Aplikasi rekayasa genetika untuk
mempelajari fungsi alami gen
Mariana Wahjudi
Mei 2015
11-11-2009
Menentukan fungsi suatu gen
 Untuk menentukan bagaimana gen/gen-gen dan protein
yang dikodenya berfungsi dalam intact organism.
Beberapa metode pendekatan
A. Salah satu cara langsung adalah dg melihat apa yang
terjadi pada organisme bila gen tersebut hilang atau
mengekspresikan protein versi lain.
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Skrining genetis untuk mengisolasi mutant target
kemudian identifikasi gen/gen-gen yang bertanggungjawab
pada fenotip yang terlihat.
Spontaneous mutants ----- bisa ditemukan pada populasi
yang sangat besar
Lebih efficient dan cepat adalah dengan mutasi dengan
agents perusak DNA, dengan zat kimia atau radiasi.
• Pendekatan alternatif dengan insertional
mutagenesis
• Targeted Mutations
• The Normal Gene in a Cell Can Be Directly
Replaced by an Engineered Mutant Gene in
Bacteria
• Gain-of-Function Mutations
o Provide Clues to the Role Genes Play in a Cell
o Can produce a new phenotype in a cell, tissue, or organism
o Cells can be engineered to express a negative version of a protein,
resulting in a loss-of-function phenotype, they can also be
engineered to display a novel phenotype through a gain-of-function
mutation.
o Often, this mutants are generated by expressing a gene at a much
higher level than normal in cells, on a multicopy plasmid or
integrating it in multiple copies in the genome.
o Genes can also be expressed at the wrong time or at the wrong
place in an organism. Such misexpression is most often
accomplished by re-engineering the genes themselves, thereby
supplying them with the regulatory sequences needed to alter their
expression.
• Large Collections of Tagged Knockouts
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Provide a Tool for Examining the Function of Every
Gene in an Organism
produce a collection of mutant strains in which every
gene in the organism has either been systematically
deleted, or altered such that it can be conditionally
disrupted.
A large mixture of such gene knockout mutants can
then be grown under various selective test conditions
and the cells that survive can be rapidly identified by
their unique sequence tags.
By assessing how well each mutant in the mixture
fares, one can begin to assess which genes are
essential, useful, or irrelevant for growth under various
conditions.
• A Complementation Test Reveals Whether Two
Mutations Are in the Same or in Different Genes
• A large-scale genetic screen can turn up many
different mutants that show the same phenotype.
These defects might lie in different genes that
function in the same process, or they might represent
different mutations in the same gene. The mutations
thereby complement one another and restore a
normal phenotype.
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Reporter Genes Reveal When and Where a Gene Is
Expressed
o Clues to gene function can often be obtained by examining when
and where a gene is expressed in the cell or in the whole organism.
o gene expression is controlled by regulatory DNA sequences, located
upstream or downstream of the coding region, which are not
generally transcribed.
o control which cells will express a gene and under what conditions,
can also be made to drive the expression of a reporter gene. One
simply replaces the target gene's coding sequence with that of the
reporter gene, and introduces these recombinant DNA molecules
into cells. The level, timing, and cell specificity of reporter protein
production reflect the action of the regulatory sequences that belong
to the original gene.
o Several other techniques, can also be used to determine the
expression pattern of a gene, hybridization techniques such as
Northern analysis and in situ hybridization for RNA detection can
reveal when genes are transcribed and in which tissue, and how
much mRNA they produce.
 Genes Can Be Redesigned to Produce Proteins of Any
Desired Sequence
 In studying the action of a gene and the protein it encodes,
one does not always wish to make drastic changes—flooding
cells with huge quantities of hyperactive protein or eliminating
a gene product entirely. It is sometimes useful to make slight
changes in a protein's structure so that one can begin to
dissect which portions of a protein are important for its
function.
B. Eksplorasi fungsi gen berdasarkan urutan DNA.
 Didasarkan pada urutan gen atau gen-gen dan
mencoba menentukan fungsinya …….. Urutan
homologous dan menentukan kapan dan dimana
gen diekspresikan.
 Tantangannya adalah menterjemahkan urutan ke
fungsi.
Metode pendekatan
 Mencari Homology dengan gen-gen lain yang telah
diketahui fungsinya.
o
Contoh mencari databases dari protein yang telah
dikharacterisasi dengan baik proteins yang punya urutan
amino acid yang mirip dengan protein gen yang baru.
 Microarrays Monitor the Expression of Thousands of Genes
at Once
 to monitor the expression of thousands of genes simultaneously.
 to examine everything from the change in gene expression
 information about a gene's function can be deduced by identifying
genes that share its expression pattern. Using cluster analysis, one
can identify sets of genes that are coordinately regulated. Genes
that are turned on or turned off together under a variety of different
circumstances may work in concert in the cell: they may encode
proteins that are part of the same multiprotein machine, or proteins
that are involved in a complex coordinated activity
 Genes that belong to the same cluster may be involved in common
cellular pathways or processes. Using cluster analysis to identify
sets of genes that are coordinately regulated.
Literatures
 Alberts,B, A. Johnson, J. Lewis, M. Raff, K. Roberts, and
P. Walter, Studying Gene Expression and Function,
2002, in Molecular Biology of the Cell, 4th edition,
New York: Garland Science