Transcript Searching databases for sequence homology

Translation of nucleotide sequences
The central dogma of genetics is essentially a "roadmap" of how
information within the DNA is transferred to protein, the
building blocks of your body. Here is a diagram of this process,
"the central dogma of genetics":
The DNA-helix is composed of two strands (called watson and crick strands)
complementary to each other and genes occur on both strands. The genes are
always transcribed in 5’ - 3’ direction.
Once a gene has been sequenced it is important to determine the correct open
reading frame (ORF). Every region of DNA has six possible reading frames,
three in each direction.
An open reading frame starts with an atg (Met) in most species and ends with a
stop codon (taa, tag or tga).
In the W2H file window,
select the file ab001874.
Select the program MAP,
and click start.
Clostridium
paraputrificum gene
for chitinase B,
complete cds
(coding sequence).
Q. Based on the results of translation, what are the differences between the codes of
mold mitochondria and the standard code? Name one of these differences.
Different genetic codes are described here:
•The Standard Code
•The Vertebrate Mitochondrial Code
•The Yeast Mitochondrial Code
•The Mold, Protozoan, and Coelenterate Mitochondrial Code and the
Mycoplasma/Spiroplasma Code
•The Invertebrate Mitochondrial Code
•The Ciliate, Dasycladacean and Hexamita Nuclear Code
•The Echinoderm and Flatworm Mitochondrial Code
•The Euplotid Nuclear Code
•The Bacterial and Plant Plastid Code
•The Alternative Yeast Nuclear Code
•The Ascidian Mitochondrial Code
•The Alternative Flatworm Mitochondrial Code
•Blepharisma Nuclear Code
•Chlorophycean Mitochondrial Code
•Trematode Mitochondrial Code
•Scenedesmus Obliquus Mitochondrial Code
•Thraustochytrium Mitochondrial Code
Select the sequence mito.seq in the file list. The sequence is from a mitochondrial gene
for a mold cytochrome c oxidase.
The standard code
UUU
UUC
UUA
UUG
Phe
Phe
Leu
Leu
F
F
L
L
UCU
UCC
UCA
UCG
Ser
Ser
Ser
Ser
S
S
S
S
UAU
UAC
UAA
UAG
Tyr Y
Tyr Y
Stop*
Stop*
UGU
UGC
UGA
UGG
Cys C
Cys C
Stop*
Trp W
CUU
CUC
CUA
CUG
Leu
Leu
Leu
Leu
L
L
L
L
CCU
CCC
CCA
CCG
Pro
Pro
Pro
Pro
P
P
P
P
CAU
CAC
CAA
CAG
His
His
Gln
Gln
H
H
Q
Q
CGU
CGC
CGA
CGG
Arg
Arg
Arg
Arg
R
R
R
R
AUU
AUC
AUA
AUG
Ile
Ile
Ile
Met
I
I
I
M
ACU
ACC
ACA
ACG
Thr
Thr
Thr
Thr
T
T
T
T
AAU
AAC
AAA
AAG
Asn
Asn
Lys
Lys
N
N
K
K
AGU
AGC
AGA
AGG
Ser
Ser
Arg
Arg
S
S
R
R
GUU
GUC
GUA
GUG
Val
Val
Val
Val
V
V
V
V
GCU
GCC
GCA
GCG
Ala
Ala
Ala
Ala
A
A
A
A
GAU
GAC
GAA
GAG
Asp
Asp
Glu
Glu
D
D
E
E
GGU
GGC
GGA
GGG
Gly
Gly
Gly
Gly
G
G
G
G
The Standard Code
Differences from the Standard Code:
The Mold...
TGA
Trp W
Standard
Ter *
The Mold, Protozoan, and
Coelenterate Mitochondrial Code
Alternative Initiation Codons:
Trypanosoma: TTA, TTG, CTG
Leishmania: ATT, ATA
Tertrahymena: ATT, ATA, ATG
Paramecium: ATT, ATA, ATG, ATC, GTG,
GTA(?)
(Pritchard et al., 1990)
Identification of open reading frames
Q. Name some of the protein products (FT: CDSs) of the ectrmd operon.
Select the sequence ectrmd ( E. coli trmD operon and nearby regions ) in the file list,
which is an operon.
FT CDS 416. .1522
>>
/note="unidentified reading frame
FT CDS 1771. .2019 >>
/note="ribosomal protein S16 (rps P gene) (aa 1-82)”
FT CDS 2617. .3384 >>
/note="tRNA (m1G) methyltransferae (trmD gene) (aa 1-255)”
FT CDS 3426. .3773 >>
/note="ribosomal protein L19 (rplS gene) (aa 1-115)"
FT CDS complement(3849. .4232) >>
/note="unidentified reading frame (16K polypeptide) (aa 1-127)"
Comparing two sequences - local and
global alignment
You will now compare the two protein sequences 1.pep and 2.pep using the "Gap"
program (Gap Protein).
