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Rekayasa Genetika

3 SKS
Dosen:
Dr Endry dan Dr Jadid

Silabus
SB 142402: Rekayasa Genetika
• Pokok Bahasan
– Pendahuluan;
– Prinsip dasar aplikasi rekayasa genetika dan analisis DNA:
langkah-langkah kloning gen, PCR, bakteriophage, preparasi
DNA target, plasmid dan inang prokariot/eukariot, enzim
restriksi, dan deteksi sel rekombinan;
– Aplikasi rekayasa genetika: sekuensing gen dan genom, studi
ekspresi gen, studi forensic dan arkeologi.
• Pustaka
– Primrose S.B. and Twyman, R.M. 2006. Principles of Gene
Manipulation and Genomics. 7th Edition. Blackwell Publishing.
Australia
– Brown, T.A. 2010. Gene Cloning and DNA Analysis, an
Introduction. 6th Edition. Wiley-Blackwell. United Kingdom.

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Selective Breeding
• Breed only those plants
or animals with
desirable traits

• People have been using

selective breeding for
1000’s of years with
farm crops and
domesticated animals.

Classic Genetics

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• Gregor Mendel
– Was the first person
to analyze patterns of
inheritance
– Deduced the
fundamental
principles of genetics

Gregor Mendel observed phenotypes and formed

hypotheses
• How do offspring come to resemble their parents physically?

• Genetics begins with the unifying assumption that biological

inheritance is carried by structures called Genes.

• The same basic patterns of inheritance apply to most organisms.

• The inheritance of some human traits can be explained from work on

plants

• Sex-linked traits in humans is more complicated

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Terms:

• Phenotype
– An organism’s physical traits

• Genotype
– An organism’s genetic makeup

Allele
• Allele: Alternate form of a gene at same position
on pair of chromosomes that affect the same trait.
• Dominant Allele: Capital Letter--O
• Recessive Allele: lowercase letter--o
• Homozygous Dominant--OO
• Homozygous Recessive--oo
• Heterozygous--Oo

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Mendel’s Peas
– These plant are
easily
manipulated
– These plants can
self-fertilize

Stamen

Carpel

Garden pea

Dominant Recessive Dominant Recessive

Pod shape Inflated Constricted

Flower color Purple White

Pod Color Green Yellow

Flower position Axial Terminal

Seed color Yellow Green

Stem length Tall Dwarf

Seed shape Round Wrinkled

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Monohybrid Crosses
P Generation
(true-breeding
parents)
Purple White
flowers flowers

F1 Generation All plants have

purple flowers

Fertilization
among F1 plants
(F1 × F1)

F2 Generation

1/
4 of plants
3/
F2 = 3:1 ratio 4 of plants
have purple have white
flowers flowers

P plants PP PP

• Using a Punnett Gametes All P All p

square to explain
the results of a F1 plants:
monohybrid cross (hybrids) All Pp

Gametes 1/ P 1/ p
2 2

Eggs P P Sperm
F2 plants:
p PP p
Phenotypic ratio
3 purple : 1 white Pp Pp

pp
Genotypic ratio
1 PP : 2 Pp : 1 pp

Figure 9.8b

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• from the monohybrid crosses, Mendel derived 4

hypotheses….combined, we now refer to these as…

= Mendel’s Principle of Segregation

– There are alternative forms of genes, now called alleles

– For each characteristic, each organism has two genes

– Gametes carry only one allele for each inherited characteristic

– Alleles can be dominant or recessive

Mendel’s Principle of Independent Assortment

• What happens when you follow the

inheritance of more than a single trait at one time?

• How do two different traits get passed to offspring?

