Download Using the NCBI Genome Databases to Compare the

Using the NCBI Genome Databases to Compare the Genes for Human &
Chimpanzee Beta Hemoglobin
Author(s) :Susan Offner
Source: The American Biology Teacher, 72(4):252-256. 2010.
Published By: National Association of Biology Teachers
URL: http://www.bioone.org/doi/full/10.1525/abt.2010.72.4.10
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HOWTODOIT
Using the NCBI Genome Databases
to Compare the Genes for Human &
Chimpanzee Beta Hemoglobin
SUSAN OFFNER
Image: © Iorboaz | Dreamstime.com
ABSTRACT
The beta hemoglobin protein is identical in humans and chimpanzees. In this tutorial,
students see that even though the proteins are identical, the genes that code for them are
not. There are many more differences in the introns than in the exons, which indicates
that coding regions of DNA are more highly conserved than non-coding regions.
Bioserver tool at the Dolan DNA Learning Center (dnalc.org), that have
useful tools for genome analysis.
BLAST Tutorial: Beta Hemoglobin
J DNA sequencing has become very easy and rapid since the 1990s. By
Key Words: Evolution; beta hemoglobin genes; beta hemoglobin protein; humans;
now, over 100 billion base pairs of DNA have been sequenced, and more
chimpanzees.
sequence is being added at an astonishing rate. All this DNA sequence
is deposited in an enormous database maintained by NCBI. This database is free and available to the public. It can be easily searched using a
This paper presents a tutorial in which students compare the gene for human
free program called BLAST (Basic Local Alignment Search Tool; Altschul
beta hemoglobin with the gene for beta hemoglobin in the chimpanzee. This
et al., 1990). In this tutorial, you will learn how to use these databases
comparison is interesting because the two proteins are identical in amino
and BLAST to compare human and chimpanzee hemoglobin.
acid sequence but, as students will find out, the genes that code for these
Hemoglobin is a protein found in red blood cells that carries oxygen.
proteins are similar but not identical. This tutorial introduces students to
One hemoglobin molecule consists of four amino
the NCBI (National Center for Biotechnology Inforacid chains: two alpha hemoglobin chains and two
mation) genome databases and gives them some
beta hemoglobin chains. The alpha and beta chains
[Students] can then
practice in using the powerful tools associated with
are coded for by separate genes; the alpha hemothem. This is where DNA sequences from labs all
globin gene is found on chromosome 16 and the
understand
that
because
over the world are deposited. This enormous databeta hemoglobin gene is found on chromosome
base is searchable and available at no cost to anythe genetic code is
11. Each of these amino acid chains contains a
body with Internet access.
heme group that can carry one oxygen molecule.
After students complete the tutorial, they will
degenerate…two
This means that one molecule of hemoglobin can
find that, although the beta hemoglobin proteins of
carry four molecules of oxygen. One beta hemohumans and chimpanzees are identical, the genes
different genes can code
globin chain contains 147 amino acids.
that code for these proteins differ by one base pair
Humans and chimpanzees last had a common
for the same protein.
in the exons (which code for proteins) and by 15
ancestor
between 5 and 8 million years ago. Their
base pairs in the introns (which do not code for
beta
hemoglobin
proteins are identical in amino
proteins). They can then understand that because
acid sequence and, of course, in secondary, tertiary, and quaternary structhe genetic code is degenerate (more than one codon can code for the
tures. In this activity, you will explore whether the gene that codes for
same amino acid), two different genes can code for the same protein. They
the beta hemoglobin protein is identical in humans and chimpanzees.
can also appreciate that introns evolve more rapidly than exons. This is
because, although it is assumed that mutations occur in all DNA at the
same rate, a mutation in an exon that changes the order of amino acids in
the protein will probably be selected against, whereas most mutations in
introns will likely have no selection pressure against them and so are more
likely to remain in the gene pool. Thus, even though there are fewer than
twice as many base pairs of introns (1068) than of exons (538), there are
15 times more mutations in the introns than in the exons.
It is suggested that teachers complete this activity themselves before
giving it to students. Finally, there are other Web sites, including the
1. Go to ncbi.nlm.nih.gov. This is the NCBI home page through
which you can access both the DNA databases and BLAST.
2. At the top of the home page, search All Databases (default) for
HBB. HBB is the abbreviation for beta hemoglobin. Click “Go.”
3. In the middle of the page on the left, click on “Nucleotide: core
subset of nucleotide sequence records.”
4. Near the top of the page, click on “HBB (Homo sapiens).” You may
have to right-click with your mouse to open this link. A new screen
The American Biology Teacher, Vol. 72, No. 4, pages 252–256. ISSN 0002-7685, electronic ISSN 1938–4211. ©2010 by National Association of Biology Teachers. All rights reserved.
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DOI: 10.1525/abt.2010.72.4.10
252
THE AMERICAN BIOLOGY TEACHER
VOLUME 72, NO. 4, APRIL 2010
Figure 1. The nucleotide sequence of the sense strand of beta hemoglobin DNA. Notice that threeletter abbreviations are used for the amino acids, because this figure was originally published in 1980
(Lawn et al., 1980), when single-letter abbreviations were not yet used. (Reprinted from Cell, 21, “The
nucleotide sequence of the human beta-globin gene," pp. 647–651, with permission from Elsevier.
Previously reprinted in Vigue, 1987.)
will come up that gives you the nucleotide sequence of the gene that
codes for the human beta-hemoglobin protein. There is a lot of additional information on this, and you will see links to many other pages.
You can explore these after you have finished the basic tutorial.
