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BTEC 3301 Lecture Notes - Lecture 4: Wild Type, Protein Isoform, Emboss

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orchidcricket59
4 May 2018
School
University of Houston
Department
Biotechnology
Course
BTEC 3301
Professor
Alvert

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BTEC 3301
Research Task 04
20 pts.
Task 4: Sequence Alignment
1) Show your strategy/tools/results by pasting snips and highlighting the important info in
those snips. From Task 2, take both your disease sequence and the normal sequence for
the protein and perform pairwise alignments using the tools specified below.
a. Draw a manual dot plot that includes at least 10 amino acids upstream and
downstream of the mutation. If your mutated amino acid is the first aa (Met),
go ahead and change the 11th wild-type amino acid instead to a different amino
acid belonging to the same physicochemical grouping as the wild-type amino
acid (see lecture 12 slide 55).
-
L
R
R
G
V
Q
A
Q
L
N
V
G
F
C
E
Q
E
F
E
Q
L
R
R
G
V
Q
A
Q
P
L
N
V
G
F
C
E
Q
E
F
E
Q
There should have been a gap at the circled area. However, there is no gap. This
is because the nucleotide sequence change did not result in an amino acid
change.
b. Do a DotMatcher of the entire mutated and normal protein sequences using the
default parameters first. Snip the resulting dot plot (into the table below). Are
you able to see the changed amino acid as a gap in the diagonal? Change a
default parameter until you are able to see the mismatch in the 2 sequences.
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Snip your changed parameter and the resulting dot plot. Is it easy to discover
the presence of 1 aa difference between two protein sequences using dot plots?
Default parameters
Optimized parameters
Window size: 10
Threshold 23
Window size: 10
Threshold: 42
Default:
Optimized:
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c. Perform a global protein alignment using the EMBOSS Needle. Since the two
sequences are simply alleles of the same gene and there is a single amino acid
change, use the PAM substitution matrix that would be most appropriate to use.
Snip the alignment and highlight the mismatch. What is the % identity? 100 %
similarity? 100%
Used PAM10
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Document Summary

Task 4: sequence alignment: show your strategy/tools/results by pasting snips and highlighting the important info in those snips. There should have been a gap at the circled area. This is because the nucleotide sequence change did not result in an amino acid change. L r r g v q a q p l n v g f c e q e f e q: do a dotmatcher of the entire mutated and normal protein sequences using the default parameters first. Snip the resulting dot plot (into the table below). Change a default parameter until you are able to see the mismatch in the 2 sequences. Snip your changed parameter and the resulting dot plot. Optimized: perform a global protein alignment using the emboss needle. Since the two sequences are simply alleles of the same gene and there is a single amino acid change, use the pam substitution matrix that would be most appropriate to use.

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Related Questions

NEED HELP PLEASE. CAN YOU PLEASE DO IT ALL NOT JUST ANSWER ONE.

MY ANIMALS ARE: HUMAN, KANGAROO, RHESUS MONKEY, BULLFROG, LAMPREY, SNAPPING TURTLE, TUNA, AND EARTHWORM

Step 3: Cytochrome c Analysis

Scientists have found that almost all living organisms have a gene that codes for a protein called Cytochrome c. This chart lists the amino acids found in the Cytochrome c protein of some of the organisms on your cladogram.

Note: not all the organisms you used will be in the chart; as such, you can focus this analysis on the animals that are in the chart. If you only chose three animals that are in the chart, you should probably extend your amino acid analysis to include a couple animals that aren’t on your cladogram.

You will use the data provided in this chart to compare and analyze the amino acid sequences for Cytochrome c among these organisms. Be sure that your comparison considers the entire amino acid sequence, through amino acid 112, and not just the first lines of the chart. Once you have compared the sequences, you will write a discussion about your findings.

