Translation%20of%20nucleotide%20sequences

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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"
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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). 
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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).
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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

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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 Phe F UCU Ser S UAU Tyr Y
UGU Cys C UUC Phe F UCC Ser S UAC Tyr Y UGC Cys C
UUA Leu L UCA Ser S UAA Stop UGA Stop UUG Leu L
UCG Ser S UAG Stop UGG Trp W CUU Leu L CCU Pro
P CAU His H CGU Arg R CUC Leu L CCC Pro P CAC His
H CGC Arg R CUA Leu L CCA Pro P CAA Gln Q CGA Arg
R CUG Leu L CCG Pro P CAG Gln Q CGG Arg R AUU
Ile I ACU Thr T AAU Asn N AGU Ser S AUC Ile I ACC
Thr T AAC Asn N AGC Ser S AUA Ile I ACA Thr T AAA
Lys K AGA Arg R AUG Met M ACG Thr T AAG Lys K AGG
Arg R GUU Val V GCU Ala A GAU Asp D GGU Gly G
GUC Val V GCC Ala A GAC Asp D GGC Gly G GUA Val V
GCA Ala A GAA Glu E GGA Gly G GUG Val V GCG Ala A
GAG Glu E GGG Gly G
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The Standard Code
The Mold, Protozoan, and Coelenterate
Mitochondrial Code
Differences from the Standard Code The
Mold... Standard TGA Trp W Ter
Alternative Initiation Codons Trypanosoma TTA,
TTG, CTGLeishmania ATT, ATA Tertrahymena ATT,
ATA, ATG Paramecium ATT, ATA, ATG, ATC, GTG,
GTA(?) (Pritchard et al., 1990)
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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 gtgt /note"unidentified
reading frame FT CDS 1771. .2019
gtgt /note"ribosomal protein S16 (rps P gene) (aa
1-82) FT CDS 2617. .3384 gtgt /note"tRNA (m1G)
methyltransferae (trmD gene) (aa 1-255) FT CDS
3426. .3773 gtgt /note"ribosomal protein L19
(rplS gene) (aa 1-115)" FT CDS
complement(3849. .4232) gtgt /note"unidentified
reading frame (16K polypeptide) (aa 1-127)"
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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?
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BESTFIT
GAP
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BESTFIT
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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
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New settings Open gap 10 Extend gap 0
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Comparing two sequences based on exact matching
of short words Compare the two sequences 1.pep
and 2.pep with COMPARE.
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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?
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4
3
2
1
A
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Searching databases for sequencehomology
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.
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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 4.9Identities
14/41 (34), Positives 22/41 (53) Query 1
VFQQDNDPKHTSLHVRSWFQRRHVHLLDQPSQSPDLNPIEH 41
F DN P HT VR L SPDL P
Sbjct 1 IFLHDNAPSHTARAVRDTLETLNWEVLPHAAYSPDLAPSD
Y 41
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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?
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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.
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N-terminal
C-terminal
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A search in pfam (www.sanger.ac.uk/Software/Pfam/)
confirm our findings.
Sr54_arcfu
Ftsy_aquae
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Pattern searches
Pattern recognition   Cx2,4Cx12Hx3,5H 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 1331 .. ABF1_HUMAN ck 1650
len 3,703 ! Q15911 homo sapiens (human).
alpha-fetoprotein enhancer binding protein (a
zinc_zinger Cx2,4Cx12Hx3,5H
Cx2Cx12Hx4H 284 KPILM
CFLCKLSFGYVRSFVTHAVHDH RMTLS
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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 1409 .. 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
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3. Using FINDPATTERNS, identify human Swissprot
proteins that have stretches of at least 40
consecutive serine residues ( hint, use S40, 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 1531 .. AF9_HUMAN ck 6118
len 568 ! P42568 homo sapiens (human). af-9
protein. 2/2003 pat_ S40, S40 149
RSIHT SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS
SSTSF S40 150 SIHTS SSSSSSSSSSSSSSSSSSSSS
SSSSSSSSSSSSSSSSSSS STSFS S40 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
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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 1534 ..
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)x4(Y)xCxC
121 QNGGS CKDQLQSYICFC LPAFE
Aspartic acid
and asparagine hydroxylation site

Post-translational hydroxylation of aspartic
acid or asparagine 1 to form erythro-beta-hydrox
yaspartic 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 ltPDOC00021gt) 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.
26321-24(1988).
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EGF-1, EGF-2, Serine protease, Glu_Carboxylation.