5. Scroll down a little until you see, in a gray box on the left,
“Genomic regions, transcripts and products.” Click on “Reference
sequence details.”
THE AMERICAN BIOLOGY TEACHER
6. On the left side of the screen, find “Genome Reference Consortium Human Build” in a gray box. Under this, find “Genomic,”
then find “Download.” To the right of ”Download,” click on
”FASTA.” You will see that the beta hemoglobin gene contains
1606 nucleotides (“letters”), here shown in what we call FASTA
format. FASTA is a standard format, used for both genes and proteins, in which the first line begins with a symbol, followed
USING THE NCBI GENOME DATABASES
253
Figure 2. The BLAST printout page, showing the comparison between human beta hemoglobin (Query) and
chimpanzee beta hemoglobin (Subject). This figure was printed from NCBI’s BLAST site.
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THE AMERICAN BIOLOGY TEACHER
VOLUME 72, NO. 4, APRIL 2010
Figure 2. (Cont'd)
by any text. The text is sometimes longer than one line, as it is
in this example. This is OK so long as there is no “return” in the
text. After this one line of text, there is a return, followed by the
nucleotide sequence of the gene or the amino acid sequence of
the protein. In cases of proteins, the single-letter abbreviations for
each of the 20 amino acids are used.
7. Copy the entire DNA sequence. Do not copy the first line.
8. Go back to the NCBI home page (ncbi.nlm.nih.gov) by clicking
on the NCBI logo at the top of the page.
9. At the top of the page, click on “BLAST.”
10. Under “BLAST Assembled Genomes” near the top of the page,
click on “Pan troglodytes.” This will allow you to compare the
human beta-hemoglobin gene with the chimpanzee genome,
and not with the entire NCBI database. This saves time,
because beta hemoglobins have been sequenced for innumerable organisms. Other BLAST searches could allow you to
compare your sequence with the entire NCBI database or with
different subsets of it.
THE AMERICAN BIOLOGY TEACHER
11. Paste the human beta-hemoglobin sequence that you have copied
into the large box at the top of the page. This is your query. The
directions for the box say “enter an accession….”
12. Scroll to the bottom of the page and click on “Begin Search.”
13. In the middle of the page, to the right of “Request ID,” click on
“View Report” in a gray box.
14. Scroll down and look at the Graphic Summary. Notice that there
is one match to a gene on the number 11 chromosome of the
chimpanzee. Note that the gene for human beta hemoglobin is on
human chromosome number 11. At the bottom of the box, you will
see one bright red line indicating that there is one match. The fact
that the line is bright red indicates that this is a very close match.
Mouse over this line, and it will tell you, in technical terms, which
chimpanzee gene your query (human beta hemoglobin) matches.
15. Scroll down to “Alignments.” The “Query” is human beta hemoglobin. The “Subject” is chimpanzee beta hemoglobin. Notice that
whenever the gene is identical, there is a vertical line between the
identical base pairs.
USING THE NCBI GENOME DATABASES
255
16. Now go to the top of the page on the left and click on “Download.”
17. Near the top of the page under “Alignment,” click on “text.”
18. Click on “save to disk.”
19. Open the file. This will give you a comparison of the two beta
hemoglobins. You can print this out if a printout is not included
in the packet your teacher has given you (it is presented here
as Figure 2). When this is on your computer screen, you will
be able to search for the beginnings and ends of the exons in
later steps.
20. Now look at the copy of the human-beta hemoglobin gene
included in the packet your teacher has given you (Figure 1). This
copy of the gene is from 1980 and uses the three-letter abbreviations for the 20 amino acids. There are three exons and two
introns in this gene. You can identify the three exons because the
amino acids they code for are given above the base sequences.
Look at the sequences at the beginning and end of each exon. Use
a highlighter to highlight the nucleotide sequences of each of the
three exons in the human beta-hemoglobin gene on this copy of
the human beta-hemoglobin gene.
21. Take out your printout of the human–chimpanzee beta-hemoglobin
comparison from the BLAST search (Figure 2).
22. Use the find options of the computer (Edit/Find) to identify the
beginning and end of each exon of the human beta-hemoglobin
gene. Highlight the three exons on your printout of the human–
chimpanzee comparison. It is easiest to highlight the exons in the
human gene. Hint: search for six bases at the beginning and end
of each exon, which you have located in Figure 1. Six bases are
enough to identify these positions.
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THE AMERICAN BIOLOGY TEACHER
23. You can now answer the following questions (the answers are
given in italics):
a. Are the exons identical? No. Circle or highlight any differences you find. How many differences are there? One.
b. Are the introns identical? No. Circle or highlight any differences you find. How many differences are there? Fifteen.
c. Are there more differences in the exons or in the introns? In
the introns. Can you think of an explanation for this? There
would be no selection against a change in an intron.
d. How can there be a difference in the exons if the proteins are
identical? The genetic code is degenerate, so more than one codon
can code for the same amino acid.
Acknowledgment
J Thanks to Nadav Kupiek for expert preparation of the artwork.
References
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). Basic local
alignment search tool. Journal of Molecular Biology, 215, 403–410.
Lawn, R.M., Efstratiadis, A., O’Connell, C. & Maniatis, T. (1980). The nucleotide
sequence of the human beta-globin gene. Cell, 21, 647–651.
Vigue, C.L. (1987). Murphy’s law and the human beta-globin gene. American
Biology Teacher, 49, 76–81.
SUSAN OFFNER is a biology teacher at Lexington High School, in Lexington, MA
02421; e-mail: [email protected].
VOLUME 72, NO. 4, APRIL 2010