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Cladograms and Genetics Name: _______________________
Taxonomy is the study of the classification. In biology, taxonomy deals with the
classification of organisms into groups based upon their similarities and differences –
using their genetic information (DNA or protein sequences) and their anatomical
In this activity, you will use protein sequences (sequences of amino acids) to determine
the evolutionary relationship between organisms. We can use protein sequences to
determine evolutionary relationships because DNA codes for a particular protein
sequence, so differences in protein sequences mean differences in DNA sequences.
The number of amino acid substitutions between two organisms shows the differences
between the organisms themselves. The greater the number of substitutions between
two organisms, the longer ago the two organisms diverged from a common ancestor.
Scientists believe that cytochrome c has evolved at a fairly constant rate. This rate of
change is the basis for a “molecular clock.” This rate of mutation can be a helpful tool in
trying to determine how organisms have evolved.
Recent advances in biochemistry have exposed powerful molecular tools for
constructing phylogenetic trees. Protein, mRNA, rRNA, and genomic sequencing are
molecular techniques commonly used in identifying and characterizing organisms.
Molecular sequence data can be used to calibrate “molecular clocks” based on the rate
of mutations in protein and nucleic acid sequences. The rationale of molecular clocks
is that the observed number of changes in an amino acid or nucleotide sequence
may be approximately proportional to the evolutionary time elapsed. The National
Center for Biotechnology Information houses a public database of sequenced
nucleotides and proteins and develops sequence analysis shareware:
Protein data
The amino acid sequence of cytochrome c has been analyzed in over 100 eukaryotic
species, and the molecular data support the notion that cytochrome c is an
evolutionarily conservative protein. Shown below are two protein sequence
comparisons for cytochrome c: between human and either rat or yeast.
Substitutions within the primary structure of cytochrome c are relatively constant over
time, which makes characterizing cytochrome c a potentially useful molecular clock. For
example, note the cytochrome c sequence similarity between humans and rats (91%) is
much higher than between humans and yeast (64%). This indicates that the
evolutionary path leading to the human species diverged with that of yeast far before its
divergence from rat. A comparison of cytochrome c sequences from different species
has been used to order the divergence of species in relative time.
Determining the absolute timing of events using molecular clocks attracts skepticism
for several reasons. The fact that different proteins evolve at different rates makes
determining the divergence rate of a particular protein problematic. Also, changes in
generation time or metabolic rate may affect a mutation rate, making molecular clocks
less predictable.
Method of protein sequencing and alignment
A common approach to protein sequencing involves repeatedly cleaving a polypeptide
(using a protein-digesting enzyme), separating the fragments using chromatography,
and sequencing the resulting small fragments. The entire protein sequence is then
reconstructed by matching regions of sequence overlap seen in the small fragments.
Algorithms may be used to compare protein sequence data against a database. These
algorithms provide local information (within a defined region) or global information
(which accounts for gaps or other aberrations that may mask multiple regions of
similarity). This technology has greatly facilitated the grouping of related proteins into
families (an example is the serine protease family of enzymes, which includes trypsin,
chymotrypsin, and elastase).
The significance of protein sequencing
Direct protein sequencing is an indispensable tool for several reasons. For example, the
position of disulfide bonds can be resolved using protein sequencing techniques. Also,
protein modifications, such as the excision of residues or the covalent attachment of
other groups, can be detected. These changes in protein sequence and structure would
evade detection by nucleic acid sequencing efforts. One drawback to protein sequencing
is that only those genes that code for proteins are under scrutiny. This is often a small
fraction of an organism’s total amount of molecular information.
Directions: Constructing a Cladogram
1. Find the human, rhesus monkey, kangaroo, snapping turtle, bullfrog, and tuna on
the "Amino Acid Sequences in Cytochrome-C Proteins from 20 Different Species"
chart provided. Highlight or underline their names. NOTE: THE SEQUENCES
2. Compare the human amino acid sequence with each of these five animals. Do this
by counting the number of times an amino acid in that animal’s protein is
different from the same amino acid position of the human sequence. Write that
information in the table below.
