Q 2011 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION

Vol. 39, No. 1, pp. 17–20, 2011

Articles

Exploring Cystic Fibrosis Using Bioinformatics Tools

A MODULE DESIGNED FOR THE FRESHMAN BIOLOGY COURSE*

Received for publication, July 19, 2010, and in revised form, September 15, 2010

Xiaorong Zhang†
From the Department of Natural Sciences, Savannah State University, Savannah, Georgia 31404

We incorporated a bioinformatics component into the freshman biology course that allows students to
explore cystic fibrosis (CF), a common genetic disorder, using bioinformatics tools and skills. Students
learn about CF through searching genetic databases, analyzing genetic sequences, and observing the
three-dimentional structures of proteins using a number of computational tools. We also use this oppor-
tunity to discuss the genetic, molecular, biochemical, and physiological aspects of CF, in an effort to
help students appreciate the interrelationships among concepts and topics in biology within a real-world
context. This component provides the freshman students at Savannah State University an early exposure
to bioinformatics and prepares them for the upper level courses in this area. It also facilitates meaningful
and in-depth learning by linking biological concepts and processes through a case study.
Keywords: Bioinformatics, cystic fibrosis, cystic fibrosis transmembrane conductance regulator (CFTR), Cn3D.

As a part of our continuous effort to incorporate a bio-
informatics component into the undergraduate curriculum
at all levels at Savannah State University (SSU), we
developed a bioinformatics module for the freshmen biol-
ogy course (Principle of Biology I). In this course, we use
cystic fibrosis (CF) as an example to teach freshmen ba-
sic knowledge and skills in bioinformatics. As the
genetic, molecular, biochemical, and physiological bases
of CF have been thoroughly investigated and well under-
stood, this genetic disease also provides us an excellent
opportunity for us to link biological topics and reveal the
underlying relationships in biology within a real-world
context, thus helping students develop a more holistic
and coherent view of biology. Our objectives for incorpo-
rating this module into the freshman biology course are
twofold: 1) to expose freshmen at SSU to the basics of
bioinformatics and 2) to help students learn biology by
linking various topics and concepts through an investiga-
tion of CF.
BACKGROUND
CF is a well-known genetic disease, which is caused
by a mutation in the gene coding for CF transmem-
brane conductance regulator (CFTR) [1]. CFTR func-
tions mainly as a chloride channel and also controls
the regulation of other transport pathways. The flow of
chloride ions helps control the movement of water in
tissues, which is necessary for the production of thin,
freely flowing mucus [2]. A mutation in the CFTR gene
disrupts the function of the chloride channel, prevent-
ing it from regulating the flow of chloride ions and
water across cell membranes. As a result, cells that
line the passageways of the lungs, pancreas, and other
organs produce mucus that is unusually thick and
sticky. This mucus clogs the airways and glands, caus-
ing the characteristic signs and symptoms of CF [2].
Although the 3D structure of the entire CFTR has not
been experimentally determined, a model depicting the
structure of CFTR has been proposed [3]. CFTR is
made up of five domains: two membrane-spanning
domains (MSD1 and MSD2) that form the chloride ion
channel, two nucleotide-binding domains (NBD1 and
NBD2) that bind and hydrolyze ATP, and a regulatory
(R) domain. The ion channel only opens when the R-
domain has been phosphorylated and ATP is bound at
the NBDs [3]. More than 1,000 mutations in the CFTR
gene have been identified in people with CF. The most
common mutation, called DeltaF508, is a deletion of
one amino acid (phenylalanine or phe) at position 508
in NBD1 domain of CFTR protein [4]. The resulting
abnormal channel breaks down shortly after it is made,
so it never reaches the cell membrane to transport
chloride ions.

