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Mega4

Introduction:
Transforming growth factor beta (TGF-β) is a protein that controls proliferation, cellular
differentiation, and other functions in most cells. It plays a role in immunity, cancer, heart
disease, diabetes, and Marfan syndrome. TGF-beta acts as an antiproliferative factor in normal
epithelial cells and at early stages of oncogenesis.[
TGF-β is a secreted protein that exists in three isoforms called TGF-β1, TGF-β2 and TGF-β3. It
was also the original name for TGF-β1, which was the founding member of this family. The TGF-
β family is part of a superfamily of proteins known as the transforming growth factor beta
superfamily, which includes inhibins, activin, anti-müllerian hormone, bone morphogenetic
protein, decapentaplegic and
Some cells secrete TGF-β, and also have receptors for TGF-β. This is known as autocrine
signalling. Cancerous cells increase their production of TGF-β, which also acts on surrounding
cells.
The Structure of TGF-β:
The peptide structures of the three members of the TGF-β family are highly similar. They are all
encoded as large protein precursors; TGF-β1 contains 390 amino acids and TGF-β2 and TGF-β3
each contain 412 amino acids. They each have an N-terminal signal peptide of 20-30 amino
acids that they require for secretion from a cell, a pro-region (called latency associated peptide
or LAP), and a 112-114 amino acid C-terminal region that becomes the mature TGF-β molecule
following its release from the pro-region by proteolytic cleavage.The mature TGF-β protein
dimerizes to produce a 25 KDa active molecule with many conserved structural motifs. TGF-β
has nine cysteine residues that are conserved among its family; eight form disulfide bonds
within the molecule to create a cysteine knot structure characteristic of the TGF-β superfamily
while the ninth cysteine forms a bond with the ninth cysteine of another TGF-β molecule to
produce the dimer.Many other conserved residues in TGF-β are thought to form secondary
structure through hydrophobic interactions. The region between the fifth and sixth conserved
cysteines houses the most divergent area of TGF-β molecules that is exposed at the surface of
the molecule and is implicated in receptor binding and specificity of TGF-β.
The primary three are:
 TGF beta 1 - TGFB1 Online 'Mendelian Inheritance in Man' (OMIM) 190180
 TGF beta 2 - TGFB2 Online 'Mendelian Inheritance in Man' (OMIM) 190220
 TGF beta 3 - TGFB3 Online 'Mendelian Inheritance in Man'
Function
o Apoptosis
Cells can die in two ways: Through programmed cell death (including apoptosis and autophagy),
when the cell self-destructs as a result of "death signals", and through necrosis, which is death
from other causes, such as lack of oxygen or toxins.

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TGF-β induces apoptosis in numerous cell types. TGF-β can induce apoptosis in two ways:
through the SMAD pathway or the DAXX pathway.
o SMAD pathway
Smads are transcription factors that transduce extracellular TGF-ß superfamily ligand signaling
from cell membrane bound TGF-ß receptors into the nucleus where they activate transcription
TGF-ß target genes
o DAXX pathway
TGF-β may also trigger apoptosis via the death associated protein 6.DAXX associate with and
bind to the type II TGF-β receptor kinase.Death associated protein 6 also known as DAXX is a
protein which in humans is encoded by the DAXX gene.
Cell cycle:
TGF-β plays a crucial role in the regulation of the cell cycle.
TGF-β causes synthesis of p15 and p21 proteins, which block the cyclin:CDK complex
responsible for Retinoblastoma protein (Rb) phosphorylation. Thus TGF-β blocks advance
through the G1 phase of the cycle. In doing so, TGF-β suppresses expression of c-myc, a gene
which is involved in G1 cell cycle progression.[5]
Immune System:
TGF-β is believed to be important in regulation of the immune system by CD25+ Regulatory T
cell.
CD25 is the alpha chain of the IL-2 receptor[1]. It is a type I transmembrane protein present on
activated T cells, activated B cells,
introduction of phylogenetic analysis:
Molecular phylogenetics, also known as molecular systematics is the use of the structure of
molecules to gain information on an organism's evolutionary relationships. The result of a
molecular phylogenetic analysis is expressed in a phylogenetic tree.
Techniques and applications:
Every living organism contains DNA, RNA, and proteins. Closely related organisms generally
have a high degree of agreement in the molecular structure of these substances, while the
molecules of organisms distantly related usually show a pattern of dissimilarity. Conserved
sequences, such as mitochondrial DNA, are expected to accumulate mutations over time, and
assuming a constant rate of mutation provide a molecular clock for dating divergence.The
molecular clock (based on the molecular clock hypothesis (MCH)) is a technique in molecular
evolution that uses fossil constraints and rates of molecular change to deduce the time in
geologic history when two species are diverged.Molecular phylogeny uses such data to build a
"relationship tree" that shows the probable evolution of various organisms. Not until recent
decades, however, has it been possible to isolate and identify these molecular structures.
The most common approach is the comparison of homologous sequences for genes using
sequence alignment techniques to identify similarity. Another application of molecular

