GEN/KROMOSOM/PLASMID

MATERIAL GENETIK
A gene is the basic physical and
functional unit of heredity. Genes,
which are made up of DNA, act as
instructions to make molecules called
proteins. In humans, genes vary in size
from a few hundred DNA bases to more
than 2 million bases.
Gen:
* Berada di dalam inti sel (pada
eukaryotic
* Unit dasar fisik sebagai pembawa
sifat keturunan
* Didalam tubuh gen berfungsi
menginstruksikan pembentukan
molekuk-molekul protein atau
mensintesa protein
*terdiri dari rangkaian DNA. Ukuran
gen bervariasi mulai dari ratusan basa
DNA sampai 2 juta basa DNA.
NUKLEOTIDA  DNA  PASANGAN
BASA  GEN  KROMOSOM 
GENOM
  22 = region 2, band 2 (read as
"two, two", not "twenty-two")
 1 = sub-band 1
Thus the entire locus of the example
above would be read as "three P two
two point one."
The cytogenetic bands count from
the centromere out toward
the telomeres.

Compo
Explanation
nent

3 The chromosome number.

The position is on the

chromosome's short arm (a
common apocryphal
explanation is that
the p stands for petitin
French); q indicates the long
p
arm (chosen as next letter in
alphabet after p;
alternatively it is sometimes
said that q stands
for queue meaning tail in
French).

The numbers that follow the

letter represent the position
on the arm: region 2, band
2, sub-band 1. The bands
Nomenclature[edit] are visible under
a microscope when chromo
22.1 some is suitably stained.
Cytogenetic banding nomenclature Each of the bands is
numbered, beginning with 1
The shorter arm of a chromosome is for the band nearest
termed the p arm or p-arm, while the the centromere. Sub-bands
longer arm is the q arm or q-arm. The and sub-sub-bands are
chromosomal locus of a typical gene visible at higher resolution.
might be written 3p22.1, where

 3 = chromosome 3 Example of bands

 p = p-arm
A range of loci is specified in a similar
way. For example, the locus of
gene OCA1 may be written "11q1.4-
q2.1", meaning it is on the long arm of
chromosome 11, somewhere in the
range from sub-band 4 of region 1 to
sub-band 1 of region 2.
The ends of a chromosome are
labeled "pter" and "qter", and
so "2qter" refers to the terminus of the
long arm of chromosome 2.
PASANGAN BASA = BASE PAIR
A kilobase (kb) is a unit of measurement in
molecular biology equal to 1000 base pairs
of DNA or RNA

The haploid human genome (23

chromosomes) is estimated to be about 3.2
billion bases long and to contain 20,000–
25,000 distinct protein-coding genes.[2][3]
Ekspresi gen merupakan rangkaian proses
penerjemahan informasi genetik (dalam bentuk urutan
basa pada DNA atau RNA) menjadi protein, dan lebih
jauh lagi: fenotipe. Informasi yang dibawa bahan
genetik tidak bermakna apa pun bagi suatu organisme
apabila tidak diekspresikan menjadi fenotipe.
Proses ekspresi genetik mengikuti tahapan yang sama
untuk semua bentuk kehidupan, dan disebut dogma
inti (central dogma) dalam genetika. Ada tiga proses
dasar yang tercakup dalam dogma inti:

 replikasi DNA,
 transkripsi DNA menjadi RNA, dan
 translasi RNA menjadi protein atau polipeptida.
Regulation of gene expression includes
a wide range of mechanisms that are
used by cells to increase or decrease
the production of specific gene
products (protein or RNA), and is
informally termed gene regulation.
Sophisticated programs of gene
expression are widely observed in
biology, for example to trigger
developmental pathways, respond to
environmental stimuli, or adapt to
new food sources. Virtually any step of
gene expression can be modulated,
from transcriptional initiation, to
RNA processing, and to the post-
translational modification of a protein.
Often, one gene regulator controls
another, and so on, in a gene
regulatory network.
Gene regulation is essential for
viruses, prokaryotes and eukaryotes
as it increases the versatility and
adaptability of an organism by
allowing the cell to express protein
when needed. Although as early as
1951, Barbara McClintock showed
interaction between two genetic loci,
Activator (Ac) and Dissociator (Ds), in
the color formation of maize seeds, the
first discovery of a gene regulation
system is widely considered to be the
identification in 1961 of the lac
operon, discovered by Jacques
Monod, in which some enzymes
involved in lactose metabolism are
expressed by E. coli only in the
presence of lactose and absence of
glucose.