Number of amino acid differences between human and …
Rhesus Monkey
Snapping Turtle
Bull Frog
Tuna Fish
3. Use the above information to construct a cladogram using genetic information.
The fewer the differences between the species, the more closely related they are.
The cladogram on the following page uses anatomical differences, rather than
protein and DNA sequences.
The cladogram on the next page is organized using anatomical (body) features. Notes:
1. The foramen magnum is the opening in the skull where the spine attaches.
2. Plancental mammals give live birth.
3. An aminion is a support structure for embryos.
Directions for BLAST Searches
1. Go to From the drop-down menu next to the
search bar, select “Protein.”
2. In the search bar, type: cytochrome c homo sapiens. This will search for the
human sequence of cytochrome C. Click ‘search.’
3. From your search results, select the first search result, which should be a 105 aa
sequence. Click ‘FASTA’ to see the amino acid sequence.
4. Copy the FASTA sequence beginning with the term ‘>gi|11128019 …’
5. Go to Select ‘protein BLAST.’
6. Copy the FASTA sequence into the search box. Remove the line break mid-amino
acid sequence, so that it’s as one phrase.
7. For ‘search set,’ choose ‘non-redundant protein sequence.’ It may be selected as
the default. For ‘program selection,’ make sure that ‘blastp’ (protein-protein
BLAST) is selected. I would select ‘show results in a new window,’ so that you
can go back to your specified search.
8. Select ‘BLAST.’ Results may take several seconds to a minute to appear. If results
take longer than 2 minutes, let Ms. Poole know.
9. The results page has two sections. The first section is a graphical display of the
matching sequences. Scroll down to the section titled “Sequences producing
significant alignments.” The species in the list that appears below this section are
those with sequences identical to or most similar to the gene of interest. The
most similar sequences are listed first, and as you move down the list, the
sequences become less similar to your gene of interest.
If you click on a particular species listed, you’ll get a full report that includes
the classification scheme of the species, the research journal in which the gene
was first reported, and the sequence of bases that appear to align with your gene
of interest.
If you click on a particular species listed, you’ll get a full report that includes
the species’ classification scheme, the research journal in which the gene was
first reported, and the sequence of bases that appear to align with your gene of
interest. Click to open the species in a new window! (Otherwise, you wont be
able to return to your search.)
If you click on the link titled “Distance tree of results,” you will see a
cladogram with the species with similar sequences to your gene of interest
placed on the cladogram according to how closely their matched gene aligns
with your gene of interest.
Recall that species with common ancestry will share similar genes. The more
similar genes two species have in common, the more recent their common
ancestor and the closer the two species will be located on a cladogram.
Select TWO of the other organisms from the cladogram and view their
alignment with human cytochrome C, either by running a BLAST with each or by
searching through your original BLAST.
For each BLAST query, consider the following:
The higher the score, the closer the alignment.
The lower the e value, the closer the alignment.
Sequences with e values less than 1e-04 (1 x 10-4) can be considered related
with an error rate of less than 0.01%.
Do these results match the results you came to from analyzing your amino
acid sequences? From your anatomical features cladogram?
Analysis Questions
Answer the following on a SEPARATE piece of paper.
1. Turn in your LABELED cladogram with each species’ scientific name. (Yes, you
will have to look them up and have them properly formatted. For names that
include more than one species, pick one and label accordingly. Google is your
friend.) (5 points)
2. Why do scientists use scientific names – rather than common names – as a way
of referring to organisms? List and explain at least three reasons! (3 points)
3. Does the cladogram organized by genetic information (and confirmed through
BLAST) agree with the cladogram organized by anatomical features? Why or
why not? Do organisms with fewer anatomical traits in common also have fewer
amino acids in common? Explain! (2 points)
4. Chickens and turkeys are both birds and have the same sequence of amino acids
in their cytochrome-c protein. Remember the definition of species! Explain in at
LEAST a paragraph how two species can have identical cytochrome-c and still be
different species. Remember the biological definition of species! (5 points)
5. How can protein sequencing and comparison be used to evaluate organisms’
evolutionary relationships? Discuss the benefits and drawbacks of using protein
sequences, rather than DNA sequences and anatomical features, to establish
organisms’ evolutionary relationships. (5 points)