SEX LINKAGE AND SEX DETERMINATION
(genes) are located on chromosomes.
Sex Linkage - the phenotypic expression of an allele
related to the chromosomal sex of the individual
Sex Determination - a biological system that
determines the development of a sexual
characteristics in an organism
Eukaryotic Chromosomes
Eukaryotes have multiple linear chromosomes in
a number characteristic of the species. Most have
two versions of each chromosome, and so are
diploid (2N). Diploid cells are produced by haploid
(N) gametes that fuse to form a zygote. The zygote
then undergoes development, forming a new
individual.
Examples of diploid organisms are humans (23
pairs) and Drosophila melanogaster (4 pairs). The
yeast Saccharomyces cerevisiae is haploid (16
chromosomes).
Chromosome pairs in diploid organisms are
homologous chromosomes. One member of each
pair (homolog) is inherited from each parent.
Chromosomes that have different genes and do not
pair are nonhomologous chromosomes.
Animals and some plants have male and female
cells with distinct chromosome sets, due to sex
chromosomes. One sex has a matched pair (e.g.,
human females with XX) and the other has an
unmatched pair (human male with XY).
Autosomes are chromosomes other than sex
chromosomes.
Sex Chromosomes: A chromosome that men and
women have different amounts of. (X and Y in
humans.)
Autosomal Chromosomes: All the other
chromosomes BESIDES the sex chromosomes.
Chromosome Theory of Inheritance
1. By the beginning of the 20th century, cytologists
had observed that chromosome number is
constant in all cells of a species, but varies
widely between species.
2. Sutton and Boveri (1902) independently
realized the parallel between Mendelian
inheritance and chromosome transmission, and
proposed the chromosome theory of
inheritance, which states that Mendelian factors
Sex Chromosomes
1. Behavior of sex chromosomes offers support for
the chromosomal theory. In many animals sex
chromosome composition relates to sex, while
autosomes are constant.
2. Independent work of McClung, Stevens, and
Wilson indicated that chromosomes are
different in male and female insects.
a. Stevens named the extra chromosome
found in females “X.”
b. In grasshoppers, all eggs have an X, and
half of the sperm produced have an X,
and the other half do not. After
fertilization, an unpaired X produces a
male, while paired X chromosomes
produce a female.
3. Other insects have a partner for the X
chromosome. Stevens named it “Y.” In
mealworms, for example, XX individuals are
female, and XY are male.
4. In both humans and fruit flies (Drosophila
melanogaster) females have two X
chromosomes, while males have X and Y.
a. Males produce two kinds of gametes with
respect to sex chromosomes (X or Y),
and are called the heterogametic sex.
b. Females produce gametes with only one
kind of sex chromosome (X) and are
called the homogametic sex.
c. In some species the situation is reversed,
with heterogametic females and
homogametic males.
5. Random fusion of gametes produces an F1
that is 1⁄2 female (XX) and 1⁄2 male (XY).
(check pdf for more photos)
Sex Linkage
1. Morgan (1910) found a mutant white-eyed male
fly, and used it in a series of experiments that
showed a gene for eye color located on the X
chromosome.
a. First, he crossed the white-eyed male with a
wild-type (red-eyed) female. All F1 flies had
red eyes. Therefore, the white-eyed trait is
recessive.
b. Next, F1 were interbred. They produced an
F2 with:
i. 3,470 red-eyed flies.
ii. 782 white-eyed flies.
c. The recessive number is too small to fit
Mendelian ratios (explanation discovered
later is that white-eyed flies have lower
viability).
d. All of the F2 white-eyed flies were male.
(see pdf for photo of x-linked inheritance)
e. Morgan’s hypothesis was that this eye color
gene is located on the X chromosome. If so,
i. Males are hemizygous, because
there is no homologous gene on the
Y. The original mutant male’s
genotype was w/Y (hemizygous with
the recessive allele).
ii. Females may be homozygous or
heterozygous. The wild-type female
in the original cross was w+/w+
(homozygous for red eyes).
iii. The F1 flies were w+/w (females) and
w+/Y (males) (females all
heterozygous, males hemizygous
dominant). iv.
iv. The F2 data complete a crisscross
inheritance pattern, with
transmission from the mutant fly
through his daughter (who is
heterozygous) to his grandson. The
F2 were:
w+ Y males would possess a single X
w+ w+/ w+ w+/ Y
Red-eyed females Red-eyed males
w w+/ w w/ Y
Red-eyed females White-eyed males
v. Morgan’s hypothesis was confirmed
by an experiment reciprocal to the
original cross. A white-eyed female
(w/w) was crossed with a wildtype
male (w+/Y).
Results of the reciprocal cross:
(1) All F1 females had red eyes (w+/w).
(2) All F1 males had white eyes (w/Y).
vi. These F1 results are different from
those in the original cross, where all
the F1 had red eyes. When the F1
from the reciprocal cross interbred,
the F2 were:
w+ Y
w+ w+/ w+ w+/ Y
Red-eyed females Red-eyed males
w w/ w w/ Y
