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PANORAIA KYRIAZOPOULOU
ER A SM U S + P L A C E M E N T 2 0 15 - 2 0 1 6
Analysis of 2 unique Greek MHC haplotypes
What is the MHC?
The MHC – Major Histocompatibility Complex is a genetic region comprised of tightly linked genes.
It has been referred to as ovine leukocyte antigen (OLA) or sheep lymphocyte antigen and now following the nomenclature system for the MHC of vertebrates (Klein et al., 1990), it is designated as ‘Ovar-Mhc’ (‘Ovar’ representing Ovis aries).
What is the purpose of the MHC?
Purpose:Encoding molecules playing a central role in
immunological response.Codes for specialised antigen-presenting receptor
glycoproteins, known as histocompatibility molecules or MHC molecules.
These molecules bind processed peptide antigens and present them to T lymphocytes triggering immune responses.
Communication between cells during immune response.
Why do we study the MHC?
• Alleles of different MHC genes have been found to contribute in disease resistance/susceptibility.
• The MHC alleles are markers of biodiversity.
• Selection for productivity traits/ disease research.
• Mate choice for many vertebrates through olfactory cues.
MHC Genes
CLASS I GENESClassical and non-classical genes.
Present peptides to CD8+ cytotoxic T cells.
Interact with natural killer (NK) cells to prevent NK-mediated cell lysis (Reyburn et al., 1997).
Controversy over the number of classical class I loci - at least four distinct polymorphic loci have been identified (Miltiadou et al., 2005).
MHC Genes
CLASS II GENESClass II genes have antigen peptide presenting
role to the TCR on CD4+ helper T cells.
In the HLA complex, these include five sets of the classical genes DP, DM, DO, DQ, and DR and non-classical genes such as LMP, TAP and TAPBP.
Not all sets contain genes for both chains, although some contain many pseudogenes (Tizard, 2004).
MHC GenesOvar-DR genes DR genes highly polymorphic molecules encoded by these genes are expressed in
higher levels.The DR heterodimer consists of an α-chain and a β-
chain, encoded by DRA and DRB genes. Class II region of the MHC of sheep (OLA).
DRA genesDRA gene is considered to be almost monomorphic. A significantly divergent DRB1 allele (Ovar-
DRB1*0901), was linked to polymorphism at the DRA locus in domestic sheep (Ballingall et al., 2010).
MHC Genes
Ovar-DR genes
DRB genesDRB locus is the most polymorphic among the
MHC genes. Ovar-DRB genes exist in multiple copies, some
functional and others non-functional. Four Ovar-DRB loci have been described (Scott et
al. 1991b). Majority of nucleotide polymorphism at Class II loci
locates in the second exon and adjoining intron 2.
MHC Genes
Ovar-DQ genes
DQA genesTwo loci, DQA1 and DQA2, exon 2A number of haplotypes the DQA1 gene appears
absent (DQA1 null; Fabb et al. 1993) and is replaced by a second locus more closely related to DQA2 (DQA2-like; Hickford et al. 2004)
DQA2-like allelic lineage appears functional (Ballingall et al., 2015).
MHC Genes
Ovar-DQ genes
DQB genesDQB1 and DQB2- exon 2 (Wright and Ballingall,
1994). Difficulty in assigning sequences to separate loci
because of high similarity between the two DQB genes.
Purpose of study
Characterising the polymorphism in the Ovar-MHC region in a rare Greek sheep breed ~Argos.
Genes studied: DRB1, DRA, DQA and DQB (partial as well as full-length allelic sequences).
Materials and methods
Argos breed:Originally from Asia Minor. Single pure flock in Messinia.Six flocks near Argos with over 50% purity.Isolated pure specimens in mixed flocks.
Materials and methods
Breed details:Officially recognised breedArea of distribution: Messinia, PeloponnesePopulation size: 100 animalsRisk status: endangeredColour: white with black headFat tailedWeight ram: 70 kg; ewe: 59 kgHeight ram: 85 cm; ewe: 70 cmUse: milk, meatProductivity milkyield: 140-160kg; littersize: 1.5-1.8
Materials and methods
At the start of this study, blood was taken from the 2 Argos sheep.
