TATAP MUKA 12

LO 52: menjelaskan analisis struktur dan fungsi gen

LO 53: menjelaskan ekspresi gen dalam konteks genom
LO 54: menjelaskan produksi protein
LO 55: menjelaskan rekayasa protein
LO 56: menjelaskan diagnosis infeksi
LO 57: menjelaskan DNA profiling
Learning Outcome (LO)
studying gen
Gene
structure
Gene
expression
Aplication of Genetic engineering
Coding
sequences
DNA
binding
proteins
Control
sequences mRNA
transcript
levels
Effects of
deletion &
mutation
protein production
Inserting coding
sequence in the
correct reading frame
Fusion
protein
Native
protein
May be
cleaved
by CnBr
Bacteria
Yeasts
Insects
Mammalian
cells
Therapeutics
rBST
Enzymes
Medical and forensic
Infection
Diagnosis
Transgenic organisms (GMO)
Plants
Animals
Alter the organisms
genetic makeup
Genes from a
different
organism
Beneficial
changes
Agricultural Biotechnology
Basic Research
Medical applications
involves investigation of
by studying
to produce
by
aim is to
by incorporating
to effect
in hosts
such as
useful for
which
can be can be
by identifying
DNA
profiling
Three main areas involved
LO 52: menjelaskan analisis struktur dan fungsi gen
Menemukan daerah penting gen
Teknik yang didasarkan atas elektroforesis gel dapat digunakan
untuk menentukan daerah gen yang terlibat pengikatan protein
regulator.
Teknik tersebut meliputi:
gel retardation
DNA footprinting (DNase protection method)
chromatin immunoprecipitation (ChIP) assay
Start sites transkripsi gen tertentu perlu ditemukan secara
eksperimental, karena start site transkripsi mungkin tidak
terlihat dari data urutan basa gen yang diperoleh.
Transcription start sites dapat diidentifikasi dengan teknik:
primer extension
S1 mapping
Prosedur
Persiapkan peta restriksi DNA
terklon dan fragmen-fragmen
restriksi.
Tambahkan protein yang diteliti
(RNA polimerase, protein represor,
atau molekul pengatur lainnya)
Biarkan terjadi pengikatan
DNA/protein
Jika fragmen kemudian dielektroforesis, hibrida DNA/protein akan
bermigrasi lebih lambat daripada fragmen kontrol tanpa protein.
Teknik ini memberikan informasi tentang lokasi situs pengikatan
molekul DNA pada protein.
Keberhasilan teknik gel retardation tergantung pada ketepatan
peta restriksi dan ukuran fragmen yang dihasilkan. Mengapa????
Gel retardation (gel-shift) assays
http://bioweb.wku.edu/courses/biol350/
Transcriptome17/Review.html
LO 52: menjelaskan analisis struktur dan fungsi gen
Prosedur:
(a) Molekul DNA dilabel di salah satu ujungnya dengan
32
P.
(b) Pprotein regulator ditambahkan dan dibiarkan mengikat
ke situs tersebut. Reaksi kontrol tanpa protein juga
dilakukan.
(c) DNase I digunakan untuk memotong untai DNA. Kondisi
dipilih sedemikian rupa sehingga rata-rata hanya satu
nick per molekul DNA. Bagian DNA yang dilindungi oleh
protein terikat tidak akan dipotong.
(d) Hasil reaksi tersebut kemudian dielektroforesis pada gel
sekuensing. Bila dibandingkan dengan reaksi kontrol
(jalur 1), reaksi uji (jalur 2) menunjukkan posisi dari
protein pada DNA.
Fig. 10.1 DNA footprinting
DNA footprinting (DNase protection)
Cara yang lebih akurat untuk mengidentifikasi daerah
DNA yang mengikat protein adalah teknik DNA
footprinting (DNase protection). Teknik ini sederhana dan
elegan bergantung pada fakta bahwa wilayah DNA yang
membentuk kompleks dengan protein tidak akan sensitif
terhadap serangan DNase I.