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phylogeny is in DNA barcoding,


DNA barcoding is a taxonomic method that uses a short genetic marker in an organism's DNA to
identify it as belonging to a particular species
where the species of an individual organism is identified using small sections of mitochondrial
DNA. Another application of the techniques that make this possible can be seen in the very
limited field of human genetics, such as the ever more popular use of genetic testing to
determine a child's paternity, as well as the emergence of a new branch of criminal forensics
focused on evidence known as genetic fingerprinting.
aims and objectives:
1-sequence analysis by blast
2-multiple alignment using clustral w
3-phylogeny:
phylogenetic tree of growth factor by using mega 4
4-guture insight for phylogeny and sequence analysis of growth factor
Procedure:
1-collection of all growth factors sequences available
2-sequence alignment via blast and clustralw
3-phylogenetic tree by maximum liklihood method and maximum arsimony method using
mega4.
Material and methods:
BLAST
To run, BLAST requires a query sequence to search for, and a sequence to search against (also
called the target sequence) or a sequence database containing multiple such sequences. BLAST
will find subsequences in the database which are similar to subsequences in the query. In
typical usage, the query sequence is much smaller than the database, e.g., the query may be
one thousand nucleotides while the database is several billion nucleotides.
The main idea of BLAST is that there are often high-scoring segment pairs (HSP) contained in a
statistically significant alignment. BLAST searches for high scoring sequence alignments
between the query sequence and sequences in the database using a heuristic approach that
approximates the Smith-Waterman algorithm. The exhaustive Smith-Waterman approach is too
slow for searching large genomic databases such as GenBank. Therefore, the BLAST algorithm
uses a heuristic approach that is less accurate than the Smith-Waterman algorithm but over 50
times faster. The speed and relatively good accuracy of BLAST are among the key technical
innovations of the BLAST programs.

Mega4:

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We announce the release of the fourth version of MEGA software, which expands on the
existing facilities for editing DNA sequence data from autosequencers, mining Web-databases,
performing automatic and manual sequence alignment, analyzing sequence alignments to
estimate evolutionary distances, inferring phylogenetic trees, and testing evolutionary
hypotheses. Version 4 includes a unique facility to generate captions, written in figure legend
format, in order to provide natural language escriptions of the models and methods used in the
analyses. This facility aims to promote a better understanding of the underlying assumptions
used in analyses, and of the results generated. Another new feature is the Maximum Composite
Likelihood (MCL) method for estimating evolutionary distances between all pairs of sequences
simultaneously, with and without incorporating rate variation among sites and substitution
pattern heterogeneities among lineages. This MCL method also can be used to estimate
transition/transversion bias and nucleotide substitution pattern without knowledge of the
phylogenetic tree. This new version is a native 32-bit Windows application with multi-threading
and multi-user supports, and it is also available to run in a Linux desktop environment (via the
Wine compatibility layer) and on Intel-based Macintosh computers under the Parallels program.
The currentversion of MEGA is available free of charge at http://www.megasoftware.net.

Clustalw:

Clustal is a widely used multiple sequence alignment computer program.The latest version is
There are two main variations:

 ClustalW: command line interface


 ClustalX: This version has a graphical user interface. [3] It is available for Windows, Mac
OS, and Unix/Linux.

This program accepts a wide range on input format. Included NBRF/PIR, FASTA,
EMBL/Swissprot, Clustal, GCC/MSF, GCG9 RSF, and GDE.The output format can be one or
many of the following: Clustal, NBRF/PIR, GCG/MSF, PHYLIP, GDE, or NEXUS.

Procedur:

I. Confirm similarity to one or more previously characterized Hops using BLAST analysis.

1. Conduct BLASTP analyses to determine whether a given protein is similar to any


previously characterized Hops.
2. If there IS NO significant similarity to one or more previously characterized Hops,
and and the newly identified Hop has been confirmed by criteria other than
sequence similarity (see Criteria for Hop name assignment), go to Name
structure and Selection: How to name a Hop for guidelines on naming novel Hop
proteins.