*This work is supported by a NSF-HBCU-UP to SSU (award LECTURE MATERIALS

#:0928454).
† To whom correspondence should be addressed. Department
of Natural Sciences, Savannah State University, Savannah, Georgia
31404, USA. Tel.: 912-358-4442; Fax: 912-353-3186.
E-mail: zhangx@savannahstate.edu.
This paper is available on line at http://www.bambed.org
Principle of Biology I is a required course for biology,
marine sciences, environmental sciences, and chemistry
majors at SSU. Multiple sections of Principle of Biology
I are offered each semester, including the summer
17 DOI 10.1002/bmb.20460
18
TABLE I
Internet addresses (URLs) for the websites used in laboratory
exercises
Database/server/
software URL
NCBI http://www.ncbi.nlm.nih.gov/
Online Mendelian http://www.ncbi.nlm.nih.gov/omim/
Inheritance in Man
Pfam Database pfam.sanger.ac.uk/
ExPaSy-Prosite http://expasy.org/prosite/
Cystic Fibrosis http://www.genet.sickkids.on.ca/app
Mutation Database
Cn3D (downloaded http://www.ncbi.nlm.nih.gov/
from Cn3D home Structure/CN3D/cn3d.shtml
page)
Phobius server http://phobius.sbc.su.se/
semester. For the past several years (2006–2010), all the
sections of the course were taught by the author, and
the any course module developed by the author was
incorporated into all the sections to ensure the consis-
tency from section to section. Approximately 150–200
students are enrolled into this course each year, and a
majority of students are freshmen. Life—The Science of
Biology by Sadava et al. [5] has been used as the text-
book. Principle of Biology I covers topics on the chemi-
cal basis of life, cells and energy, and genes and hered-
ity. Some basic concepts of genomics and bioinformatics
have been introduced into the current edition (9th edition)
in a new chapter, called ‘‘Genomes.’’ In this textbook, CF
is used as an example of an autosomal recessive trait in
the topic of Mendelian genetics, and it is also briefly dis-
cussed on topic of gene mutations. However, other
aspects of CF are not discussed in the book. Thus, we
developed the lecture materials by collecting original
research papers and Internet resources on CF. We used
Online Mendelian Inheritance in Man (OMIM) (Table I) at
National Center for Biotechnology Information (NCBI) as
a starting place, which provides a comprehensive, au-
thoritative, and timely compendium of genetic diseases,
including CF. OMIM also provides hyperlinks to other
genetic online resources and references. We organize
and deliver these materials in a way that the materials
are aligned closely with topics covered in the course. For
example, in the chapter on Cell Membranes, we explain
the domain organization and topology of CFTR within a
cellular membrane, and how a CFTR functions as a chlo-
ride channel and regulates the transport of chloride ions
and water; In the chapter From DNA to Protein, we dis-
cuss different mutations that have occurred in the CFTR
gene, and how the amino acid sequence of the CFTR
protein is altered through transcription and translation;
we relate the structures of the CFTR protein to the lec-
ture topics on protein folding and functional domains.
Throughout the semester, we revisit CF again and again
to relate it to many concepts and topics in biology, so
that students can make sense of the abstract biological
concepts and processes through a real-world example.
BIOINFORMATICS LABS
The lab section accompanying Principle of Biology I
had consisted of traditional one-time or short-term
BAMBED, Vol. 39, No. 1, pp. 17–20, 2011
laboratory exercises. The traditional approach often failed
to demonstrate the connectedness and interrelatedness
of biology. Thus, we designed a few lab exercises for
students to learn about CF using bioinformatics tools,
such as databases and software. These exercises were
designed to reinforce the concepts discussed in the
lecture sessions.
Exercise 1: Explore the Genetic Sequences of the
CFTR Gene and Its Encoded Protein by Searching
Genetic Databases
The goal of this exercise is to help students explore
the genomic, cDNA, and protein sequences of CFTR.
Students are able to retrieve all the information about the
CFTR gene (GeneID: 1080) by searching the NCBI site
(Table I), including the graphic view of its chromosomal
location (7q31.2), its gene structure (27 exons and 26
introns), and its genomic sequence. Students are also
able to access the cDNA (accession #: NM_000492) and
protein (accession #: P13569) sequence data. In addition,
students need to search the CF Mutation Database (Ta-
ble I) to examine the mutations associated with the
CFTR gene, including DeltaF508. This exercise can be
linked to several lecture topics, such as central dogma,
eukaryotic gene structure and intron splicing, and gene
mutations. Through this exercise, students are expected
to understand how the information in a gene flows into
its protein product through transcription and translation
and how a gene mutation leads to alterations in the
amino acid sequence of the protein that may result in a
serious genetic disease.
Exercise 2: Visualization of the 3D Structures of CFTR
Using Cn3D
This activity requires students to use Cn3D, a program
that can be easily downloaded from the NCBI site and
used to view the 3D structures of biomolecules stored in
NCBI’s Molecular Modeling Database (MMDB) (Table I).
Although there are no structures of the entire CFTR pro-
tein stored in MMDB or other structure databases,
MMDB contains the structures of CFTR NBD1 domain
with the phe508 (wild-type) and DeltaF508 mutation,
which were determined by using crystallography and
hydrogen/deuterium exchange mass spectrometry [6, 7].
This hands-on exercise was designed to enhance stu-
dent learning and understanding of the abstract con-
cepts of protein structure and protein folding pathway.
Viewing the structures of NBD1 under different represen-
tation styles (e.g. wireframe, space-filling, ribbon, and
ball-and-stick), students are able to examine all the
structural features of a protein, such as secondary struc-
tures (a-helix and b-sheet), chemical bonds, shape, sur-
face, etc. Especially through a direct observation of the
structures of wild-type and mutated NBD1 domain of
CFTR, students can visualize how a deletion of an amino
acid results in a change in the 3D structure of a protein,
and causing a serious disease. CFTR, therefore, repre-
sents an excellent example of illustrating the close rela-
tionships between the structure and function of a protein.
 19