Gene Activation
PLASMID
A plasmid is a small DNA
molecule within a cell that is
physically separated from a
chromosomal DNA and can
replicate independently.

They are most commonly found

in bacteria as small circular,
double-stranded DNA molecules;
however, plasmids are
sometimes present in archaea
and eukaryotic organisms.

In nature, plasmids often carry genes

that may benefit the survival of the
organism, for example antibiotic
resistance.
While the chromosomes are big and
contain all the essential genetic
information for living under normal
conditions, plasmids usually are very small
and contain only additional genes that may
be useful to the organism under certain
situations or particular conditions.
Artificial plasmids are widely used as
vectors in molecular cloning, serving to
drive the replication of recombinant DNA
sequences within host organisms.
Plasmids are considered replicons, a unit of
DNA capable of replicating autonomously
within a suitable host.
Plasmids can be transmitted (trasnform)
from one bacterium to another (even of
another species) via conjugation or
conduction
transformation
In molecular biology, transformation is the
genetic alteration of a cell resulting from the
direct uptake and incorporation of exogenous
genetic material from its surroundings
through the cell membrane(s). For
transformation to take place, the recipient
bacteria must be in a state of competence,
which might occur in nature as a time-limited
response to environmental conditions such as
starvation and cell density, and may also be
induced in a laboratory.[1]
Transformation is one of three processes for
horizontal gene transfer, in which exogenous
genetic material passes from bacterium to
another, the other two being

conjugation
(transfer of genetic material between two
bacterial cells in direct contact) in which the
genetic material passes through the
intervening medium, and uptake is completely
dependent on the recipient bacterium.[1]
transduction
Transduction is the process by which foreign DNA is introduced into a cell by a
virus or viral vector.[1] An example is the viral transfer of DNA from one
bacterium to another.[2] Transduction does not require physical contact between
the cell donating the DNA and the cell receiving the DNA (which occurs in
conjugation), and it is DNase resistant (transformation is susceptible to DNase).
Transduction is a common tool used by molecular biologists to stably introduce a
foreign gene into a host cell's genome.

When bacteriophages (viruses that infect bacteria) infect a bacterial cell, their
normal mode of reproduction is to harness the replicational, transcriptional, and
translation machinery of the host bacterial cell to make numerous virions, or
complete viral particles, including the viral DNA or RNA and the protein coat.
This host-to-host transfer of genetic
material is called horizontal gene transfer,
and plasmids can be considered part of the
mobilome.
The size of the plasmid varies from 1 to
over 200 kbp,[2] and the number of
identical plasmids in a single cell can range
anywhere from one to thousands under
some circumstances.
The relationship between microbes and
plasmid DNA is neither parasitic nor
mutualistic, because each implies the
presence of an independent species living
in a detrimental or commensal state with
the host organism.
Rather, plasmids provide a mechanism for
horizontal gene transfer within a
population of microbes and typically
provide a selective advantage under a
given environmental state. Plasmids may
carry genes that provide resistance to
naturally occurring antibiotics in a
competitive environmental niche, or the
proteins produced may act as toxins under
similar circumstances, or allow the
organism to utilize particular organic
compounds that would be advantageous
when nutrients are scarce.[3]
Crown gall/AGROBACTERIUM TUMEFACIENS
Crown gall is a bacterial disease of the
stems and roots of many woody and
herbaceous plants, including fruit,
vegetables and ornamental plants.
Infection with this disease causes knobbly
swellings (galls) on stems, roots, trunks
and branches.
What is crown gall?
Crown gall is a disease caused by the
bacterium Agrobacterium tumefaciens
(synonym Rhizobium radiobacter), which
enters the plant through wounds in roots
or stems and stimulates the plant tissues
to grow in a disorganised way, producing
swollen galls. Galls are present all year.
Crown gall affects many plants, both
woody and herbaceous. These are some of
the plants on which it is most commonly
found:
 Fruit: Apples, cherries, currants,
gooseberries, grapevines,
blackberries, peaches, pears, plums
and quince
 Vegetables: Beetroot, courgettes,
runner beans and swedes
 Herbaceous plants: Alcea (hollyhock),
Argyranthemum (marguerites),
 Begonia, Dahlia, Lathyrus (sweet
peas), Lupinus (lupins) and Phlox
 Woody plants: Crataegus (hawthorn),
Euonymus, Populus (poplar), Salix
(willow), Rosa and Ulmus (elm)
Note: swellings caused by crown gall
should not be confused with the harmless
nitrogen-fixing nodules produced on the
roots of many members of the pea family.
A Ti or tumour inducing plasmid is a
plasmid that often, but not always, is a
part of the genetic equipment that
Agrobacterium tumefaciens and
Agrobacterium rhizogenes use to
transduce its genetic material to plants.