White-eyed females White-eyed males
(see pdf for photos of reciprocal cross)
2. Morgan’s discovery of X-linked inheritance
showed that when results of reciprocal crosses
are different, and ratios differ between progeny
of different sexes, the gene involved is likely to
be X-linked (sex-linked).
3. This was strong evidence that genes are
located on chromosomes. Morgan received
the 1933 Nobel Prize for Physiology or
Medicine for this work.
Sex-Linked Characteristics
 X-linked characteristics
 Z-linked characteristics
 Y-linked characteristics
X-Linked White Eyes in Drosophila
 Appearance of rare phenotypes was
associated with the inheritance of particular
chromosomes
 Sex-linked genes are located on the X
chromosome
 The wild-type eye color of Drosophila is dull
red, but pure lines with white eyes are
available.
 Allele for white is recessive
 White-eyed females would possess two X
chromosomes and one Y and that red-eyed
chromosome
X-Linked Color Blindness in Humans
 Human eye detects only three colors—red,
green and blue
 Affected woman passes the X-linked
recessive trait to her sons but not to her
daughters
  Affected man passes the trait to his
grandsons through his daughters but never
to his sons
 Pattern of inheritance exhibited by X-linked
recessive characteristics is sometimes called
crisscross inheritance.
Colourblindness - result from a defect in one of
three genes found on X chromosome
*Red-green color blindness is inherited as an X-
linked recessive trait in humans
(see pdf for reciprocal cross and possible
inheritance of colorblindness allele)
Carrier – an individual that has two alleles, one
damaged one and one regular
Z-Linked Characteristics
 In organisms with ZZ-ZW sex determination
 males are the homogametic sex (ZZ)
 Females are the heterogametic sex (ZW)
 Same as that of X-linked characteristics,
except that the pattern of inheritance in
males and females is reversed
 Z-linked characteristic is the cameo
phenotype in Indian blue peafowl
(see photos in pdf)
Y-Linked Characteristics
 Exhibit a distinct pattern of inheritance
 Present only in males
 All male offspring of a male with a Y-linked
trait inherit the trait
 Relatively little genetic information on the Y
chromosome
Recognizing Sex-linked Inheritance
 Alleles on sex chromosomes are inherited in
predictable patterns
 Y-linked trait can be inherited only from the
paternal grandfather (the father’s father),
never from the maternal grandfather
 X-linked characteristics also exhibit a
distinctive pattern of inheritance
Sex-Linkage
When writing alleles that are sex-linked, we use a
convention like that more complicated one from back
at the beginning: we write the X or Y normally, and
make the allele itself a superscript.
 If the A gene is on the X chromosome, then
genotypes can have one of these alleles: XA,
Xa, and Y.
 If there’s no allele, the Y becomes sort of like
the “free square” in the middle of a bingo
board. It doesn’t affect the phenotype at all.
 So a man who is XAY will have the dominant
version of the trait, and a man who is XaY will
have the recessive version of the trait. He
doesn’t have two little-a’s, true, but there’s
also no dominant allele to “drown out” the
recessive allele.
 if the gene is on the Y chromosome, we could
have the alleles X, YA, or Ya.
 if the gene is on the Y chromosome, we could
have the alleles X, YA, or Ya.
 Phenotype:
o XX - Will not have the trait, whatever
it is. •
o XYA - Dominant phenotype
o XYa - Recessive phenotype
 Phenotypes for people with a gene on the X
chromosome
o XBXB – Dominant
o XBXb – Dominant
o XbXb – Recessive
o XBY – Dominant
o XbY - Recessive
 Punnett Squares for sex-linked traits work
like normal, except that you use the
superscripts.
Sex Determination
Some mechanisms of sex determination include:
a. Genotypic sex determination, in which sex is
governed by genotype.
b. Genic sex determination, in which sex
chromosomes are not involved.
Genotypic Sex Determination Systems
Genotypic sex determination may occur two different
ways:
a. In the Y-chromosome mechanism of sex-
determination (e.g., in mammals), the Y
chromosome determines sex, conferring
maleness.
Dosage Compensation Mechanism for X-Linked
b. In the X chromosome-autosome balance
Genes in Mammals
system (e.g., Drosophila, Caenorhabditis
elegans) the ratio between number of X
chromosomes and number of sets of
autosomes determines sex. Y is required for
male fertility, but does not determine sex.
Sex Determination in Mammals
1. Mammals use the Y-chromosome mechanism
of sex-determination, in which the Y
chromosome determines sex by conferring
maleness.
2. Sex of mammals is determined by a gene on
the Y chromosome, testis-determining factor.
In the absence of this gene, gonads develop
into ovaries.
Evidence for the Y Chromosome Mechanism of
Sex Determination
1. Understanding of the Y chromosome
mechanism of sex determination came from the
study of individuals with unusual chromosome
complements. In humans these aneuploidies
include:
a. XO individuals, who are sterile females
