BM1106 MINIPROJECT REPORT
CHLAMYDIAL MOMP GENE &
ITS USE IN MEDICAL STRAIN TYPING
Full Name :Julia Bte Abdullah
Matric Number : 982019Y02
E-mail Address : sci80019@nus.edu.sg
Date Submitted : 5th April 1999
The objectives of this project are :
1.
2.
3.
4.
5.
6.
To retrieve literature using PubMed at ncbi.nlm.nih.gov
To retrieve DNA sequence using Entrez .
To use EditSeq in DNAStar for DNA sequence analysis.
To perform multiple sequence alignment and view alignment report
To view amplification summary
To state the best PCR primer design and conditions deemed appropriate for PCR.
INTRODUCTION:
1.BIOLOGICAL & MEDICAL SIGNIFICANCE OF CHLAMYDIA
SPECIES
Chlamydia is a sexually-transmitted disease (STD) caused by bacterial infection. It is
popularly known as “ the silent epidermic” because three-quarters of the women and
half of the men who contracted the disease have no symptoms initially until
complications arise later. Although Chlamydia can produce serious consequences, it's
not as well known as other STDs, such as gonorrhea or syphilis.
It is a gram negative obligate intracellular bacteria that cause acute and chronic disease
in mammalian and avian species.
Chlamydia - General Characteristics




Obligate intracellular parasite
Gram-negative bacteria
"Energy parasites" - can't synthesize own ATP
Virulence factors : Intracellular growth
Inhibition of phagolysosome fusion
Endotoxin
Chlamydia - Multiplication


Elementary bodies: small dense cells; rigid cell walls; ~0.3um in diameter;
extracellular form - attaches to and enters host cell
Reticulate bodies: ~8 hours later within host cell; ~1um in diameter, divide by
binary fission for 24-48 hours within phagosome --> differentiate into elementary
bodies --> host cell lysis releases elementary bodies .
The genus Chlamydia is comprised of four species: C. trachomatis, C. pneumoniae, C.
precorum and C. psittaci (1- 4). For this project, we will be dealing with mainly
Chlamydia Trachomatis.
C. trachomatis is divided into 15 serovars (5-8). Serovars A, B, Ba and C are agents of
trachoma (9), leading cause of preventable blindness, endemic in the third worlds.
Serovars L1-L3 are the agents of lymphogranuloma venereum. Serovars D-K are a
common cause of sexually transmitted genital infection worldwide: cervitis,
endometritis/salpingitis(10) in females and uretrities(11) in both males and females.
Endometritis/salpingitis can lead to agglutination of salpinx with a higher risk of
extrauterine pregnancy and infertility. Genital infection can cause acute infection and
persistent infection occasionally without any clinical signs. Generally, these infections
are treatable once they are diagnosed, however, without any treatment the infections
can progress to severe chronic inflamantation leading to infertility, ectopic pregnancy,
induced abortion or child delivery. Moreover, infants to infected mothers can be infected
during birth leading to conjunctivitis or pneumonia (12-14).
The serology of C.trachomatis is more interesting in cases of chronic infections than in
acute infections.
C. pneuomoniae is an important respiratory pathogen in human and cause up to 10% of
community-acquired pneumonia cases. It has been associated with acute respiratory
diseases, pneumonia, asthma, bronchitis, pharyngitis, acute chest syndrome of sickle
cell disease, coronary heart disease and Guillain-Barre syndrome (15-17).
C. psittaci infects a diverse range of host species from molluscs through birds to
mammals and also causes severe pneumonia. In animals, C. psittaci and C. pecorum
are capable of inducing diverse disease syndroms like pneumonia, enteritis,
polyserositis, encephalitis and conjunctivitis.
Serological testing, now an established approach in many countries, has been shown to
provide a comprehensive answer for the detection of C. trachomatis infection. In
suspected deep-seated infections, serum sampling reduces the necessity for invasive
procedures which are required for antigen direct detection. In cases of lower urogenital
infections, collection limitations such as effectiveness of scrape sampling procedure,
specimen handling and transportation difficulties have to be weighed. Above all, there
remains the issue that most Chlamydial infections are asymptomatic. Therefore, an
infection may persist for a long time, ascend the upper genital tract causing deep and
chronic infections , and increase the probability of false negative results in direct antigen
detection.
2. CHLAMYDIAL MAJOR OUTER MEMBRANE PROTEIN
(MOMP) AND ITS GENE
In the search for a vaccine against Chlamydia infection (Chlamydia Trachomatis
specifically), scientists have found out that they can make use of the major outer
membrane protein ( MOMP ) of chlamydia and its gene. In fact there is a Chlamydia
Trachomatis Genome Project The goal of the Chlamydia Genome Project is to
determine the DNA sequence of the chromosome of Chlamydia trachomatis, serovar D
(D/UW-3/Cx), trachoma biovar, and L2/434/Bu, LGV biovar. The project is a
collaborative effort involving scientists at the University of California at Berkeley and
Stanford University .