Genomic DNA was prepared. RNA was extracted and brought to the Moredun
Research Institute.
Materials and methods
DRB1 genotyping:
Primers used for sequence based genotyping were 455/329 (Ballingall and Tassi, 2010, and unpublished data).
PCR cycling profile: 1 cycle 5 minutes, 94o C,
35 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C followed by 4 min at 72°C.
Materials and methods
DRB1 genotyping:
For the mother (Animal 251 or Argos 28) the 3-prime end was amplified by designing a forward allele-specific primer 462 (Unpublished data).
Nested approach :primers 455 and 201 annealing temperature at 55°C for 30 cycles
then 2ul PCR product added into the second round with the new primer and 201
annealing temperature of 60 °C for 35 cycles.
Materials and methods
DRB1 Full-length genotyping:primers 204/207 and 205/207 (Ballingall et al.,
2008) internal primers 222/223 control primers 414/415
PCR cycling profile: 1 cycle 5 minutes, 94o C,
55 cycles of 1min at 94°C, 1 min at 60°C, 1 Min at 72°C
followed by 4 min at 72°C
Materials and methods
DQA/DQB genotyping
Primers: DQA1: 314/315 DQA2: 316/317
DQA2-like: 316/317 DQB1: 363n/ 365n
DQB2:363n/406
PCR cycling profile: 1 cycle 5 minutes, 94o C,
35 cycles of 30 s at 94°C, 30 s at 58°C, 30 s at 72°C followed by 4 min at 72°C.
DQA/DQB full-length genotyping:
Primers: DQA1/2: 283/241 244/241 347/349 DQB1/2: 245/247 246/248
PCR cycling profile: 1 cycle 3 minutes, 94o C,
35 cycles of 30 s at 94°C, 30 s at 55°C, 30 s at 72°C followed by 3 min at 72°C.
Materials and methods Class I genotyping:
Primers: 415/409
PCR cycling profile: 1 cycle 5 minutes, 94o C,
30 cycles of 30 s at 94°C, 30 s at 55°C, 30 s at 72°C followed by 4 min at 72°C.
Class I full-length genotyping:
Primers: 417/411 417/412 410/412 413/411
413/412
PCR cycling profile: 1 cycle 5 minutes, 94o C,
30 cycles of 30 s at 94°C, 30 s at 55°C, 30 s at 72°C followed by 4 min at 72°C.
Materials and methods
Efficiency of PCR was checked on a 1% agarose .
Remaining PCR product purified.Bidirectional sequencing.
After receiving the first results the selected PCR fragment was cloned, screened and digested with RSAI and sent for sequencing.
Materials and methods
Levels of hybridisation were very high for the Class I using a PCR profile of 30 cycles.
New cDNA was prepared and PCR using primers 416/409 was repeated.
The samples were removed after 12, 14, 16, 18, 20, 22, 25 and 28 cycles and ran on agarose gels 1%.
Click icon to add pictureAgarose gel with PCR sample removed after 18, 20 and 22 cycles
18 cycles
20 cycles
22 cycles
Click icon to add pictureAgarose gel with PCR sample removed after 25 and 28 cycles
25 cycles
28 cycles
Materials and methods
The gel fragment for 16, 18 and 20 cycles was cut out and cleaned up.
PCR screening showed that the transformation worked effectively and samples from colonies selected were sent for sequencing.
The results received after this procedure showed significantly lower levels of hybridisation.
Results
New DQA1 and DQA2 alleles found.
New DQB1 and 2 DQB2 alleles found.
5 new classical Class I alleles found.
2 new non-classical Class I alleles found.
High levels of diversity
Results
Optimal PCR cycles for lower levels of hybridisation in Class I : 16-20
Hybrids at 30 cycles: 11/30 and PCR errors
Hybrids at 16-20 cycles: 1/40, few PCR errors
This may mean some alleles that appear in low frequencies might be missed.