Amino Acid Sequences in Cytochome-C Proteins

1-30

Amino Acid Position Number

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Human, Chimpanzee

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

I

M

K

C

S

Q

C

H

T

V

E

K

Rhesus Monkey

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

I

M

K

C

S

Q

C

H

T

V

E

K

Horse

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

V

Q

K

C

A

Q

C

H

T

V

E

K

Pig, Cow, Sheep

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

V

Q

K

C

A

Q

C

H

T

V

E

K

Dog

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

V

Q

K

C

A

Q

C

H

T

V

E

K

Gray Whale

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

V

Q

K

C

A

Q

C

H

T

V

E

K

Rabbit

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

V

Q

K

C

A

Q

C

H

T

V

E

K

Kangaroo

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

V

Q

K

C

A

Q

C

H

T

V

E

K

Penguin

-

-

-

-

-

-

-

-

G

D

I

E

K

G

K

K

I

F

V

Q

K

C

S

O

C

H

T

V

E

K

Snapping Turtle

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

V

Q

K

C

A

Q

C

H

T

V

E

K

Bullfrog

-

-

-

-

-

-

-

-

G

D

V

E

K

G

K

K

I

F

V

Q

K

C

A

Q

C

H

T

C

E

K

Tuna

-

-

-

-

-

-

-

-

G

D

V

A

K

G

K

K

T

F

V

Q

K

C

A

Q

C

H

T

V

E

N

31-60

Amino Acid Position Number

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

Human, Chimpanzee

G

G

K

H

K

T

G

P

N

L

H

G

L

F

G

R

K

T

G

Q

A

P

G

Y

S

Y

T

A

A

N

Rhesus Monkey

G

G

K

H

K

T

G

P

N

L

H

G

L

F

G

R

K

T

G

Q

A

P

G

Y

S

Y

T

A

A

N

Horse

G

G

K

H

K

T

G

P

N

L

H

G

L

F

G

R

K

T

G

Q

A

P

G

F

T

Y

T

D

A

N

Pig, Cow, Sheep

G

G

K

H

K

T

G

P

N

L

H

G

L

F

G

R

K

T

G

Q

A

P

G

F

S

Y

T

D

A

N

Dog

G

G

K

H

K

T

G

P

N

L

H

G

L

F

G

R

K

T

G

Q

A

P

G

F

S

Y

T

D

A

N

Gray Whale

G

G

K

H

K

T

G

P

N

L

H

G

L

F

G

R

K

T

G

Q

A

V

G

F

S

Y

T

D

A

N

Rabbit

G

G

K

H

K

T

G

P

N

L

H

G

L

F

G

R

K

T

G

Q

A

V

G

F

S

Y

T

D

A

N

Kangaroo

G

G

K

H

K

T

G

P

N

L

N

G

I

F

G

R

K

T

G

Q

A

P

G

F

T

Y

T

D

A

N

Penguin

G

G

K

H

K

T

G

P

N

L

H

G

I

F

G

R

K

T

G

Q

A

E

G

F

S

Y

T

D

A

N

Snapping Turtle

G

G

K

H

K

T

G

P

N

L

N

G

L

I

G

R

K

T

G

Q

A

E

G

F

S

Y

T

E

A

N

Rattlesnake

G

G

K

H

K

T

G

P

N

L

H

G

L

F

G

R

K

T

G

Q

A

V

G

Y

S

Y

T

A

A

N

Bullfrog

G

G

K

H

K

V

G

P

N

L

Y

G

L

I

G

R

K

T

G

Q

A

A

G

F

S

Y

T

D

A

N

Tuna

G

G

K

H

K

V

G

P

N

L

W

G

L

F

G

R

K

T

G

Q

A

E

G

Y

S

Y

T

D

A

N

61-90

Amino Acid Position Number

61

62

63

64

65

66

67

38

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

Human, Chimpanzee

K

N

K

G

I

I

W

G

E

D

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Rhesus Monkey

K

N

K

G

I

T

W

G

E

D

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Horse

K

N

K

G

I

T

W

K

E

E

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Pig, Cow, Sheep

K

N

K

G

I

T

W

G

E

E

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Dog

K

N

K

G

I

T

W

G

E

E

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Gray Whale

K

N

K

G

I

T

W

G

E

E

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Rabbit

K

N

K

G

I

T

W

G

E

D

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Kangaroo

K

N

K

G

I

I

W

G

E

D

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Penguin

K

N

K

G

I

T

W

G

E

D