LO 52: menjelaskan analisis struktur dan fungsi gen
LO 52: menjelaskan analisis struktur dan fungsi gen
Mengidentifikasi daerah penting gen
Start sites transkripsi gen tertentu perlu diidentifikasi secara
eksperimental, karena start site transkripsi mungkin tidak terlihat
dari data urutan basa gen yang diperoleh.
Start sites transkripsi dapat diidentifikasi dengan teknik:
primer extension
cDNA disintesis dari primer yang menghibridisasi dekat ujung 5 mRNA.
Dengan mengukur fragmen yang dihasilkan, ujung 5 mRNA dapat
diidentifikasi.
Jika reaksi sekuensing paralel dijalankan dengan menggunakan klon genomik
dan primer yang sama, start site transkripsi gen dapat ditentukan.
S1 mapping
In S1 mapping the genomic fragment that includes the TC start site is labelled
and used as a probe.
The fragment is hybridised to the mRNA and the hybrid then digested with
single-strand-specific S1 nuclease.
The length of the protected fragment will indicate the location of the TC start
site relative to the end of the genomic restriction fragment.
This assay measures the distance between an end label and the point to which reverse
transcriptase can copy the RNA. A short fragment of DNA, complementary to RNA, shorter
than the RNA and labeled at the 5' end, is hybridized to the RNA. It will now serve as a
primer for synthesis of the complementary DNA by reverse transcriptase. The size of the
resulting primer extension product gives the distance from the labeled site to the 5' end of
the RNA (or to the nearest block to reverse transcriptase)
The nucleotide in DNA that
encodes the 5' end of mRNA is
almost always the start site for
transcription. Thus methods to
map the 5 end of the mRNA
are critical first steps in
defining the promoter.
primer extension
This assay measures the distance between an end label (at a specific known site on DNA)
and the end of a duplex between RNA and the labeled DNA. A fragment of DNA
(complementary to the RNA) that extends beyond the 5' end of the RNA is labeled at a
restriction site within the RNA-complementary region. The labeled DNA is hybridized to
RNA and then digested with the single-strand specific nuclease S1. The resulting
fragment of protected DNA is run on a denaturing gel to determine its size. Note that
this fragment runs from the labeled site to the nearest interruption between the DNA
and the RNA. This could be the beginning of the RNA, or it could be an intron, or it could
be an S1 sensitive site.
S1 mapping
Investigating gene expression
Teknologi DNA rekombinan dapat digunakan untuk
mempelajari ekspresi gen dalam dua cara utama.
Pertama, gen yang telah diisolasi dan dikarakterisasi
dapat dimodifikasi dan efek modifikasi diteliti
(deletion analysis)
Kedua, probe yang telah diperoleh dari urutan
terklon dapat digunakan untuk menentukan level
mRNA untuk protein tertentu dalam berbagai
kondisi (Dot-blot analysis)
Kedua pendekatan, dan ekstensinya, telah memberikan
banyak informasi yang berguna tentang bagaimana
ekspresi gen diatur dalam berbagai jenis sel.
LO 53: menjelaskan ekspresi gen dalam konteks genom
1) In this hypothetical example a gene
has five suspected upstream
controlling regions (1 to 5, hatched).
2) The gene promoter is labelled P.
3) Often the lacZ gene is used as a
reporter gene for detection of gene
expression using the X-gal system.
4) Deletions are created using an enzyme
such as Bal 31 nuclease.
5) In this example four deletion
constructs have been made (labelled
A to D).
6) In A, region 1 has been deleted, with progressively more upstream sequence
removed in each construct so that in D regions 1, 2, 3, and 4 have been deleted
and only region 5 has been retained.
7) The effects of these deletions can be monitored by the detection of -
galactosidase activity and, thus, the positions of upstream controlling elements
can be determined.
8) As an alternative to using Bal 31, restriction fragments can be removed from
the controlling region.