exhibiting Turner syndrome. Most XO
fetuses die before birth. Surviving Turner
syndrome individuals become noticeable at
puberty, when secondary sexual
characteristics fail to develop. Other traits
include: (i) Below average height (ii) Weblike
necks (iii) Poorly developed breasts (iv)
Immature internal sexual organs (v) Reduced
ability to interpret spatial relationships.
b. XXY individuals, who are male and have
Klinefelter syndrome. Other traits include:
(i) Above average height (ii) Breast
development in about 50% of XXY
individuals (iii) Subnormal intelligence in
some cases.
c. XYY individuals are male, and tend to be
taller than average. Fertility is sometimes
affected.
d. XXX individuals are usually normal women,
although they may be slightly less fertile and
a few have below average intelligence.
e. Higher numbers of X and/or Y chromosomes
are sometimes found, including XXXY,
XXXXY, and XXYY. The effects are similar to
Klinefelter syndrome.
1. Gene dosage varies between the sexes in
mammals, because females have two copies
of X while males have one. Early in
development, gene expression from the X
chromosome must be equalized to avoid
death. Different dosage compensation
systems have evolved in different organisms.
2. In mammals, female somatic cell nuclei
contain a Barr body (highly condensed
chromatin) while male nuclei not. The Lyon
hypothesis explains the phenomenon:
a. Barr body is a condensed and (mostly) inactivated X
chromosome. Lyonization of one chromosome leaves
one transcriptionally active X, equalizing gene dose
between the sexes.
b. An X is randomly chosen in each cell for inactivation
early in development (in humans, day 16
postfertilization).
c. Descendants of that cell will have the same X
inactivated, making female mammals genetic mosaics.
Examples are:
i. Calico cats, in which differing descendant cells
produce patches of different color on the
animal.
ii. Women heterozygous for an X-linked allele
responsible for sweat glands, who have a
mosaic of normal skin and patches lacking
sweat glands (anhidrotic ectodermal
displasia).
d. Lyonization allows extra sex chromosomes to be
tolerated well. No such mechanism exists for autosomes,
and so an extra autosome is usually lethal.
e. The number of Barr bodies is the number of X
chromosomes minus one
f. X-inactivation involves three steps:
i. Chromosome counting (determining number
of Xs in the cell).
ii. Selection of an X for inactivation.
iii. Inactivation itself.
g. Counting the chromosomes involves the X-inactivation
center (XIC in humans, Xic in mice). Experiments in
transgenic mice show that:
i. Inactivation requires the presence of at least
two Xic sequences, one on each X
chromosome.
ii. Autosomes with an Xic inserted are randomly
inactivated, showing that Xic is sufficient for
chromosome counting and initiation of
lyonization
h. Selection of an X for inactivation is made by the X-
controlling element (Xce) in the Xic region. There are
different alleles of Xce, and each allele has a different
probability that the X chromosome carrying it will be
inactivated.
 i. The gene Xist is required for X inactivation. Uniquely, it
is expressed from the inactive X.
i. The Xist gene transcript is 17-kb. Although it
has no ORFs, it receives splicing and a poly(A)
tail.
ii. During X inactivation, this RNA coats the
chromosome to be inactivated and silences
most of its genes.
iii. Inactivation itself is not well understood, but it
is known that it initiates at the Xic and moves
in both directions, ultimately resulting in
heterochromatin.
Genic Sex Determination
1. Other eukaryotes use a genic system instead
of entire sex chromosomes.
2. A single allele determines the mating type
(e.g., MATa and MATa in Saccharomyces
cerevisiae).
3. Yeast mating types have identical
morphologies, but are able to fertilize gametes
only from the opposite mating type.
Environmental Sex Determination Systems
A few species use environmental sex
determination systems, in which environmental
factors affect the sex of progeny. Some types of
turtles are an example. Eggs incubated above 32°
develop into females, while those below 28° become
males. Eggs between these temperatures produce a
mix of the two sexes. Details will vary with each
species using this system.
In this system, the environment triggers a
developmental pathway which is under genetic
control.
Analysis of Sex-Linked Traits in Humans
1. X-linked traits, like autosomal ones, can be
analyzed using pedigrees.
2. Human pedigree analysis, however, is
complicated by several factors:
a. Data collection often relies on family
recollections.
b. If the trait is rare and the family small, there may
not be enough affected individuals to establish a
mechanism of inheritance.
c. Expression of the trait may vary, resulting in
affected individuals being classified as normal.
d. More than one mutation may result in the same
phenotype, and comparison of different pedigrees
may show different inheritance for the “same” trait.
X-Linked Recessive Inheritance