A safe and effective vaccine may require the use of a recombinant polypeptide
The MOMP gene may be the source of such a vaccine. Research has demonstrated
that unique serological determinants are associated
with the MOMP protein and the identification of the genetic sequence for the
C.trachomatis MOMP gene provided the basis on which production and
optimization of a MOMP- based vaccine has centered. The MOMP gene sequence may
also be suitable for the derivation of nucleic acid probes useful in the specific detection
of C. trachomatis and its related species. Therefore a primary vaccine can be
constructed to combat Chlamydial infections.Various
genes are located on the outer membrane protein, all of which are classified and
numerically for to its own particular strain.
3. MOLECULAR TYPING OF THE MOMP GENE USING RFLP AND ITS
USEFULNESS
Restriction Fragment Length Polymorphism (RFLP) is the technique whereby the
cleavage of DNA and DNA fragments analysis is used to differentiate organism. Also
known as DNA fingerprints, RFLP involved cutting DNA with restriction enzymes and
separation according to its size with gel electrophoresis. Restriction enzymes recognise
a specific sequence of bases and cut the DNA strand only at those places that have that
sequence.It is then blot onto nylon sheet and radioactive DNA probe is added for the
interpretationof the bands. Each individual banding pattern is unique and can be used
todifferentiate strains from one another. RFLPs have been sequenced to determine
what sequence variation is causing the polymorphism. One of the advantages of
working with RFLPs is that the sequence need not be known; all that is needed is a
genomic clone that can be used to detect the polymorphism. The RFLPdetection and
collection of genetic data can be very rapid with this method.However, without
sequence information, the interpretation of complicated RFLP allele systems can be
very difficult. In the lab, we have seen many different types of RFLP polymorphisms.
Interpretation of the type of the polymorphism is based on our knowledge of DNA
sequence variation and how chromosomes have evolved and not on any certainty of the
cause of the specific polymorphism.
Methods
Firstly, retrieval of DNA sequences were from the Nucleotide databases in ENTREZ at
PubMed at ncbi.nlm.nih.gov. 10 DNA sequences of “Chlamydia Trachomatis and
MOMP and complete codons” were chosen .
REFERENCE NUMBER
STRAIN
AF063204
K/UW31/CX
AF063203
Ja/IU-A795
AF063202
J/UW36/CX
AF063201
Ia/IU-4168
AF063200
I/UW57/CX
AF063199
G/UW57/CX
AF063198
E/IU-51538
AF063197
D/IU-83786
AF063196
D/IU-72403
AF063194
Ba/Apache-2
DNASTAR was installed. EDITSEQ in DNASTAR was opened and each DNA sequence
was opened, copied and pasted from ENTREZ to EDITSEQ. Each sequence was saved
as a separate file in EDITSEQ.
Next, MEGALIGN in DNASTAR was opened. The sequences which were saved in
EDITSEQ was entered. Alignment by Clustal method was done. The phylogenetic tree
is as follows:
From the above diagram, we can see that strains Ia, I, Ja, J and K are related to one
another just as strains Ba, D, E and G are. This is further proven when the sequence
distances were viewed. The percentage of similarities are very close for strains Ia, I,
Ja, J and K and strains Ba, D, E and G. Below is the sequence distances of the 10
strains used obtained from the View Sequence Distances.
Next, a region of multiple sequence alignment to show the conserved regions was
produced. The highlighted blue region represents the conserved sequences which were
present in most of the strains and the unshaded white region was the region with
variable sequences of Chlamydia Trachomatis.
At the PRIMERSELECT, we view the locations of the primer pairs of the 10 selected
strains. There are 7 primary pairs, 55 located and 15 alternates.
In silico amplification was done next. This is by viewing amplification summary. It
shows the conditions for the PCR to be carried out.
From this table, the primer location was divided into upper and lower regions and the
temperature for each is clearly distinguished. It describes in detail the conditions for
each primer as well such as primer locations.
Last but not least, MAPDRAW was done to show where the restriction enzymes had
cut. There are 8 sites cut by the enzymes Alu1 and Ear 1. This process is also known
as in silico restriction mapping. In silico restriction mapping was
performed to analyse for the possible enzymes-cut-sites to produce DNA fragments.
In conclusion, from this project we have shown that it is possible to make use of stored
data in PubMed and DNASTAR package and utilized it in such a way that it is possible
to predict the outcome of our ‘Chlamydia Genome Project’ to produce a vaccine
hopefully in the near future! Also, using sophisticated computational software means we
may even omit in vitro or in vivo experimentation. Even though it is still far too early to
do so, nevertheless it is possible.
REFERENCES
1. http://www.ncbi.nlm.nlh.gov/
2. http://sequence-www.stanford.edu/group/chlamydia/index.html
3. http://chlamydia-www.berkeley.edu.4231/descript.html
4. http://www.niaid.nih.gov/default.htm