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3. If there IS significant similarity to one or more previously characterized Hops


(roughly defined as a BLAST expect value of less than 10-5 and with alignment
extending over 60% of the length of the protein) follow the steps below to assign
a subgroup classification, or contact the PPI site administrator and a subgroup
classification can be generated for you.

II. Sequence alignment in ClustalW

A. Obtain a file listing all sequences in the Hop family of interest

1. Go to the list of assigned Hop names and obtain the sequences for all members
of the appropriate family by clicking on the family name.
2. Save the list of sequences as a text file. Note that the sequences are listed in
FASTA format.
3. Add the sequence of the newly identified Hop to the list (also in FASTA format)
and save the file.

B. Generate alignment file

ClustalW is a general purpose tool for alignment of multiple sequences. It is available through
numerous websites, including the EMBL-EBI site described here. (The HopA family is used to
illustrate the various procedures)

Go to ClustalW at EMBL-EBI. The following window will appear

1. ClustalW will generate the files listed in the window below. Click on the link to the
sequence alignment file (designated by an .aln extension). Save the .aln file as a text file,
renaming it if desired

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III. Clustering analysis and genetic distance calculation in MEGA.


MEGA (Molecular Evolutionary Genetics Analysis) is a free software package for comparative
sequence analysis

A. Download and install MEGA

1. Go to the MEGA 4 download site, provide the requested information, and


download the program. An .exe file will appear on your desktop.
2. Open the .exe file and follow the instructions for installation of MEGA4.
B. Convert Clustal alignment file to MEGA format
3. Open the installed MEGA program. The following window will appear

4. Under File on the menu select "Convert to MEGA format". The following window
will appear:

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5. Select the output file you saved from ClustalW as "Data file to convert:" and for
"Data format" select ".aln (CLUSTAL)". Click OK.
The converted file will appear in the "Text File Editor and Format Converter"
window.
6. Scroll through the converted file to check format.
If line numbers are present, either manually remove them or return to ClustalW
and generate a new .aln file without line numbers.
If any extraneous symbols are present following the last sequence, delete them
7. Save the output file with a .meg extension

C. Perform clustering analysis in MEGA

8. Return to the initial MEGA window (shown above in III.B.1) and select "click me
to activate a data file".
9. Select the .meg file that you saved in step III.B.5. An "Input Data" window will
appear. Under "Data Type" select "Protein Sequences" and click OK.
10. The window shown below will appear, with the open data file indicated at the
bottom

11. Generate a phylogenetic tree from the active data file using Phylogeny >
Bootstrap Test of Phylogeny.

At this point, a number of options can be selected, including UPGMA Tree,


Neighbor-Joining Tree, Minimum Evolution and Maximum Parsimony. Similarly,

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in the "Analysis Preferences" window for UPGMA, Neighbor-Joining, or Minimum


Evolution, models can be selected under Models>Amino Acid. Users are
encouraged to generate trees using a variety of these options. The general
clustering patterns should be similar, regardless of method.

The method that best approximates those described in Lindeberg et al, 2005
(using MEGA 4 rather than MEGA 2.1) involves Neighbor-Joining using the Amino
Acid: p-distance model (the "Analysis Preferences" window for Neighbor-Joining
is shown below)

Result:

The Bootstrap consensus tree resulting from Neighbor-Joining analysis for the HopA

family is shown below.

Discussion:

Genetic distance between the new Hop and established subgroups is Calculated.

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References:
1. Gojobori T, Li WH, Graur D. 1982. Patterns of nucleotide substitution in pseudogenes
and functional genes. J Mol Evol. 18:360–369.
2. Hall BG. Phylogenetic trees made easy: A how-to manual. Sunderland (MA): Sinauer
Associates.
3. Kumar S, Dudley J. 2007. Bioinformatics for biologists in the genomics era.
Bioinformatics. 10.1093/bioinformatics/ btm239.
4. Kumar S, Tamura K, Nei M. 2004. MEGA3: an integrated software for Molecular
Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 5:150–163.
5. Saitou N, Nei M. 1987. The Neighbor-Joining method—a new
method for reconstructing phylogenetic trees. Mol Biol Evol. 4:406–425.
6. Tamura K, Nei M. 1993. Estimation of the number of nucleotide substitutions in the
control region of mitochondrial- DNA in humans and chimpanzees. Mol Biol Evol. 10:
512–526.
7. Tamura K, Nei M, Kumar S. 2004. Prospects for inferring very large phylogenies by using
the Neighbor-Joining method. Proc Natl Acad Sci USA. 101:11030–11035.
8. Thompson JD, Higgins DG, Gibson TJ. 1994. ClustalW— improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-specific
gap penalties and weight matrix choice. Nucleic Acids Res.

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