Fig. 10.2 Deletion analysis in the study of
gene expression
deletion analysis
LO 53: menjelaskan ekspresi gen dalam konteks genom
1) Samples of total RNA from synchronous cell
cultures of Chlamydomonas reinhardtii grown
under batch culture and turbidostat (control)
culture conditions were spotted onto a
membrane filter.
2) The filter was probed with a radiolabelled cDNA
specific for an mRNA that is expressed under
conditions of flagellar regeneration.
3) (a) An autoradiograph was prepared after
hybridisation.
4) Batch conditions (i) show a periodic increase in
transcript levels with a peak at 15 h.
5) Control samples (ii) show constant levels.
6) Data shown in (b) were obtained by counting
the amount of radioactivity in each dot.
7) This information can be used to determine the
effect of culture conditions on the expression of
the flagellar protein.
Fig. 10.3 Dot-blot analysis of
mRNA levels.
Dot-blot analysis
LO 53: menjelaskan ekspresi gen dalam konteks genom
Ekspresi gen dalam konteks genom
Although much emphasis is still placed on the analysis of
individual genes, advances in gene manipulation technology have
opened up the study of genomes to the point where this is
emerging as a discipline in its own right.
Often called simply genomics, the emphasis here is on a holistic
approach to how genomes function.
Therefore, we are now much more likely to assess the function of
a gene within the context of its role in the genome, as opposed to
considering gene structure and expression in isolation.
The development and use of DNA microarrays (sometimes
referred to as DNA chips) for investigating gene structure and
expression is a nice example of how a new technology can move
an area of science forward dramatically.
Lets look at this aspect to illustrate the point.
LO 53: menjelaskan ekspresi gen dalam konteks genom
Fig. 10.4 A DNA microarray
on a glass slide. A spot on
the slide with a DNA sequence
is called a field. Each of the 48
small squares contains a 45
45 array of DNA sequences,
giving a total of 97 200 fields.
Fig. 10.5 DNA microarray technology. The 97 200 field array (a) has 48
blocks of 45 45 fields (b). Samples to be analysed (often cDNA
prepared from mRNA and labelled with fluorescent dyes) are pumped
onto the array and allowed to hybridise. Excess is washed off and the
array is read in a laser-activated scanning device. The pattern of
hybridisation is presented on a computer display, part of which is
indicated in (c). The different levels of signal in each field indicate the
level of gene expression and are correlated with the genes on the
microarray using computer analysis.
LO 53: menjelaskan ekspresi gen dalam konteks genom
An array is generated with the sequences of
interest attached to the support matrix.
Samples of DNA are generated from the two
cell types. Often this is achieved by synthesising
cDNA, which is tagged with a fluorescent dye
(a). The cDNA samples are hybridised
separately to the microarray (b) and the signals
analysed. Results are compared as shown in (c).
A black spot indicates no expression of the gene
represented by the field. In this example a
positive result is shown by the blue circles.
Comparison of the results shows where genes
are differentially expressed. Thus, the gene in
position A1 is expressed in cell A but not in B,
whilst B1 is the reverse. Some genes (e.g. C2)
are not expressed in either of the cell types,
and some (e.g. E5) are expressed in both.
Fig. 10.11 Transcriptome analysis in two
cell types using a microarray
LO 53: menjelaskan ekspresi gen dalam konteks genom
In this example, (a) mRNA from the two
cells is used to prepare cDNA that is
labelled with two different fluorescent
dyes. (b) The cDNAs are mixed and
hybridised to the array. The pattern of
signal detected is shown in (c). The
various colours produced indicate the
levels of transcript in each of the cells.
This can give an indication of
differential expression of genes using a
single microarray.
Fig. 10.12 Transcriptome analysis using
pooled cDNAs to investigate differential
gene expression
LO 53: menjelaskan ekspresi gen dalam konteks genom
The synthesis and purification of proteins from cloned genes is one of the most
important aspects of genetic manipulation, particularly where valuable
therapeutic proteins are concerned.
Many such proteins have already been produced by recombinant DNA (rDNA)
techniques and are already in widespread use; we will consider some examples
later in this chapter.