FUCTION OF PLASMID IN GENETIC

ENGINEERING

Multiple Cloning Site

If the main purpose of a plasmid is to
serve as a vehicle for genes of interest, we
need to be able to insert the human
insulin gene into the plasmid. This is the
purpose of a multiple cloning site. A
multiple cloning site is the location in a
plasmid where a sequence of DNA,
typically a gene, can be inserted. We'll
discuss in greater detail how a gene can
be inserted into the multiple cloning site
in another lesson.
Restriction enzymes & DNA ligase
Key points:
 Restriction enzymes are DNA-cutting enzymes. Each
enzyme recognizes one or a few target sequences and cuts
DNA at or near those sequences.

 Many restriction enzymes make staggered cuts, producing

ends with single-stranded DNA overhangs. However, some
produce blunt ends.

 DNA ligase is a DNA-joining enzyme. If two pieces of DNA

have matching ends, ligase can link them to form a single,
unbroken molecule of DNA.

 In DNA cloning, restriction enzymes and DNA ligase are used

to insert genes and other pieces of DNA into plasmids.

How do you cut and paste DNA?

In DNA cloning, researchers make many copies of a piece of
DNA, such as a gene. In many cases, cloning involves inserting
the gene into a piece of circular DNA called a plasmid, which
can be copied in bacteria.

How can pieces of DNA from different sources (such as a

human gene and a bacterial plasmid) be joined together to
make a single DNA molecule? One common method is based
on restriction enzymes and DNA ligase.

 A restriction enzyme is a DNA-cutting enzyme that

recognizes specific sites in DNA. Many restriction enzymes
make staggered cuts at or near their recognition sites,
producing ends with a single-stranded overhang.
 If two DNA molecules have matching ends, they can be joined
by the enzyme DNA ligase. DNA ligase seals the gap between
the molecules, forming a single piece of DNA.
Restriction enzymes and DNA ligase are often used to insert
genes and other pieces of DNA into plasmids during DNA
cloning.

 Restriction enzymes
 Restriction enzymes are found in bacteria (and other
prokaryotes). They recognize and bind to specific
sequences of DNA, called restriction sites. Each
restriction enzyme recognizes just one or a few
restriction sites. When it finds its target sequence, a
restriction enzyme will make a double-stranded cut in
the DNA molecule. Typically, the cut is at or near the
restriction site and occurs in a tidy, predictable pattern.

As an example of how a restriction enzyme recognizes and

cuts at a DNA sequence, let's consider EcoRI, a common
restriction enzyme used in labs. EcoRI cuts at the following
site:
 5'-...GAATTC..

.-3' 3'-...CTTAAG...-5'

EcoRIsite

When EcoRI recognizes and cuts this site, it always does so in

a very specific pattern that produces ends with single-
stranded DNA “overhangs”:
An EcoRI enzyme binds to an EcoRI site in a piece of DNA and
makes a cut on both strands of the DNA. The pattern of the cut
is:

5'-...G|AATTC...-3' 3'-...CTTAA|G...-5'

Thus, it produces an overhang of 5'-AATT-3' on each end of

the cut DNA.

If another piece of DNA has matching overhangs (for instance,

because it has also been cut by EcoRI), the overhangs can
stick together by complementary base pairing. For this
reason, enzymes that leave single-stranded overhangs are
said to produce sticky ends. Sticky ends are helpful in cloning
because they hold two pieces of DNA together so they can be
linked by DNA ligase. Not all restriction enzymes produce
sticky ends. Some are “blunt cutters,” which cut straight down
the middle of a target sequence and leave no overhang. The
restriction enzyme SmaI is an example of a blunt cutter:
 A SmaI enzyme binds to the SmaI restriction site, which is:

5'-...CCCGGG...-3' 3'-...GGGCCC...5'

It makes a cut right in the middle of this sequence on both

strands, producing blunt ends. The cut sites are:

5'-...CCC|GGG...-3' 3'-...GGG|CCC...5'

Blunt-ended fragments can be joined to each other by DNA

ligase. However, blunt-ended fragments are harder to ligate
together (the ligation reaction is less efficient and more likely
to fail) because there are no single-stranded overhangs to
hold the DNA molecules in position.