FIG. 1. 3D structures of NBD1 of a normal CFTR (a) and a CFTR with DeltaF508 mutation (b). These structures were
retrieved from MMDB at NCBI, viewed and edited with Cn3D. PDB IDs for these structures are 2BBO for F508 NBD1 and 1XMJ
for Delta508 NBD1. The polypeptide backbone is represented by a wire with a-helices shown as green arrows pointing toward the
C-terminus (c) and b-sheets shown as yellow arrows pointing to the C-terminus. Mg2þ and ATP (in ball-and-stick form) are also
shown. In the F508 NBD1, phenylalanine at position 508 is labeled; in DeltaF508 NBD1, glycine at position 509 is labeled.

Figure 1 shows the 3D structures of a normal NBD1 and Based on the combined results of pfam database
a mutated NBD1 with DeltaF508, which are stored in searches and Phobius analysis, students drew a sketch
MMDB and viewed with Cn3D. Although the deletion of of the domain organization and topology of CFTR in a
Phe at position 508 does not lead to a global change in cellular membrane (Fig. 2b), which helped students
the conformation of NBD1, it does cause local structural
changes restricted to amino acid residues 509–511,
which lead to a serious genetic disorder [6, 7].

Exercise 3: Analysis of Amino acid Sequence

of CFTR Protein
In this exercise, students use bioinformatics programs
to analyze the amino acid sequence of CFTR to make
several predictions. First, students need to identify the
important domains in CFTR by searching the Pfam data-
base (Table I), a large collection of protein domains at
Sanger Institute [8]. Students are able to identify two
membrane-spanning domains (MSD1 and MSD2) and
two nucleotide-binding domains (NBD1 and NBD2) that
bind and hydrolyze ATP, which are arranged in an order
of MSD1-NBD1-MSD2-NBD2 from N- to C-terminus.
Similar results are also obtained by searching PROSITE,
a database of protein domains, families, and functional
sites [9].
Students also use the Phobius program [10] (Table I) to
predict transmembrane a-helices in CFTR. Phobius is a
program of predicting transmembrane helices in an inte-
gral protein. It is also capable of predicting the most
probable orientations of transmembrane helices and
FIG. 2. a: Analysis of human CFTR protein using Phobius
intra- or extracellular regions of the protein. A Phobius program. This graphic output indicates the location of pre-
analysis of CFTR is shown in Fig. 1a as an example of dicted transmembrane segments, its predicted topology, and
student work. Phobius predicts 12 transmembrane a-hel- the reliability of the prediction. The blue line is the probability
ices, which form two membrane-spanning domains that a certain position is outside of the cell, the green line is the
(MSD1 and MSD2), and a long intracellular loop between probability it resides in the cytoplasmic side of the membrane,
and the gray line is the probability that this residue is in the
the two membrane-spanning domains. Both N- and C- membrane. b: Diagram showing the architecture and topology
terminal are predicted to be located on the inside of a of CFTR based on the sequence analysis of CFTR protein. Dou-
cell membrane. ble lines represent phospholipid bilayer.
20
understand what specific role of each domain of CFTR
plays in the transport of chlorides ions and water, and
how the function of CFTR is closely related to its domain
structure.
ASSESSMENTS OF LEARNING OUTCOMES
This bioinformatics component definitely increased stu-
dent awareness of bioinformatics as an emerging field.
Results of our survey in Spring 2010 indicated that none
of the entering freshmen had heard of bioinformatics and
that they first became aware of the field and its essential
role in modern biology through this course. We were also
under the impression that students in general had shown
increased enthusiasm for learning biology, which was
reflected from their active participation in the class and
their expression of interests in taking more courses in
this area, such as Introduction to Biocomputing and Mo-
lecular Evolution. Student performance was evaluated
mainly through examinations, laboratory reports, and a
class project. For example, students were given a set of
questions for each lab which was designed to assess the
level of their understanding of the concepts and skills
they learned. All of the students were able to complete
the assignments with help from the instructor. For the
projects, each student was asked to focus on a particu-