1. Human traits involving recessive alleles on the
X chromosome are X-linked recessive traits. A
famous example is hemophilia A among
Queen Victoria’s descendants.
2. X-linked recessive traits occur much more
frequently among males, who are hemizygous.
A female would express a recessive X-linked
trait only if she were homozygous recessive at
that locus.
3. Some characteristics of X-linked recessive
inheritance:
a. Affected fathers transmit the recessive allele to all
daughters (who are therefore carriers), and to none
of their sons.
b. Father-to-son transmission of X-linked alleles
generally does not occur.
c. Many more males than females exhibit the trait.
d. All sons of affected (homozygous recessive)
mothers are expected to show the trait.
e. With a carrier mother, about 1⁄2 of her sons will show
the trait and 1⁄2 will be free of the allele.
f. A carrier female crossed with a normal male will have
1⁄2 carrier and 1⁄2 normal daughters.
4. Other X-linked recessive traits are Duchenne
muscular dystrophy and two forms of color
blindness.
X-Linked Dominant Inheritance
1. Only a few X-linked dominants are known.
2. Examples include:
a. Hereditary enamel hypoplasia (faulty and
discolored tooth enamel)
b. Webbing to the tips of toes.
c. Constitutional thrombopathy (severe
bleeding due to lack of blood platelets).
3. Patterns of inheritance are the same as X-
linked recessives, except that heterozygous
females show the trait (although often in a
milder form).
Y linked inheritance (Holandric traits)
 There are far fewer Y-linked than X-linked
genetic disorders
 This is not surprising given that the Y
chromosome is smaller and has many less
genes than the X chromosome.
 Y-linked inheritance shows a pattern of
transmission of the mutant phenotype from
father to son, and it is never observed in
females.
 An example of a Y linked phenotypic trait is
hairy ears.
Sex limited inheritance
 Y-linked inheritance is often confused with
sexlimited inheritance.
 Sex-limited traits can only occur in one sex
because the feature affected is unique to that
sex.
 For example, premature baldness is an
autosomal dominant trait, but presumably as a
result of female sex hormones, the condition is
rarely expressed in the female, and then
usually only after menopause.
X-inactivation
 During the growth and development of
females’ cells, one X chromosome is
inactivated in each body cell.
 The inactivated X chromosome is visible in a
female’s cells as a Barr body.
 Which of the two X chromosomes becomes
inactive in a cell is a matter of chance,
therefore heterozygous females express
different alleles in different cells.
 This is generally not noticeable in the
phenotype – for example a woman
heterozygous for the recessive condition
haemophilia A will produce sufficient clotting
factor VIII.
 Tortoise shell cats are an example where X
inactivation is visible in the phenotype as one
of the genes which controls coat colour is sex-
linked.
 One of the genes that controls coat colour in
cats is sex-linked.
 It has alternative alleles Xo (orange) and Xb
(black)
 If Xo are inactivated will produce dark fur. If Xb
is inactivated will produce orange fur.
Pedigree Analysis
The technique of looking through a family tree (of
humans or other organisms) for the occurrence of a
particular characteristic in one family over a number
of generations.
 Can be used to determine the likely mode of
inheritance:
o Autosomal dominant
o Autosomal recessive
o X-linked dominant
o X-linked recessive
 When looking at pedigrees, incomplete
penetrance is occasionally observed.
o Incomplete penetrance describes the
situation where a proportion of a
population with a particular genotype
does not show the expected phenotype.
o Complete penetrance of a phenotype
means that all individuals with a
particular genotype will show the affected
phenotype.
Autosomal Dominant Pattern
An idealised pattern of inheritance of an autosomal
dominant trait includes the following features:
 both males and females can be affected
 all affected individuals have at least one affected parent
 transmission can be from fathers to daughters and sons,
or from mothers to daughters and sons
 once the trait disappears from a branch of the pedigree,
it does not reappear
 in a large sample, approximately equal numbers of each
sex will be affected.
Examples include:
 Huntington disease
 Achondroplasia (a form of dwarfism)
 Familial form of Alzheimer disease
 Defective enamel of the teeth
 Neurofibromatosis (the ‘Elephant man’ disease)
Autosomal Recessive Pattern
An idealised pattern of inheritance of an autosomal
recessive trait includes the following features:
 both males and females can be affected
 two unaffected parents can have an affected child
 all the children of two persons with the condition must
also show the condition
 the trait may disappear from a branch of the pedigree, but
reappear in later generations
  over a large number of pedigrees, there are
approximately equal numbers of affected females and
males.