DNA ligase
If you’ve learned about DNA replication, you may already
have met DNA ligase. In DNA replication, ligase’s job is to join
together fragments of newly synthesized DNA to form a
seamless strand. The ligases used in DNA cloning do basically
the same thing. If two pieces of DNA have matching ends, DNA
ligase can join them together to make an unbroken molecule.

Fragment 1 of DNA:
 5'-...G 3'-...CTTAA

Fragment 2 of DNA:

AATTC...-3' G...-5'

The single-stranded regions of the two molecules can stick

together by hydrogen bonding, but there are still gaps in the
backbone:

5'-...G|AATTC...-3' 3'-...CTTAA|G...-5'

DNA ligase seals the gaps to make an unbroken molecule of

DNA:

5'-...GAATTC...-3' 3'-...CTTAAG...-5'

How does DNA ligase do this? Using ATP as an energy source,

ligase catalyzes a reaction in which the phosphate group
sticking off the 5’ end of one DNA strand is linked to the
hydroxyl group sticking off the 3’ end of the other. This
reaction produces an intact sugar-phosphate backbone.

Building a recombinant plasmid

Let's see how restriction digestion and ligation can be used to
insert a gene into a plasmid. Suppose we have a target gene,
flanked with EcoRI recognition sites, and a plasmid, containing a
single EcoRI site:
 We start off with a target gene and a circular plasmid. The target
gene has two EcoRI restriction sites near its ends. The plasmid has
one EcoRI site in it, lying just after a promoter that drives
expression in bacteria. The sequence of the EcoRI sites is:

5'-GAATTC-3' 3'-CTTAAG-5'

Our goal is to use the enzyme EcoRI to insert the gene into the
plasmid. First, we separately digest (cut) the gene fragment and
the plasmid with EcoRI. This step produces fragments with sticky
ends:
 We separately digest (cut) the gene fragment and the plasmid
with EcoRI. This step produces fragments with sticky ends. All of
the ends have an overhang of four nucleotides, with the sequence
5'-AATT-3'. That's because EcoRI's cut pattern is:

5'-G|AATTC-3' 3'-CTTAA|G-5'

Next, we take the gene fragment and the linearized (opened-up)

plasmid and combine them along with DNA ligase. The sticky ends
of the two fragments stick together by complementary base
pairing:
 Next, we take the gene fragment and the linearized (opened-up)
plasmid and combine them along with DNA ligase. The sticky ends
of the two fragments stick together by complementary base
pairing. However, there are still gaps in the sugar-phosphate
backbones of the DNA double helix at the junction sites where the
gene and plasmid DNA meet.

Once they are joined by ligase, the fragments become a single

piece of unbroken DNA. The target gene has now been inserted
into the plasmid, making a recombinant plasmid.
Restriction digests and ligations
involve many molecules of DNA
In the example above, we saw one outcome of a ligation
between a gene and plasmid cut with EcoRI. However, other
outcomes could happen in this exact same ligation. For
instance, the cut plasmid could recircularize (close back up)
without taking in the gene. Similarly, the gene could go into
the plasmid, but flipped backwards (since its two EcoRI sticky
ends are identical).
 Left: recombinant plasmid produced when gene goes in
forwards ("pointing" away from the promoter that is already
in the plasmid).

Middle: non-recombinant plasmid produced when the cut

plasmid simply closes back up (its ends ligate with each
other).

Right: recombinant plasmid produced when gene goes in

backwards ("pointing" back towards the promoter that is
already in the plasmid).

Restriction digests and ligations like this one are performed

using many copies of plasmid and gene DNA. In fact, billions
of molecules of DNA are used in a single ligation! These
molecules are all bumping into one another, and into DNA
ligase, at random in different ways. So, if multiple products
can be made, all of them will be made at some frequency –
including ones we don't want.
How can we avoid the "bad" plasmids? When we transform
bacteria with DNA from a ligation, each one takes up a
different piece of DNA. We can check the bacteria after
transformation and use only the ones with the correct
plasmid. In many cases, plasmid from transformed bacteria is
analyzed using another restriction digest to see if it contains
the right insert in the right orientation.