JalView

Alignment as a reflection of evolution

Task 1

We have to make a sequence alignment of 6 sequences of proteins from family HSP70. I used the program "seqret" (EMBOSS) to load these sequences of proteins in format FASTA. Then I merged into one file and made an alignment by Jalview.

Here You can see some information about proteins I used:
Table 1.

Entry	Entry name	Protein name	Organism	Supergroup
Q8TQR2	DNAK_METAC	Chaperone protein DnaK (HSP70)	Methanosarcina acetivorans (strain ATCC 35395 / DSM 2834 / JCM 12185 / C2A)	Archaea
Q18GZ4	DNAK_HALWD	Chaperone protein DnaK (HSP70)	Haloquadratum walsbyi (strain DSM 16790 / HBSQ001)	Archaea
Q13E60	DNAK_RHOPS	Chaperone protein DnaK (HSP70)	Rhodopseudomonas palustris (strain BisB5)	Bacteria
Q73GL7	DNAK_WOLPM	Chaperone protein DnaK (HSP70)	Wolbachia pipientis wMel	Bacteria
Q74ZJ0	HSP7F_ASHGO	Heat shock protein homolog SSE1	Ashbya gossypii (strain ATCC 10895 / CBS 109.51 / FGSC 9923 / NRRL Y-1056) (Yeast) (Eremothecium gossypii)	Eukaryota
Q8SQR8	HSP7F_ENCCU	Heat shock protein homolog SSE1	Encephalitozoon cuniculi (strain GB-M1) (Microsporidian parasite)	Eukaryota

*HSP70 = Heat shock protein 70 (Heat shock 70 kDa protein)

The alignment was made by Jalview by program Tcoffee with Defaults, colored by ClustalX with Identity Threshold = 100%. There are labels (identity 80%, plurality 100%, gaps): C - positions which are conserved 80% or more, F - absolutely functionally conserved, G - positions with gaps. To see the whole alignment press here.

About functionally conserved:
1. L and I are aiphatic amino acid residues with tne same numbers of atoms;
2. T and S have an alcohol group (-OH);
3. K and R are positively charged amino acid residues

Picture 1. The part of alignment of 6 sequences of proteins from family HSP70 wis labels.

The datas about alignment was taken by program "infoalign" (EMBOSS).

Table 2. Data when the conserved 100%.

Name	SeqLen	AlignLen	GapLen	% of GapLen	Ident	% of Ident	Similar	% of Similar
DNAK_METAC_1-617	617	847	230	27,15	31	3,66	0	0
DNAK_HALWD_1-641	641	847	206	24,32	31	3,66	0	0
DNAK_RHOPS_1-633	633	847	214	25,27	31	3,66	0	0
DNAK_WOLPM_1-640	640	847	207	24,44	31	3,66	0	0
HSP7F_ASHGO_1-697	697	847	150	17,71	31	3,66	0	0
HSP7F_ENCCU_1-658	658	847	189	22,31	31	3,66	0	0

Table 3. Data when the conserved 70%.

Name	SeqLen	AlignLen	GapLen	% of GapLen	Ident	% of Ident	Similar	% of Similar
DNAK_METAC_1-617	617	847	230	27,15	284	33,53	12	1,42
DNAK_HALWD_1-641	641	847	206	24,32	282	33,29	9	1,06
DNAK_RHOPS_1-633	633	847	214	25,27	294	34,71	4	0,47
DNAK_WOLPM_1-640	640	847	207	24,44	296	34,49	4	0,47
HSP7F_ASHGO_1-697	697	847	150	17,71	129	15,23	53	6,26
HSP7F_ENCCU_1-658	658	847	189	22,31	91	10,74	57	6,73

Table 4. Data when the functionally conserved 100%. (calculate by python script with matrix BLOSUM62)

Name	SeqLen	AlignLen	GapLen	% of GapLen	Ident	% of Ident	Similar	% of Similar
DNAK_METAC_1-617	617	847	230	27,15	31	3,66	110	12,00
DNAK_HALWD_1-641	641	847	206	24,32	31	3,66	110	12,00
DNAK_RHOPS_1-633	633	847	214	25,27	31	3,66	110	12,00
DNAK_WOLPM_1-640	640	847	207	24,44	31	3,66	110	12,00
HSP7F_ASHGO_1-697	697	847	150	17,71	31	3,66	110	12,00
HSP7F_ENCCU_1-658	658	847	189	22,31	31	3,66	110	12,00

Table 5. Average values.

AlignLen	GapLen	%	Identity
			identity 100%	%	plurality 100%	%	identity 70%	%
847	199,33	23,53	31	3,66	246	29,04	31	3,66

Task 2

I used a sequence of protein called Human Insulin. With help of "msbar" (EMBOSS) I have done 7 generations of mutant insulin (7 artificial point mutations in every generation), then I combined these mutant generations and the origin one in the file (see "Bash script").

The alignment was made by Jalview by program Tcoffee with Defaults, colored by ClustalX with Identity Threshold = 100%. To see the whole alignment press on picture or here.

Picture 2. The alignment made by program.

Table 5. The list of artificial mutations in insulin.

Position	Type of point mutation	Generation
2	insert of S	p3 -> p4
3	deletion of A	p4 -> p5
9	insert of N	p1 -> p2
10	insert of L	p1 -> p2
11	insert of L	p1 -> p2
12	insert of L	p6 -> p7
21	insert of N	origin -> p1
29	deletion of A	p2 -> p3
29	insert of A	p5 -> p6
30	insert of Y	p2 -> p3
31	insert of A	p2 -> p3
36	insert of V	p5 -> p6
75	replacement R to S	p3 -> p4
78	replacement E to R	origin -> p1

Picture 3. The alignment made by program with some changes which I did.

I had to change some items in the alignment (To see the whole alignment press here):

• There wasn't any amino acids before A in the origin sequence (insgead of M at the start of sequence), so I decided that there were an insert of S (position 2, p3 -> p4) and a deletion of A (position 3, p4 -> p5). Also, I fixed a computer alignment and changed the insert of S (position 2, p3 -> p4) and replacement A to S (position 3, p4 -> p5).
• According the origin sequence the are insert of H (position 69, p2 -> p3) and conserved K (position 70). So, I fixed a computer alignment again and changed the replacement K to H (position 69, p2 -> p3) and insert of K (position 70, p2 -> p3).

Task 3

I used the nucleotide sequence of the gene of the protein called Human Insulin. With help of "msbar" (EMBOSS) I have done 7 generations of mutant insulin gene. Than with help "transeq" (EMBOSS) I made the transcriptions and constructed alignment of the respective mutant proteins (you can see the script). The alignment was made by Jalview by program Tcoffee with Defaults, colored by ClustalX with Identity Threshold = 60%.