Examples include:
 Albinism
 Cystic fibrosis
 Thalassaemia
 Tay-Sachs disease
 Phenylketonuria
 Red hair colour

X linked Dominant Pattern

An idealised pattern of inheritance of an X-linked
dominant trait includes the following features:
 a male with the trait passes it on to all his daughters and
none of his sons
 a female with the trait may pass it on to both her
daughters and her sons
 every affected person has at least one parent with the
trait
 if the trait disappears from a branch of the pedigree, it
does not reappear
 over a large number of pedigrees, there are more
affected females than males

Examples include:
 Vitamin D resistant rickets
 Incontinentia pigmenti, a rare disorder that results in
the death of affected males before birth

X linked Recessive Pattern

An idealised pattern of inheritance of an X-linked
recessive trait includes the following features:
 all the sons of a female with the trait are affected
 all the daughters of a male with the trait will be carriers of
the trait and will not show the trait; the trait can appear in
their sons
 none of the sons of a male with the trait and an unaffected
female will show the trait, unless the mother is a carrier
 all children of two individuals with the trait will also show
the trait
 in a large sample, more males than females show the
trait.

Examples include:
 Ichthyosis, an inherited skin disorder
 One form of red–green colour-blindness
 One form of severe combined immunodeficiency
disease
 Haemophilia
 Fragile X syndrome
 Duchenne muscular dystrophy
 RECOMBINANT DNA TECHNOLOGY
Recombinant DNA is a DNA molecule
constructed by joining two DNA segment derived
from different sources. The segments should be cut
by the same restriction enzyme.
The technology that are used in DNA cloning,
which permits researcher to prepare large number of
identical DNA molecule is called Recombinant DNA
technology.
Basics steps for DNA cloning
Vector + DNA fragment
Recombinant DNA
Introduce within a host cell
Replication of recombinant DNA within host cells
Isolation, Sequencing, and manipulation of purified
DNA fragments
Restriction Enzyme
A restriction enzyme recognizes a specific
nucleotide pair sequence in DNA called a restriction
site and cleaves the DNA within or near that
sequence.
Types of restriction endonuclease:
Cutting DNA molecule by restriction enzyme
Many restriction enzymes make staggered cuts
in the two DNA strand at their recognition site,
generating fragments that have single stranded
“Tail” at both ends. At room temparature these single
stranded regions often called “Sticky end” can
transiently base pair with those on other DNA
fragments generated by the same restriction
enzyme.
A few restriction enzymes cut both DNA strand
generating fragements with “blunt” ends in which all
the nucleotide at the fragment ends are base pared
to nucleotides in the complementary strand. The
blunt ends do not form hydrogen bond with each
other.
(see pdf for photo)
Cloning Vector
A cloning vector is a genome that can accept the
target DNA and increase the number of copies
through its own autonomous replication.
Features of cloning vector:
I. Can independently replicate themselves and
the foreign DNA segments they carry
II. Contain a number of unique restriction
endonuclease cleavage site
III. Carry selectable marker to distinguish host cell
that carry vectors from host cells that do not
contain a vector
IV. Relatively ease to recover from the host cell.
Some cloning vector with example:
Plasmid
Plasmids are circular, double-stranded DNA
molecules that are separate from a cell
chromosomal DNA. They are located outside of the
chromosome and they can replicate autonomously
within a host
Plasmid cloning vector must have these three
features:
 An “Ori” ( origin of replication) sequence, needed
for the plasmid to replicate.
 A selectable marker- so that bacterial cell with
the plasmid can be distinguished easily from cell
that lack of plasmid
 One or more uniqe restriction enzyme cleavage
sites that present just once in the vector for the
insertion of the DNA fragment to be cloned
(see pdf for photo)
pBR322
One of the first versatile plasmid vectors developed.
It is the ancestor of many of the common plasmid