lar genetic disorder based on his or her own interests.
The project assessed each student’s ability to independ-
ently search databases to retrieve genetic sequences
and other information associated with the disorder and
analyze the genetic data using bioinformatics tools and
software. All of the students were required to write a pa-
per on their projects to present their findings. Through
the project, students had investigated a wide range of
genetic disorders using bioinformatics tools, including
sickle cell anemia, Huntington disease, albinism, breast
cancer, and many others. Seventy to eighty percent of
the students were able to complete their projects satis-
factorily.
CONCLUSIONS AND FUTURE PLANS
We introduced bioinformatics to the freshmen biology
course at SSU using CF as an example. Students were
exposed to several important bioinformatics concepts
and skills through learning CF, such as database search-
ing, 3D structure visualization, and protein sequence
analysis. This component was also designed to enhance
students’ understanding of biology by linking concepts
and topics through teaching CF. For example, we rein-
forced several important biological concepts in the con-
text of CF, including central dogma, gene mutation, pro-
BAMBED, Vol. 39, No. 1, pp. 17–20, 2011
tein folding, protein domain structure, ion channel, etc.
We expect that this bioinformatics component would
help our students learn biology as well as prepare them
for the upper-level bioinformatics courses such as Intro-
duction to Biocomputing, Molecular Evolution and Intro-
duction to Genomics. Unfortunately, we were unable to
explore a broader range of bioinformatics tools due to
time constraints in this course. We plan to continue to
explore CF in the second part of freshman biology (Prin-
ciple of Biology II). Students will investigate the molecular
evolution of CFTR genes using bioinformatics tools. For
example, students can identify the CFTR genes in other
species (orthologs) and build evolutionary trees of spe-
cies using these CFTR genes. Such evolutionary analysis
of CFTR genes can be linked to the topics on molecular
evolution and phylogenetic analysis in the course Princi-
ple of Biology II.
REFERENCES
[1] J. M. Rommens, M. C. Iannuzzi, B. Kerem, M. L. Drumm, G.
Melmer, M. Dean, R. Rozmahel, J. L. Cole, D. Kennedy, N. Hidaka
(1989) Identification of the cystic fibrosis gene: Chromosome walk-
ing and jumping. Science 245, 1059–1065.
[2] D. C. Gadsby, P. Vergani, L. Csanády (2006) The ABC protein
turned chloride channel whose failure causes cystic fibrosis. Nature
440, 477–483.
[3] D. N. Sheppard, M. J. Welsh (1999) Structure and function of the
CFTR chloride channel. Phys. Rev. 79, S23–S45.
[4] J. L. Bobadilla, M. Macek, J. P. Fine, P. M. Farrell (2002) Cystic fi-
brosis: A worldwide analysis of CFTR mutations—Correlation with
incidence data and application to screening. Hum. Mutat. 19, 575–
606.
[5] D. Sadava, D. M. Hillis, H. C. Heller, M. R. Berenbaum (2010) Life:
The Science of Biology, 9th ed., Sinauer Associates, Inc. and Free-
man and Company.
[6] H. A. Lewis, X. Zhao, C. Wang, J. M. Sauder, I. Rooney, B. W.
Noland, D. Lorimer, M. C. Kearins, K. Conners, B. Condon, P. C.
Maloney, W. B. Guggino, J. F. Hunt, S. Emtage (2005) Impact of the
deltaF508 mutation in first nucleotide-binding domain of human
cystic fibrosis transmembrane conductance regulator on domain
folding and structure. J. Biol. Chem. 280, 1346–1353.
[7] H. A. Lewis, C. Wang, X. Zhao, Y. Hamuro, K. Conners, M. C. Kear-
ins, F. Lu, J. M. Sauder, K. S. Molnar, S. J. Coales, P. C. Maloney,
W. B. Guggino, D. R. Wetmore, P. C. Weber, J. F. Hunt (2010) Struc-
ture and dynamics of NBD1 from CFTR characterized using crystal-
lography and hydrogen/deuterium exchange mass spectrometry. J.
Mol. Biol. 396, 406–430.
[8] R. D. Finn, J. Mistry, B. Schuster-Böckler, S. Griffiths-Jones, V. Hol-
lich, T. Lassmann, S. Moxon, M. Marshall, A. Khanna, R. Durbin, S.
R. Eddy, E. L. L. Sonnhammer, A. Bateman (2006) Pfam: Clans,
web tools and services. Nucleic Acids Res. 34, D247–D251.
[9] N. Hulo, A. Bairoch, V. Bulliard, L. Cerutti, B. A. Cuche, E. de Cas-
tro, C. Lachaize, P. S. Langendijk-Genevaux, C. J. A. Sigrist (2008)
The 20 years of PROSITE. Nucleic Acids Res. 36, D245–D249.
[10] L. Käll, A. Krogh, E. L. L. Sonnhammer (2007) Advantages of com-
bined transmembrane topology and signal peptide prediction—The
Phobius web server. Nucleic Acids Res. 35, W429–W432.