A Dihybrid Cross

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RRYY rryy
Dihybrid Cross
Gametes RY ry

RrYy

Eggs RY RY Sperm
RrYy RrYy
rY rY
RRYY

Ry RrYY RrYY Ry

ry RRYy rrYY RRYy ry

RrYy RrYy RrYy RrYy Yellow round

9/
16
rrYy RRyy rrYy Green round
3/
16
Rryy Rryy Yellow
3/
16 wrinkled
rryy 1/ Green
16
wrinkled

9:3:3:1

• Mendel’s principle of independent assortment

– Each pair of alleles segregates independently of the

other pairs during gamete formation

P a B

P a b

Genotype: PP aa Bb

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Using a Testcross to Determine an Unknown

Genotype

• A testcross is a mating
Testcross:
between:
– An individual of Genotypes P_ pp
unknown Two possibilities for the purple flower:
genotype and
– A homozygous PP Pp
recessive
Gametes P P p
individual
P Pp p Pp pp

Offspring All purple 1 purple : 1 white

Incomplete Dominance in Plants and People

Red White
RR rr

• In incomplete
Gametes R r
dominance F1
hybrids have an Pink
Rr
appearance in
between the Gametes
1/
2 R 1/
2 r

phenotypes of
the two parents Eggs 1/
2
R
Red
1/
2 R Sperm
1/
1/
2
r RR 2 r
Pink Pink
Rr rR
White
rr
Figure 9.18

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Types of cells
• Not all cells of an organisms have the same
number of chromosomes.
• Two types of cells:
– Somatic Cells
– Gametes

Somatic Cells
• Non-sex Cells. • Diploid
• These cells do not carry
genetic information for
sexual reproduction. • Means double number.

• Contain a full compliment of

chromosomes • Designated 2n

• Characteristic to their • All somatic cells in an

species. organism have the 2n or
diploid number of
• Referred to as the diploid chromosomes.
number of chromosomes.

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Gametes
• Sex Cells • Haploid
• Cell which carry genetic
information for sexual • Means single number.
reproduction.

• Contain one half the compliment • Designated n

of chromosomes characteristic to
their species.
• Referred to as the haploid
number of chromosomes.
• All gametes formed by an
organism have the n or
haploid number of
• chromosomes.

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Human Life Cycle

• Adults produce gametes--egg and sperm.
• Gametes fuse to produce zygote.
• Zygote grows and develops to produce baby.
• Meiosis--process of division that produces gametes.
• Mitosis--process of replication and division required
for growth.
• Adults, zygote and baby--2n. 2n=diploid
• Gametes--n. n=haploid

Mitosis
• Process of division that produces two daughter cells
with identical chromosomal content of parent cell.

• Mitosis is one stage of the cell cycle.

• Cell cycle--cycle of stages a cell goes through in

order to grow and divide.

• Stages: I=Interphase, Growth 1=G1, DNA

synthesis=S, Growth 2=G2, Mitosis=M

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The Human cell cycle

• Interphase--G1, S, G2
• Mitosis--M
• G1--growth
• S--DNA Synthesis,
replication
• G2--growth
• M:
– mitosis-- nuclear division
– cytokinesis--cell division

Stages of Division- Mitosis

• Prophase--nuclear envelope breakdown,
chromosome condensation, spindle formation.
• Metaphase--chromosomes are lined up precisely on
the metaphase plate, or middle of the cell.
• Anaphase--spindle pulls sister chromatids apart.
• Telophase--chromatids begin to decondense and
become chromatin. Spindle disappears.
• Cytokinesis--divide cell and organelles. Actin ring,
or cleavage furrow splits cell.

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Gamete Production -Meiosis

• In order to reproduce we must produce
gametes.
• Gametes are sperm and egg.
• Why is that siblings are not identical?
• Meiosis blends DNA from parental
contributions to produce a mixed up “half” or
haploid, set of DNA.
• Crossing over is critical for producing haploid
DNA with genetic diversity.

The Process of Meiosis

Interphase

– Haploid gametes are Centrosomes

(with
produced in diploid centriole
pairs)
organisms
– Two consecutive
divisions occur,
meiosis I and meiosis
II, preceded by
interphase
Nuclear
envelope Chromatin

Chromosomes duplicate

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Prophase -I
Replicated pairs of chromosomes line
up side by side.

These pairs are called Homologous--

both have same gene order (gene for
eye color, hair color, etc).