vectors used in biochemistry laboratories
Features of pBR322 :
 contain an origin of replication (ori) and a gene
(rop) that helps to regulate the number of copies
of plasmid DNA in a cell
 There are two selectable marker genes, confers
resistance to ampicillin and tetracyclin
 Contain a number of unique restriction sites that
are useful constructing recombinant DNA
Advantage of pBR322 :
I. Small size enables easy purification and
manipulation
II. Two selectable marker allow easily selection of
recombinant DNA
Disadvantage of pBR322 :
I. It has very high mobility
II. Not a very high copy number is present as is
expected from a good vector
(see pdf for photo)
Bacterial Artificial Chromosome (BAC)
A bacterial artificial chromosome (BAC) is a DNA
construct, based on a functional fertility plasmid,
used for transforming and cloning in bacteria usually
E. coli .
Main features of BAC
 Ori S – The origin of replication
 Rep E – for plasmid regulation and regulation of
copy number
 Par A and Par B – for partitioning F plasmid DNA
to daughter cells during division and ensure
stable maintenance of BAC
 Selectable marker- for antibiotic resistance in
this chloramphenicol resistance
 T7 and SP6- are universal promoter to ensure
gene expression from the cloned fragment
(see pdf for photo)
Yeast Artificial Chromosome (YAC)
The vector used for cloning the largest possible DNA
inserts is the yeast artificial chromosome (YAC).
YACs have the largest capacity of any cloning vector
and can propagate with inserts measured in the
megabase length range.
A YAC has the following features:
 A yeast telomere at each end to seal the ends of
chromosome. They are required for maintenance
of linear chromosome.
 A yeast centromere allowing regulated
segregation during mitossis.
 A selectable marker on each arm for detecting
and maintaining the YAC in yeast.
 An origin of replication sequence ARS that allows
the vector to replicate in a yeast cell.
 An origin of replication (ori) that allows vector to
replicate and a selectable marker such as amp
 A cloning region that contains one or more
restriction site, the restriction cutting in this site.
(see pdf for photo)
Shuttle vector
A plasmid that has both bacterial and eukaryotic
origin of replication and so can propagate in either
kind of cell.
(see pdf for photo)
Expression vector
An expression vector is a cloning vector containing
the necessary regulatory sequence to allow
transcription and translation of a cloned gene or
genes.
Features of expression vector:
 A promoter upstream of the multiple cloning site
 A transcription terminator downstream of the
multiple cloning site and
 A DNA sequence encoding Shine Dalgarno
sequence for translation initiation located
between the promoter and the multiple cloning
site
Basic steps for recombinant DNA
1. Isolation and purification of DNA
Both vector and target DNA molecule can be
prepared by a variety of routine method of
heating the cell extracts in the presence of
detergents and removing proteins by phenol
extraction. In some cases the target DNA is
synthesized in vitro.
2. Cleavage of DNA at particular sequence
Cleaving DNA to generate fragment of
defined length or specific end point is done
by restriction enzyme usually endonuclease
 3. Ligation of DNA fragments  Then by using terminal transferase which is a
DNA polymerase that add deoxynucleotides to
A recombinant DNA molecule is usually
free 3’ end without the need of template.
formed by ligating cleaved DNA to vector
DNA. DNA fragments are typically joined by
 To this a synthetic poly C is hybridized which is
using DNA ligase
used as primer for the synthesis of the
4. Introduction of recombinant DNA into complentary strand of the cDNA.
compatible host cell
 It is necessary to protect the cDNA from
Suitable host cells are selected and rDNAis unwanted digesstion by restriction enzymes.
introduced into these host cells. Therefore the cDNA is treated with a
The direct uptake of foreign DNA by a host modification enzymes. Therefore the cDNA is
cell is called genetic transformation. treated with a modification enzyme that
rDNAcan also be packaged into virus particle methylates specific bases within the restriction
and transferred into host cell by transfection. enzyme sequence