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Snapping Turtle

K

N

K

G

I

T

W

G

E

E

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Rattlesnake

K

N

K

G

I

I

W

G

D

D

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

V

F

Bullfrog

K

N

K

G

I

T

W

G

E

D

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

Tuna

K

S

K

G

I

V

W

N

N

D

T

L

M

E

Y

L

E

N

P

K

K

Y

I

P

G

T

K

M

I

F

91-112

Amino Acid Position Number

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

Human, Chimpanzee

V

G

I

K

K

K

E

E

R

A

D

L

I

A

Y

L

K

K

A

T

N

E

Rhesus Monkey

V

G

I

K

K

K

E

E

R

A

D

L

I

A

Y

L

K

K

A

T

N

E

Horse

A

G

I

K

K

K

T

E

R

E

D

L

I

A

Y

L

K

K

A

T

N

E

Common Zebra

A

G

I

K

K

K

T

E

R

E

D

L

I

A

Y

L

K

K

A

T

N

E

Pig, Cow, Sheep

A

G

I

K

K

K

G

E

R

E

D

L

I

A

Y

L

K

K

A

T

N

E

Dog

A

G

I

K

K

T

G

E

R

A

D

L

I

A

Y

L

K

K

A

T

K

E

Gray Whale

A

G

I

K

K

K

G

E

R

A

D

L

I

A

Y

L

K

K

A

T

N

E

Rabbit

A

G

I

K

K

K

D

E

R

A

D

L

I

A

Y

L

K

K

A

T

N

E

Kangaroo

A

G

I

K

K

K

G

E

R

A

D

L

I

A

Y

L

K

K

A

T

N

E

Penguin

A

G

I

K

K

K

S

E

R

A

D

L

I

A

Y

L

K

D

A

T

S

K

Snapping Turtle

A

G

I

K

K

K

A

E

R

A

D

L

I

A

Y

L

K

D

A

T

S

K

Rattlesnake

T

G

L

K

K

K

K

E

R

T

D

L

I

A

Y

L

K

E

A

T

A

K

Bullfrog

A

G

I

K

K

K

G

E

R

Q

D

L

I

A

Y

L

K

S

A

C

S

K

Tuna

A

G

I

K

K

K

G

E

R

Q

D

L

V

A

Y

L

K

S

A

T

S

-

More information:

Cytochrome c is a protein used in cellular respiration by most eukaryotes, which is why it's a very common protein to use for comparisons. You don't need to know anything about cellular respiration or the protein itself to do this analysis; just remember that a protein is a specific chain of amino acids.

Which amino acids are found, and the order in which they're found (their sequence), determine the shape, structure, and function of the protein. Also remember that an amino acid sequence is directly determined by the DNA sequence, so similarities in one also indicate similarities in the other.

Please note that the entire sequence of 112 amino acids represents one protein, and this sequence data is all one chart, not four. As such, your analysis should consider the sequence as a whole, and not amino acids 1 – 30, then amino acids 31 – 60, etc.

Be careful not to read too much into the differences you find. Since this analysis only looks at one protein, you will not be able to connect differences here to specific differences in traits between organisms. In other words, it might be that all animals with a placenta have a “V” at a particular location, while animals without a placenta have either a “C” or a “K.” That similarity helps us confirm that those animals with a placenta are more closely related to each other than to the others, but the “V” does not code for the placenta, nor is it related to that particular trait at all.

How to Analyze the Data

While you analyze the data that you are given to identify similarities and differences, be sure to use specific data to draw your comparisons, hypotheses, and discussions from. It is especially helpful to quantitatively compare data. A good way to do this, or at least to start, is to pick one species as a starting point or ‘baseline’ and then compare the amount of differences between that and each other species. Be careful, though, about the conclusions you draw – if species B and C each have 5 differences from species A, then they could have anywhere from 0 to 10 differences with each other!