Q. What is the quality value when comparing 1.pep and 2.pep? What quality values
are obtained with 10 randomisations? May we conclude that the alignment
corresponds to a pair of evolutionary related proteins?
Q. Are the results from "Gap" and "Bestfit" different? What alignment is more
significant?
GAP
BESTFIT
BESTFIT
Aligning two sequences - Gap extension penalty. Alignment of
genomic sequence with mRNA
Select the following two sequences:
V00594 (Human mRNA for metallothionein) and
J00271 (corresponding genomic sequence).
Q. Based on the alignment, how many exons are there in this gene? Compare your result to
what's in the annotation section for J00271.
Default settings
Open gap= 50
Extend gap= 3
New settings
Open gap= 10
Extend gap= 0
Comparing two sequences based on exact matching of short words
Compare the two sequences 1.pep and 2.pep with COMPARE.
Identification of repeats in a human chromosome
analyze the sequence chr21.seq with COMPARE.
Q. What is the relationship between the number of diagonals in the plot and the
number of repeats? Approximately how many nucleotides are there in the repeated
sequence?
5
4
3
2
1
A
Searching databases for sequence
homology
BLAST search in the W2H environment.
In this exercise we will identify homologs of the 54 kDa subunit of the mammalian
signal recognition particle (SRP54).
Q. Name one mammalian ortholog to the mouse srp54 according to the blast output.
BLAST and the role of the substitution matrix in the detection
of remote similarity.
Go to BLAST at the NCBI.
1. VFQQDNDPKHTSLHVRSWFQRRHVHLLDQPSQSPDLNPIEH
2. IFLHDNAPSHTARAVRDTLETLNWEVLPHAAYSPDLAPSDY
First use the default matrix BLOSUM62, then the PAM250 matrix.
Q. The remote similarity between these two proteins is detected only with one of
the matrices. Which one?
Score = 31.2 bits (69), Expect = 4.9
Identities = 14/41 (34%), Positives = 22/41 (53%)
Query: 1 VFQQDNDPKHTSLHVRSWFQRRHVHLLDQPSQSPDLNPIEH 41
+F DN P HT+ VR
+ + +L
+ SPDL P ++
Sbjct: 1 IFLHDNAPSHTARAVRDTLETLNWEVLPHAAYSPDLAPSDY 41
BLAST and sequence filtering.
Use the protein with accession P19934 (E. coli TolA protein).
Q. From the result of the search with filtering: Give an example of a region
filtered in the query sequence (examine the alignments)?
Give an example of a sequence with a high score that is found in the output
without filtering but is not found in the output with filtering?
Multiple sequence alignment
PILEUP
Apply pileup on a set of related proteins from two different families, sr54 and ftsy.
Q. What protein family (sr54 or ftsy) has a C-terminal portion* which is lacking in the
other family ?
Q. What protein family has a N-terminal portion which is lacking in the other family ?
Q. Can you identify a highly conserved sequence motif in the alignment, i.e a
subsequence shared by all members of the the srp54 and ftsy protein family ?
* Protein sequences have a polarity, the N- (NH2 or amino-) terminus is at the left and
the C-(COOH or carboxy-) terminus at the right in a linear sequence. This is
sometimes denoted like:
N-GHYTRWYYQIIQ-C where "GHYTRWYYQIIQ" is the actual amino acid sequence.
A concept like "the N-terminal half" of this (very short) protein would then be the
sequence "GHYTRW".
Proteins are typically made up of two or more functional domains. The SRP54 and
Ftsy proteins are examples of this. The multiple sequence alignment allows us to
identify the differences between the proteins with respect to domain organisation.
N-terminal
C-terminal
A search in pfam (www.sanger.ac.uk/Software/Pfam/) confirm our
findings.
Sr54_arcfu
Ftsy_aquae
Pattern searches
Pattern recognition
Cx{2,4}Cx12Hx{3,5}H
The search pattern defined means: A cysteine residue followed by any two to
four amino acids, a cysteine residue, any 12 amino acids, histidine, any three
to five amino acids and finally histidine. ).
Q. Give an example of a human protein in Swissprot described as a zinc finger protein
and that is identified with findpatterns!
! FINDPATTERNS on SwissProt:* allowing 0 mismatches ! Using patterns from:
zincfinger September 22, 2003 13:31 ..
ABF1_HUMAN ck: 1650 len: 3,703 ! Q15911 homo sapiens (human). alpha-fetoprotein
enhancer binding protein (a
zinc_zinger
Cx{2,4}Cx12Hx{3,5}H
Cx{2}Cx{12}Hx{4}H
284: KPILM CFLCKLSFGYVRSFVTHAVHDH RMTLS
2.Search Swissprot human proteins for the sequence "GDSGGP.
Examine the result carefully. Are you able to identify serine proteases?