5. Replication and expression of recombinant  Then ligate each end of the cDNA a short
DNA in host cells restriction site linker. This will produce blunt end
6. The inserted gene doing along with the at the end of the DNA.
vector will replicate inside the host so that
many copies of the desired gene is  The next step is to treat the cDNA with restriction
synthesized. For expression of the desired enzymes that are specific to the blunt ends. This
gene, expression vector is used. will result with sticky end.
7. Identification of host cell that contain
recombinant DNA of interest  The final step is to ligate the sticky ends of the
cDNA with the lamda phage arms that have
Vectors are usually contain selction marker
complementary sticky ends, thereby inserting
through which we can identify the host cell
the double strand cDNA into the vector.
that have taken up foreign DNA
cDNA library
Screening of cDNA library
A collection of cDNA clones that represent all the
There are several way to screen a cDNA library
mRNAs expressed in a cell type is called cDNA
library. i. Using a DNA probe with a homologous sequence
Generating a cDNA library: ii. Using an oligonucleotide probe bassed on a
known amino acid sequence
 Isolation of mRNA- All mRNA have a poly A tail.
By using a column that contains a short poly T iii. Using an antibody against the protein of interest
sequence it is possible to isolate the mRNA for
both tRNA and rRNA. iv. Plus/Minus or differential screening
(see pdf for photo)
 Isolated mRNA is treated with an enzyme
reverse transcriptase, that will create cDNA
intermediate from the mRNA.
By hybridizing the poly A of the mRNA with oligo
T’d a primer is created. Reverse transcriptase
recognizes this template and will add bases to 3’
end.
 The mRNA-cDNA complex is treated with an
alkali or RNase H which hydrolyzes the mRNA
but not the cDNA.
 EVOLUTIONARY GENETICS Types of Natural Selection
Natural selection as a force of evolution a. Stabilizing selection
b. Disruptive selection
What is Darwin’s natural selection?
c. Directional selection
The differential success (survival and reproduction)
of individuals within the population that results from
their interaction with their environment.
“Survival of the most adaptable, elimination of
‘inferior’ individual”
Two conditions (assumptions):
1. There is variation in populations. Variation is
heritable.
2. In every generation some organisms are
more successful at surviving and
reproducing than others. Survival and
reproduction are not random, but are related
to variation among individuals. Organisms
with best characteristics are ‘naturally Heritability is an essential feature of NS
selected.’
 Heritability: individual’s characteristics are
If 2 conditions are met then the population will passed from one generation to the next.
change from one generation to the next. Evolution Measured as proportion of the variation in a
will occur. trait in a population that is due to variation in
genes.
Evidence of natural selection
 Mendel’s genetics and inheritance
 Evolution of beak shape in Finches.
Mendel’s conclusions in Pea Experiment:
 Peter and Rosemary Grant’s (and
colleagues) work on Medium Ground 1. Flower color is a heritable trait
Finches Geospiza fortis.
2. For each inherited characteristic, an
Is variation in beak size correlated with organism has two units, one from each
variation in fitness? parent (one each from egg and sperm). The
unit may be the same or different.
In 1978, there was a
severe drought, small 3. When the two units are different, one is fully
seeds declined more than
large seeds.
expressed, another one has no noticeable
effect of the organism’s outward
Small beak birds have appearance.
difficulty to find seeds, and
suffered heavy mortality, Dominant: the unit is expressed (purple)
especially females.
Recessive: not expressed (white).
Conclusion: Natural selection indeed caused
evolution in beak size Genes are the units of inheritance.
What are genes?  Reassortment of genes provided by two
parents in the offspring
Genes: discrete subunit of chromosome, carry
 Increases dramatically the variation within a
genetic information
population by creating new combinations of
Chromosomes: the threadlike structures where DNA existing genes.
is contained
Asexual reproduction: less variation (only
DNA: Deoxyribonucleric acid. All DNA is composed mutation)
of the same 4 nucleotide (ATGC), differ in sequence.
What do we mean by genetic variation?
Alleles: alternate forms of a gene (A, a).
 Range (variance) of phenotypes, as in
Locus: the position of an allele occupies on a Darwin’s Finch example
chromosome
 Different chromosomal arrangements
Homozygous (AA, aa) vs heterozygous (Aa) (cytogenetics)

Dominance (A vs a), incomplete dominance (Aa  DNA sequence differences among

shows different trait to AA or aa) individuals

How are genes transmitted?  Electrophoresis--> electromorphs =

allozymes
Genotype: the sum of genes carried by the
individual.  Molecular markers: RFLP, RAPD, etc.

Gene pool: total collection of genes across all Evolution is a change of gene frequencies within
individual in the population at any one time a population (or species) over time

Phenotype: the observed expression of genotype
(color etc)
Phenotypic plasticity
Phenotypic plasticity: the ability of a genotype to give
a range of phenotypic expressions under different
environmental conditions.
Genetic variation is the ingredient for NS
Genetic variation within a population is absolutely
necessary for natural selection to occur.
If all individuals are identical within a population,
then their fitness will all be the same.
Source of genetic variation
 Mutation: inheritable changes in a gene or a
chromosome
o Gene mutation: (point mutation)
o Chromosome mutation: deletion, duplication,
inversion, translocation
 Individuals do not evolve, populations evolve
 Focus on gene pool, collective
But why do we see populations are still the ‘same’
over many generations?
The Hardy-Weinberg Principle:
Gene frequencies will remain the same in
successive generations of a sexually reproducing
population if the following five conditions hold:
 Random mating
 There is no mutation
 The population is very large
 There is no selection
 There is no migration (isolated from other
populations)