Sister chromatid from one pair interact

with a Sister chromatid from another
pair.

One sister is from father, one sister

from mother, but they have same gene
order.

Prophase -I
• This interaction is called
Synapsis.

• Synapsis results in the

formation of a Tetrad (4
sisters together).

• Crossing over swaps sections

of homologous genes.

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Meiosis - I
Figure 2.9 (1)

Prophase I

Metaphase I

Anaphase I

Telophase I

Meiosis - II
Figure Prophase
2.9 (2) II

Metaphase II

Anaphase II

Telophase II

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• Meiosis I
Meiosis I: Homologous
chromosomes separate
Telophase I
Prophase I Metaphase I Anaphase I
and Cytokinesis
Sites of crossing over Microtubules attached to Sister chromatids Cleavage
Chromosomes remain attached furrow
Spindle

Sister Tetrad Centromere

chromatids

Homologous Tetrads line up Pairs of homologous Two haploid cells form:

chromosomes chromosomes chromosomes are still
pair and exchange split up double
segments

• Meiosis II
Meiosis II:
Sister chromatids separate

Prophase II Telophase II
Metaphase II Anaphase II
and Cytokinesis

Sister chromatids Haploid daughter cells

separate forming

During another round of cell division, the sister chromatids finally separate; four haploid
daughter cells result, containing single chromosomes

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Sexual life cycles

• Haploid Gametes join to form a zygote
• Somatic cells divide by Mitosis to produce adult organism
• Meiosis produces gametes in sex cells

Genes on sex chromosomes determine Sex and sex-

linked traits
• Micrograph of the chromosomes of an organism paired and
numbered.
• Used to check for chromosomal abnormalities in individuals.

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Sex Determination
• All embryos start on a neutral or
"indifferent" path. The 4 week old
embryo is indifferent
• By 7 weeks, the SRY (sex-related)
gene encoded by the short arm of the
Y chromosome begins to roar!

• Testis determining factor converts

progesterone to testosterone

Sex Determination
• Indifferent embryos have two sets of
ducts:
• Müllerian ducts--will be come the
future oviducts--thus female.

• Wolfian ducts--will become the future

vas deferens--thus male
– dependent on testosterone for its
continued development

• The testes also produce an anti-

Müllerian hormone that promotes
regression of the Müllerian ducts
– without SRY, the indifferent embryo will
naturally develop into a female

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Sex Determination

Two copies of DAX (double X) inactivate SRY, thus this

individual would be genetically male, but look female.

Sex Determination

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SexFigure
Determination
2.16 (3)

Sex Chromosomal Disorders

• Turner Syndrome – XO only one sex
chromosome
• Short, thick neck and stature
• Do not undergo puberty, or menstruate,
• no breast development
• Kleinfelter Syndrome – XXY
• Testis and prostate underdeveloped
• No facial hair
• Brest development
• Long arms and legs: big hands and feet
• Can be mentally retarded

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An XY Individual with Androgen Insensitivity

Syndrome

Androgen Insensitivity Syndrome is a sex reversal condition where XY

individuals look female. These individuals have the Y chromosome and
functional SRY. These individuals have testis which generate AMH and
testosterone. However, the genetic mutation results in a lack of the
testosterone receptor. Estrogens are made in the adrenal gland which drive
phenotypic development. As adults, these individuals have testes in the
abdomen and lack a uterus and oviducts.

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Issues of sex determination

• So, clearly being Male or Female isn’t as cut and dry
as people have been led to believe!

• 17 out of 1,000 people are really neither

– XY, but Female anatomy
– XX, but Male anatomy
– Both Female and Male anatomy

• Other genes, such as testosterone receptor on

chromosomes other than X and Y chromosomes
have a role in sexual development

Issues of sex determination

• So, some people fall between Male and Female
– Due to chromosomal variation
– Variations in SYR gene
– Testosterone receptor gene

• All of the different variations are known as intersex

– These people are altered surgically to make them
conform to one sex or the other

• Should there be five genders instead of two?

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