Restriction Endonuclease mapping
Part
II: Construct Restriction Maps of several
subspecies of A. mellifera likely to
be found in Newfoundland hives. Identify Restriction
Endonucleases that could be used to identify species, based
on PCR fragment data in Part I.
1) Retrieve complete mtDNA genome
sequence for Apis mellifera mellifera voucher
1410: accession
KY926884
Retrieve complete mtDNA genome from [two] additional
subspecies of A. mellifera, as instructed.
2) For these subspecies:
Choose Complete Record / File / GenBank:
GenBank:
Create File as KY926884.gb
[File
*.gb is a flatfile that
includes coordinates for genes]
Choose Complete Record / File / GenBank:
FASTA: Create
file as: KY926884.FASTA
[File *.FASTA is the complete sequence, with
leading string to identify it]
3) Start a new Alignment.
Open MEGA:
Click on ALIGN icon;
choose Align / Edit;
select "Insert sequence from
file";
select KY926884.fasta and the other
subspecies *.fasta files.
The primer pair amplifies a
region that includes the genes NADH2,
Cox1 & the 5' end of Cox2,
near the 5' end of the genome.
Three tRNA
genes: last one tRNA-Trp
runs [1722
- 1793] = 71 + 1 =
72 bp
Second ORF: "cytochrome c
oxidase subunit 1" [1794
- 3354] = 1560 + 1 = 1561 bp
One tRNA gene:
tRNA-Leu runs
[3355 - 3424] = 69
+ 1 = 70 bp
Third ORF: "cytochrome
c oxidase subunit 2"
[3618 - 4295] = 677 + 1
= 678 bp
Two tRNA genes: tRNA-Asp
runs
[4294 - 4362]
= 68 + 1 =
69 bp
Region spans [1722 - 4362] = 2640 +1 = 2,641 bp
4)
Align all sequences
by eye. Trim all sequences to the 2,641
bp target: remove 5' end, remove 3'
end
5) Identify all
Restriction Sites in each
sequence with RestrictionMapper
v.3. [Parameters TBD]
Construct a restriction map for
the [three] subspecies as
directed.
4) Compare maps. identify Restriction
sites that could be used to distinguish the subspecies in
your set.
Important note: the mtDNA
Genetic Code for invertebrates differs in
several respects from the "Universal" code. Note
especially that AUA, AUU, and in Apis honeybees
AUC can act as Start (Initiation) Codons. This means
that the first amino acid in Apis ORFs can
be Isoleucine (Ile, I) instead of
the expected Methionine (Met, M).
Also, UGA codes for Tryptophan (Trp,
W) instead of acting as a Stop Codon.