Once you’ve compared each animal to a baseline animal, you should also compare other pairs of species so that you can discuss their specific relationships. If you only have a few animals with data to analyze in part C, you could simply make this comparison for every pair. If you have 6 or 7 animals, then you could pick just a few pairs to compare.

There are other legitimate ways to form quantitative data – for instance, you could focus on specific locations where most animals have the same amino acid, but some are different. These types of analysis tend to be harder to interpret, though. If you choose to use them, I suggest that you still complete at least some of the above types of analysis as well.

The discussion topics that I'm looking for are:

Use the chart to compare the amino acid sequences for the organisms. Do this in a way that lets you obtain quantitative (numerical) data; see above for suggestions.

Hypothesize about how closely related the different organisms are, based on the amino acid data in the chart.

In particular, focus on organisms that are also found on your phylogenetic tree, but you may choose to discuss others, too. That would be especially helpful if you used only three or four of the organisms that are also in the chart, or if one or more organisms that you did not include in your tree jump out at you as being especially similar or especially different from the ones you did include.

This does not need to be in the form of a formal hypothesis; that is, you do not need to put this in “if/then/because” form.

Because “how closely related” can be interpreted in many ways, it’s often helpful to put this in relative terms: “Animal D is the closest relative to Animal A.” “Animals C and B are more closely related to each other than either one is to Animal D.”

Discuss how this analysis compares to the relationships that were shown on your phylogenetic tree.

To start, does the Cytochrome c data agree or disagree with the relationships shown on the tree?

Remember that "discuss" means more than simply identifying whether the relationships are the same or different as what is shown. You can also suggest why they're likely the same, or what possible differences could imply, if anything. See the note at the end of “More Information” above – don’t read TOO MUCH into any one piece of data!

If your amino acid analysis included animals that weren’t on your cladogram, you could use your amino acid data to speculate where they likely “fit” on the cladogram.

The phrase “common ancestor” should show up somewhere in this discussion.

Please be aware that Step 3 should be written as a short essay, not a list or set of questions and answers. You may include a chart or table for the quantitative data you identified, but the key findings should also be written in sentence form. Once you’ve finished writing this, please proofread for spelling, grammar, and writing clarity.

Step 3 should be a sizeable portion of this portfolio. I expect that your analysis should be about 250 – 400 words long in order to thoroughly analyze the data and discuss the questions.

For the following strand of DNA, use the table below to provide: bases for the complementary mRNA and the associated amino acids. What do you notice about the genetic code for proline in the peptide? How would the loss of the first G or switching that G to a T affect the structure and function of this protein?

DNA: GGC CTA CAA GCC GGA ACA GTT ACC GCG TAA CGA

RNA: ??? ??? ??? ??? ??? ??? ??? ??? ??? ??? ???

Amino Acid:??? ??? ??? ??? ??? ??? ??? ??? ??? ??? ???
(3 Letters)

Genetic Code Table (mRNA)

1st position
(5' end)

2nd position (middle)

 3rd position
(3' end)
U C A G
U Phe F
Phe F
Leu L
Leu L 		Ser S
Ser S
Ser S
Ser S		 Tyr Y
Tyr Y
STOP
STOP		 Cys C
Cys C
STOP
Trp W 		U
C
A
G
C Leu L
Leu L
Leu L
Leu L		 Pro P
Pro P
Pro P
Pro P		 His H
His H
Gln Q
Gln Q		 Arg R
Arg R
Arg R
Arg R		 U
C
A
G
A Ile I
Ile I
Ile I
Met M 		Thr T
Thr T
Thr T
Thr T		 Asn N
Asn N
Lys K
Lys K		 Ser S
Ser S
Arg R
Arg R		 U
C
A
G
G Val V
Val V
Val V
Val V		 Ala A
Ala A
Ala A
Ala A		 Asp D
Asp D
Glu E
Glu E		 Gly G
Gly G
Gly G
Gly G		 U
C
A
G

Instructions

BACKGROUND

The amino acid sequence for a protein that is associated with the onset of Huntington's Disease in humans is 582 amino acids long and is provided below. This disease is autosomal dominant and results in a prolonged degeneration of nerve cells in the brain.