Are there proteins that are not proteases?
! FINDPATTERNS on SwissProt:* allowing 0 mismatches ! Using patterns from: serineproteases
September 22, 2003 14:09 ..
2ACC_HUMAN ck: 2269 len: 414 ! Q9y5p8 homo sapiens (human). serine/threonine protein
phosphatase 2a, 48 k
serine_proteases GDSGGP
331: SLLRD GDSGGP ELSDW
ACH1_LONAC ck: 3441 len: 213 ! P23604 lonomia achelous (giant silkworm moth) (saturnid moth).
achelase i
serine_proteases GDSGGP
186: RDQCQ GDSGGP LYHNR
ACH2_LONAC ck: 5072 len: 214 ! P23605 lonomia achelous (giant silkworm moth) (saturnid moth).
achelase ii
serine_proteases GDSGGP
187: RDQCQ GDSGGP LYHNG
Q62284 ck: 7458 len: 135 ! Q62284 mus musculus (mouse). gamma-7s nerve growth factor (y-ngf)
(fragmen
serine_proteases GDSGGP
85: KDTCK GDSGGP LICDG
3. Using FINDPATTERNS, identify human Swissprot proteins that have stretches of
at least 40 consecutive serine residues ( hint, use S{40,} as search pattern.
Q. Give one example of a protein that has least 40 consecutive serines (strange
protein!).
! FINDPATTERNS on SwissProt:*_HUMAN allowing 0 mismatches ! Using patterns from:
serine40 September 22, 2003 15:31 ..
AF9_HUMAN ck: 6118 len: 568 ! P42568 homo sapiens (human). af-9 protein. 2/2003
pat_
S{40,}
S{40}
149: RSIHT SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSTSF
S{40}
150: SIHTS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS STSFS
S{40}
151: IHTSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS TSFSK
Databases searched: SWISS-PROT, Release 60.0, Released on 24Mar2003, Formatted on
24Mar2003 Total finds: 3 Total length: 4,795,187 Total sequences: 9,172 CPU time:
42.26
Motifs
Search for protein motifs in the Swissprot sequence fa7_human.
Q. What motifs are found with MOTIFS?
MOTIFS from: /d/u/paul/work/fa7_human.pep
Mismatches: 0 September 22, 2003 15:34 .. fa7_human.pep
Check: 4382 Length: 466 ! ID FA7_HUMAN STANDARD; PRT; 466 AA.
______________________________________________________________________________
Asx_Hydroxyl Cx(D,N)x4(F,Y)xCxC
Cx(D)x{4}(Y)xCxC
121: QNGGS CKDQLQSYICFC LPAFE
****************************************************
Aspartic acid and asparagine hydroxylation site
***************************************************
Post-translational hydroxylation of aspartic acid or asparagine [1] to form erythrobeta-hydroxyaspartic acid or erythro-beta-hydroxyasparagine has been identified in a
number of proteins with domains homologous to epidermal growth factor (EGF).
Examples of such proteins are the blood coagulation protein factors VII, IX and X,
proteins C, S, and Z, the LDL receptor, thrombomodulin, etc. Based on sequence
comparisons of the EGF-homology region that contains hydroxylated Asp or Asn, a
consensus sequence has been identified that seems to be required by the
hydroxylase(s). -Consensus pattern: C-x-[DN]-x(4)-[FY]-x-C-x-C [D or N is the
hydroxylation site] -Note: this consensus pattern is located in the N-terminal of
EGF-like domains, while our EGF-like cysteine pattern signature (see the relevant
entry <PDOC00021>) is located in the C-terminal. -Last update: January 1989 / First
entry. [ 1] Stenflo J., Ohlin A.-K., Owens W.G., Schneider W.J. J. Biol. Chem.
263:21-24(1988).
K ey
SIGNAL
From
1
To
20
Lengt h
20
Des c ri pt i on
POTENTIAL .
PROPEP
21
60
40
CHAIN
61
212
152
FACTOR VII LIGHT CHAIN.
CHAIN
213
466
254
FACTOR VII HEAVY CHAIN.
DOMAIN
66
95
30
GLA-RICH.
DOMAIN
106
142
37
EGF-LIKE 1, CALCIUM-BINDING
(POTENTIAL ).
DOMAIN
147
188
42
EGF-LIKE 2.
DOMAIN
213
466
254
SITE
212
213
2
ACT_SITE 253
253
BY SIMILARITY .
ACT_SITE 302
302
BY SIMILARITY .
ACT_SITE 404
404
BY SIMILARITY .
BINDING
398
SUBSTRATE (BY SIMILARITY ).
398
SERINE PROTEASE.
CLEAVAGE (BY FACTOR XA, FACTOR XIIA,
FACTOR IXA, OR THROMBIN).
EGF-1, EGF-2, Serine protease, Glu_Carboxylation.