ATG ATG ACG GCG TGT GAC

L S L E T T R K E I V M A M M M T A C D 2 1 7 0 2 1 8 0 2 1 9 0 E x o n 1 8 /E x o n 1 9 2 2 1 0 2 2 2 0

* ______________ <-X18R * * * CTG TCT GCC ATC ACC AAG CCC TGG GAA GTC CAG AGC AAG GTT GCT CTG TTG GTG GCG GCT

L S A I T K P W E V Q S K V A L L V A A 2 2 3 0 2 2 4 0 2 2 5 0 2 2 6 0 E x o n i 9 / E x o n 2 0 XI 9F-> _________ W 88-> ____________________ <-X19R

GAG TTC TGG GAA CAA GGG GAC TTG GAA AGA ACA GTT CTG GAT CAG CAG CCT ATT CCG ATG E F W E Q G D L E R T V L D Q Q P I P M

2 2 9 0 2 3 0 0 2 3 1 0 2 3 2 0 2 3 3 0 2 3 4 0 < - X 2 0 R _______________ X 20F->_______________ ATG GAC CGG AAC AAG GCA GCC GAG CTC CCC AAA CTA CAG GTT GGC TTC ATC GAC TTC GTG

M D R N K A A E L P K L Q V G F I D F V 2 3 5 0 E x o n 2 0 / E x o n 2 1 2 3 7 0 2 3 8 0 2 3 9 0 2 4 0 0

T * * * * ________ < -W 1 0 0 /W 1 0 1 -> _____ TGC ACG TTC GTG TAC AAG GAG TTT TCC CGT TTC CAC GAA GAG ATC CTG CCC ATG TTC GAC

C T F V Y K E F S R F H E E I L P M F D 2 4 1 0 2 4 2 0 2 4 3 0 2 4 4 0 2 4 5 0 2 4 6 0

- A

CGA CTG CAG AAC AAC AGG AAG GAA TGG AAG GCC TTG GCT GAT GAG TAC GAG GCC AAG CTG R L Q N N R K E ^ K A L A D E Y E A K L

2 4 7 0 2 4 8 0 2 4 9 0 2 5 0 0 E x o n 2 1 / E x o n 2 2 * * <-X21R <-R 4/R 4B _____ AAG GCC CTG GAG GAG GAG AAG CAG CAA CAA GAG GAC AGG ACG ACA GCC AAG AAA GCG GGC

K A L E E E K Q Q Q E D R T T A K K A G 2 5 3 0 2 5 4 0 2 5 5 0 2 5 6 0 2 5 7 0 2 5 8 0 _________ * * <-X22R______

ACA GAG ATT TGC AAC GGT GGC CCC GCA CCC AAG TCT TCC ACC TGC TGC ATC CTG TGA GCT T E I C N G G P A P K S S T C C I L *

CAAX-MOTIF

Figure 3.24. Nucleotide sequence o f PDEB cDNA showing the positions o f the introns 16-21. The primers used to amplify across each intron are shown on the sequence. The nucleotides at the extreme 3' and 5' end o f each exon are shown in bold type. The map positions o f each intron is conserved in mouse and in man.

...EXON 16...CTACTTCAA gtgggtggccccccccccc...

.cccggggcgtcgactctttgttcttggggttcagbAAGAGGAC...EXON 17...GATAGTCATlgtgagcagtgggggcgacaggcagccccccagctggtcgatc

tcagacgtccccccacaaaaactagaagaccccc... ...ggccagcagcgctctggagcttcgtctccgcgcag GGCCATGAT.. EXON 18...CAGAGCAAG|gtcagaagtcatgcagggcccctgcctcggggagaccaggga

gaggaagcctgcgggggg... ..gcagaggagggcaggagcctctttcctcaatttctgaccaagc

gctgaccccccaagatgactgccttttatgcacccacctcccaag GTTCGTCTG...EXON 19...CAGCCTATT gtgagtgttacttccagaatcttctacccaccaggctccttggta acctcgggcctcgcttggtgcacacagaggacccctggggact tgg^tcggagcatcagtctcactttgtggggttggtcatgtga cacgag...

-«-ggggctgcgagccgggccggggccgagtc________________________________

to ctgcaggctcagaggaggtgtctctgctcccccacaag | CCGATGATG...EXON 20...GTGTACAAG I gtgagccccagcgtgcatcggggtcacaggcgggcggccgcc

(X) gggctggcccggggctgacctgccgcagccccagcctctgcc

ccccggcagcttagggcctgaaagccgcag..

"tggtgtcag

caagggccaggccgtggagccgccacctgcccccactgcctgg________________________________

gaactgtcgagaggtgtggtccgacctcctgattctgtgtccag IGAGTTTTCC...EXON 21...CCAAGAAAGI gtctggccgcaggggtgggcacgggttagccgcc

...gccgaggcggggctggctaatcggctcaggcggg caccggggccgaggaggacggccggggagcaggccgtgctc______________________ accctcgtgctcaccctcccctcaccgtcccgtctctctcgcag |CGGGCACAG...EXON 22... cgcgagctgtaaagggacagtggggcagcagag D gcgagcctggaggcaggcaggtggctcgcggcac | ggggaccctcaggcgcgggctggcgagcgggaa g. cagggaagggcc... 3.

I

Figure 3.25. Exon/Intron boundaries and genomic sequences mapping the six exons at the extreme 3' end of canine PDEB.

Characterisation o f p d e b in norm al dogs