In many cases a bacterial host cell can be used for the expression of cloned
genes, but often a eukaryotic host is required for particular purposes.
Eukaryotic proteins are often subjected to post-translational modification
(PTM) in vivo, and it is important that any modifications are achieved in an
expression system if a functional protein is to be produced.
Produksi proteins
LO 54: menjelaskan produksi protein
(a) The coding sequence for the
cloned gene (shaded) is not
preceded by bacterial coding
sequence; thus, the mRNA
encodes only insert-specified
amino acid residues. This
produces a native protein,
synthesised from its own N
terminus.
(b) The gene fusion contains
bacterial codons (hatched);
therefore, the protein
contains part of the bacterial
protein. In this example the
first three N-terminal amino
acid residues are of bacterial
origin (hatched). The
ribosome-binding site, or
ShineDalgarno sequence, is
marked SD.
Fig. 11.1 Native and fusion proteins.
LO 54: menjelaskan produksi protein
Fig. 11.2 The importance of reading frame. A simple sentence is used for illustration.

(a) The message has been cloned downstream from the start site and is readable, as itis in
the correct reading frame
(b) A deletion of one base at the start is enough to knock out the sense completely.
(c) Addition of an extra base also causes problems.
LO 54: menjelaskan produksi protein
LO 55: menjelaskan rekayasa protein
One of the most exciting applications of gene manipulation lies in the field of
protein engineering.
This involves altering the structure of proteins via alterations to the gene
sequence and has become possible because of the availability of a range of
techniques, as well as a deeper understanding of the structural and functional
characteristics of proteins.
This has enabled workers to pinpoint the essential amino acid residues in a
protein sequence; thus, alterations can be carried out at these positions and
their effects studied.
The desired effect might be alteration of the catalytic activity of an enzyme by
modification of the residues around the active site, an improvement in the
nutritional status of a storage protein, or an improvement in the stability of a
protein used in industry or medicine.
Proteins that have been engineered by the incorporation of mutational changes
have become known as muteins.
Protein engineering
(a) The requirement for mutagenesis in vitro is
a single-stranded DNA template containing
a cloned gene (heavy line). An
oligonucleotide is synthesised that is
complementary to the part of the gene
that is to be mutated (but which
incorporates the desired mutation). This is
annealed to the template (the mutation is
shown as an open star).
(b) The molecule is made double-stranded in a
reaction using DNA polymerase and ligase,
which produces a hybrid wild-type/mutant
DNA molecule with a mismatch in the
mutated region.
(c) On introduction into E. coli the molecule is
replicated, thus producing double-stranded
copies of the wild-type (WT) and mutant
(M) forms. The mutant carries the original
mutation and its complementary base or
sequence (filled star).
Fig. 11.3 Oligonucleotide-directed
mutagenesis
LO 55: menjelaskan rekayasa protein
In this procedure, some knowledge of the gene
and protein sequence is required.
A change is then introduced into the gene,
as shown in (a).
In this case the codon GAC is altered to GAA
by changing the third base in the codon DNA
sequence. This causes a change in the amino
acid sequence, shown in (b) as a change
from aa X to aa Y.
This causes a change in the way that the
protein folds, shown in (c).

In this example the active site (shown by the
arrows) becomes slightly larger in the altered
molecule.
Fig. 11.4 Protein engineering by the
rational design method
LO 55: menjelaskan rekayasa protein
In addition to genetic conditions that affect the individual, rDNA technology is
also important in the diagnosis of certain types of infection.
Normally, bacterial infection is relatively simple to diagnose, once it has taken
hold.
Thus, the prescription of antibiotics may follow a simple investigation by a
general practitioner.
A more specific characterisation of the infectious agent may be carried out
using microbiological culturing techniques, and this is often necessary when the
infection does not respond well to treatment.
Viral infections may be more difficult to diagnose, although conditions such as
Herpes infections are usually obvious.
Diagnosis of infection
LO 56: menjelaskan diagnosis infeksi
Despite traditional methods being applied in many cases, there may be times
when these methods are not appropriate.
