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2008 Spring BioDB Homework 2
This problem set is due by 2PM, April 8, 2008. You shall upload your answers to
your web site as instructed by your TA. For all questions, please make a refer?ence
such as screen-shot to indicate the source of your answer.
Please read the following abstract before you start working on this homework.
Gelatinases (MMP-2 and -9) and their natural inhibitors as prognostic indicators
in solid cancers.
Neoplastic growth and dissemination involve increased proteolytic activity that
is able to escape the regulative elements. Matrix metalloproteinases (MMPs),
particularly gelatinases A and B (MMP-2 and -9), play a role in tumor invasion and
angiogenesis, and they participate in cancer progression in several neoplasias. The
expression of tissue inhibitors of gelatinases, TIMPs-1 and -2, has also been shown to
be associated with the clinical course in some cancers. The prognostic value of these
markers, however, seems to vary a great deal in different neoplastic diseases. In this
review, the impact of the gelatinases and their inhibitors on the clinical course in
several solid cancers is evaluated based on the growing data from recent clinical
studies. The clinical data most often explore the overexpression of mRNA or
immunoreactive protein in tumor tissue, or measure the concentration of the
circulating proteinase or its inhibitor in pretreatment or follow-up serum samples. The
growing amount of recent clinical data suggests that the impact of gelatinases on
treatment decisions should be tested in clinical trials.
- Biochimie. 2005 Mar-Apr;87(3-4):287-97. Review.
1.
Use GeneID to predict splice sites and start and stop codons in the human
MMP-2 sequence. Identify the real sites among the predictions. Do they tend
to show high scores?
First, I use Esembl to retrieve the MMP2 genomic sequence.
Then I use GeneID to predict genes.
This is the result.
# Gene 1 (Forward). 13 exons. 640 aa. Score = 61.69
I use WORD to calculate the real number of nucleotide, and
then compare with the prediction number of GENEID.
2.
Now, use geneID to predict the set of possible exons. Compare your results
with the exon predictions with the real exons identified in the Ensembl
database.
The number of GENEID exon prediction is 13, the same with
Ensembl.
3.
Using a promoter prediction server, predict what transcription factors may
regulate MMP-2 gene expression. Can you correlate these transcription factors
with the functions described in the abstract above? Give a short explanation.
I use TRANSFAC.
Transcription factor AP-2alphaA、p53 may regulate the
expression of MMP2.
I use GENECARD to find the function of TFAP2A:
transcription factor 2, AP-2 alpha, retinoic acid responsive gene, 52 kDa, regulating
the expression of genes required for development of tissues of ectodermal origin,
activating CDKN1A (WAF1) and other genes such as MCAM, MMP2, involved in
the progression of human melanoma and potentially involved in anterior eye chamber
development.
P53 down-regulates the expression of MMP2
p53 is a DNA-binding protein containing DNA-binding, oligomerization and
transcription activation domains. It is postulated to bind as a tetramer to a p53-binding
site and activate expression of downstream genes that inhibit growth and/or invasion,
and thus function as a tumor suppressor.
4.
Design a pair of PCR primers that can use in a typical real-time quantitative
PCR analysis with SYBR green for measuring human MMP-2 gene
expression.
5.
Conduct a restriction enzyme digestion analysis on the MMP-2 cDNA
sequence.
6.
Conduct a phylogenetic analysis of MMP2s found from at least 6 different
organisms. Which specie’s MMP-2 is the most closely related one to human
MMP-2?
Ans : P.troglodytes is the most closely species.
7.
Retrieve the protein interacting network of human MMP-2.
8.
Retrieve any microarray expression analysis on human MMP-2 in various
cancer tissues. Give a clear explanation on how to find it. Based on your
findings, MMP-2 is highly expressed in what cancer type in human?
I use CGAP to find expression analysis of MMP2.
Ans: Cartilage、Skin cancer
9.
Retrieve the following article and use DAVID to redo the functional
annotation analysis on genes listed in table 3. “Identification of gene
signatures for invasive colorectal tumor cells, A.H. Wiese et al. Cancer
Detection and Prevention 31 (2007) 282-295” Can you find MMP2 in your
analysis?
Ans: I can’t find MMP2.
10.
Identify a possible SNP in human MMP-2 gene.