INSTRUCTIONS

For this assignment, use any method you determine most feasible (i.e. programming in MATlab, R script, Crystal Ball, Witchcraft/Hocus Pocus, etc.) to decipher the amino acid composition of this protein and answer the 15 questions associated with this protein and its associated amino acid structure.

You will be calculating proportions (percentages - %) for this assignment. Calculate all proportions to 1 decimal place and make sure you have rounded up or down correctly. If unsure, use a spreadsheet program such as Excel to display your proportional calculations to 1 decimal place.

Spelling matters (Incorrect spelling will be graded as such - incorrect).

Huntington Disease Associated Protein Amino Acid Sequence

The first amino acid below ('m') represents the amino (NH3) terminus of this protein. The last amino acid in this sequence represents the carboxy (COOH) terminus and is the very last 'c' at the bottom of this protein sequence structure. The amino acids are numbered individually across the rows (left to right) beginning from the amino terminus (#1) to the carboxy terminus (#582).

mvskreniat

hhlyqawdpv

pslspattga

lisheklllq

inperelgsm

syklgqvsih

svwlgnsitp

lreeewdeee

eeeadapaps

spptspvnsr

khragvdihs

csqfllelys

rwilpsssar

rtpailisev

vrsllvvsdl

fternqfelm

yvtltelrrv

hpsedeilaq

ylvpatckaa

avlgmdkava

epvsrllest

lrsshlpsrv

galhgilyvl

ecdllddtak

qlipvisdyl

lsnlkgiahc

vnihsqqhvl

vmcatafyli

enypldvgpe

fsasiiqmcg

vmlsgseest

psiiyhcalr

glerlllseq

lsrldaeslv

klsvdrvnvh

sphramaalg

lmltcmytgk

ekvspgrtsd

pnpaapdses

vivamervsv

lfdrirkgfp

cearvvaril

pqflddffpp

qdimnkvige

flsnqqpypq

fmatvvykvf

qtlhstgqss

mvrdwvmlsl

snftqrapva

matwslscff

vsastspwva

ailphvisrm

gkleqvdvnl

fclvatdfyr

hqieeeldrr

afqsvlevva

apgspyhrll

tclrnvhkvt

tc

CARBOXY TERMINUS

I need these decoded in order to answer the following questions:

a.) What is the name of the least abundant amino acid in this protein and what proportion of the total amino acids in the protein does this individual amino acid represent?

b.) What is the proportion of essential amino acids included in this protein sequence?

c.)How many Tryptophan residues are present in this protein sequence?

d.) The amino acid sequence from residues 73-85 is represented by the amino acids EEEWDEEEEEEAD, how would you generally classify this small region of the protein

e.) Two amino acids are present in the same proportion in this protein, which two are those?

f.) How many positively charged amino acids are present in this protein?

g.)Which class of amino acids is the most abundant in this protein?

h.) What amino acid(s) is/are present at locations 572 and 582 in the Huntington Disease protein (Full Amino Acid Name(s))

i.) What type of structure could form between these amino acids that could help stabilize this protein's structure?

j.) How many valine residues are present in this protein?

k.)Which class of amino acids is least abundant in this protein?

l.) How may negatively charged amino acids are present in the Huntington disease protein?

m.) How many Aspartic Acid residues are present in this protein and what proportion do they represent of the total amino acids in this protein?

n.)Summed together, how many hydrophilic amino acid residues are present in this protein?

o.)What is the sequence of amino acids from positions 487-492 (First letters only)?

p.) How would you classify this area of the Huntington protein based on these amino acids?