Infection by the human immunodeficiency virus (HIV) is one case in point.
The virus is the causative agent of acquired immune deficiency syndrome
(AIDS).
The standard test for HIV infection requires immunological detection of anti-
HIV antibodies, using techniques such as ELISA (enzyme linked
inmmunosorbent assay, sometimes known as the enzyme immunoassay),
Western blot, and IFA (indirect immunofluorescence assay).
However, these antibodies may not be detectable in an infected person until
weeks after initial infection, by which time others may have been infected.
A test such as this, where no positive result is obtained even though the
individual is infected, is a false negative.
The use of DNA probes and PCR technology circumvents this problem by
assaying for nucleic acid of viral origin in the T-lymphocytes of the patient, thus
permitting a diagnosis before the antibodies are detectable.
Other examples of the use of rDNA technology in diagnosing infections include
tuberculosis (caused by the bacterium Mycobacterium tuberculosis), human
papilloma virus infection, and Lyme disease (caused by the spirochaete Borrelia
burgdorferi).
LO 56: menjelaskan diagnosis infeksi
LO 57: menjelaskan DNA profiling
Given the size of the human genome, and our knowledge of genome structure, it is
relatively easy to calculate that each persons genome is unique, the only exceptions
being monozygotic twins (twins derived from a single fertilised ovum).
This provides the opportunity to use the genome as a unique identifier, if suitable
techniques are available to generate robust and unambiguous results.
The original technique was called DNA fingerprinting, but with improved technology the
range of tests that can be carried out has increased, and today the more general term
DNA profiling is preferred.
The technique has found many applications in both criminal cases and in disputes over
whether people are related or not (paternity disputes and immigration cases are the
most common).
The basis of all the techniques is that a sample of DNA from a suspect (or person in a
paternity or immigration dispute) can be matched with that of the reference sample
(from the victim of a crime, or a relative in a civil case).
In scene-of-crime investigations, the technique can be limited by the small amount of
DNA available in forensic samples.
Modern techniques use the PCR to amplify and detect minute samples of DNA from
blood-stains, body fluids, skin fragments, or hair roots.
DNA profiling
Fig. 12.8 Genetic fingerprinting of minisatellite DNA sequences.
(a) A chromosome pair, with one
minisatellite (VNTR) locus highlighted.
In this case the locus is heterozygous
for VNTR length. Cutting with HinfI
effectively isolates the VNTR.
(b) The VNTR fragments produced (from
many loci) are separated by
electrophoresis and blotted.
(c) Challenging with a multi-locus probe
(MLP) produces the bar code pattern
(d) If a single-locus probe (SLP) is used,
the two alleles of the specific VNTR
are identified.
LO 57: menjelaskan DNA profiling
Fig. 12.9 A DNA profile prepared using a multi-locus probe
Samples of the suspects DNA isolated
from the victim (V; boxed) and seven
candidate suspects (17) were cut with a
restriction enzyme and separated on an
agarose gel. The fragments were blotted
onto a filter and challenged with a
radioactive probe. The probe hybridises to
the target sequences, producing a profile
pattern when exposed to X-ray film. The
band patterns from the victims sample
and suspect 5 match.
LO 57: menjelaskan DNA profiling
Samples of DNA from the mother (M), four
children (14), and the father (F) were
prepared as in Fig. 12.9. A single-locus
probe was used in this analysis. The band
patterns therefore show two maternal
bands and two paternal bands, one band
from each homologous chromosome on
which the target sequence is located. In
the case of child 1, the paternal band is
different from either of the two bands in
lane F, indicating a different father (band
labelled DF). This child was in fact born to
the mother during a previous marriage.
Courtesy of Cellmark Diagnostics.
Reproduced with permission.
Fig. 12.10 A DNA profile prepared
using a single-locus probe for
paternity testing
LO 57: menjelaskan DNA profiling
TATAP MUKA 13
Learning Outcome (LO)
LO 58: menjelaskan tanaman transgenik
LO 59: menjelaskan hewan transgenik