 Genetic recombination
Sexual reproduction: two individuals produce
haploid gametes (egg or sperm) – that combine to
form a diploid cell or zygote.
Five Causes of Evolution
 Mutations
 Gene flow - Emigration and immigration of
individuals (Flow of alleles)
 Genetic Drift – Changes in the gene pool of
a small population due to chance
 Nonrandom mating
 Natural selection
Genetic drift in a small population
Speciation
Speciation is the splitting of one species into 2
different species.
Concept of species:
 Biological Species: a species is a group of
organisms whose individuals have the
potential to interbreed and produce fertile
offspring.
o Reproductively isolated: don’t produce
fertile hybrids
o Natural conditions: artificial breeding
doesn’t count. For example, artificial
insemination, keeping 2 species locked
up together.
 Morphological species: members of the same
species look similar to each other. Many
examples of organisms that look similar but can’t
produce fertile offspring.
 No one species concept applies to all organisms
Genetic isolation mechanisms (reproductive
barriers)
 Premating mechanisms
 Habitat selection; temporal isolation;
behavior or mechanical or structure
incompatibility
 Postmating mechanisms
 Genetic barrier such as hybrids are
sterile; seed abortion; hybrid
inviability.
Mechanisms of Speciation
Several are well documented:
 Allopatric speciation (probably most
vertebrates) - geographical isolation
 Sympatric speciation (especially plants and
insects) - speciation via polyploidy
(see ppt for photos)
Adaptations reflect trade-offs and constraints
Adaptation: Individuals of a species have certain
characteristics that enable an organism to thrive in a
given environment.
Adaptations maintain or increase fitness of an
organism in a given environmental conditions.
Tradeoff
 Evolution is reflected in changes in gene
frequencies and gene phenotypes
 However, changes in gene frequencies
involve tradeoffs
 Giraffes do not graze well on grass due to
their long necks
 Snow leopards can hide in snowy mountains,
ordinary leopards can hide well in green
trees
Constraint
 There may be many possible ways that an
organism can be better adapted to a
particular condition, but other aspects of the
organism may limit the possibilities.
 In other words, adaptation may be limited by
various constraints imposed by other
aspects of the organism.
DESCENT WITH MODIFICATION: A DARWINIAN
VIEW OF LIFE
Overview: Endless Forms Most Beautiful
• A new era of biology began in 1859 when
Charles Darwin published The Origin of
Species
• The Origin of Species focused biologists’
attention on the great diversity of organisms
• Darwin noted that current species are
descendants of ancestral species
• Evolution can be defined by Darwin’s
phrase descent with modification
• Evolution can be viewed as both a pattern
and a process
Scala Naturae and Classification of Species
• The Greek philosopher Aristotle viewed
species as fixed and arranged them on a
scala naturae
• The Old Testament holds that species were
individually designed by God and therefore
perfect
• Carolus Linnaeus interpreted organismal
adaptations as evidence that the Creator
had designed each species for a specific
purpose
• Linnaeus was the founder of taxonomy, the
branch of biology concerned with classifying
organisms
• He developed the binomial format for
naming species (for example, Homo
sapiens)
Ideas About Change over Time
 The study of fossils helped to lay the
groundwork for Darwin’s ideas
 Fossils are remains or traces of organisms from
the past, usually found in sedimentary rock,
which appears in layers or strata.
 Paleontology, the study of fossils, was largely
developed by French scientist Georges Cuvier
 Cuvier advocated catastrophism, speculating
that each boundary between strata represents a
catastrophe
 Geologists James Hutton and Charles Lyell
perceived that changes in Earth’s surface can
result from slow continuous actions still
operating today
 Lyell’s principle of uniformitarianism states that
the mechanisms of change are constant over
time
 This view strongly influenced Darwin’s thinking
Lamarck’s Hypothesis of Evolution
• Lamarck hypothesized that species evolve
through use and disuse of body parts and the
inheritance of acquired characteristics
• The mechanisms he proposed are
unsupported by evidence
Darwin’s Research
As a boy and into adulthood, Charles Darwin had
a consuming interest in nature.Darwin first studied
medicine (unsuccessfully), and then theology at
Cambridge University. After graduating, he took an
unpaid position as naturalist and companion to
Captain Robert FitzRoy for a 5-year around the
world voyage on the Beagle
The Voyage of the Beagle
• During his travels on the Beagle, Darwin
collected specimens of South American
plants and animals
• He observed that fossils resembled living
species from the same region, and living
species resembled other species from
nearby regions
• He experienced an earthquake in Chile and
observed the uplift of rocks
 • Darwin was influenced by Lyell’s Principles
of Geology and thought that the earth was
more than 6000 years old
• His interest in geographic distribution of
species was kindled by a stop at the
Galápagos Islands west of South America
• He hypothesized that species from South
America had colonized the Galápagos and
speciated on the islands
Darwin’s Focus on Adaptation
• In reassessing his observations, Darwin
perceived adaptation to the environment
and the origin of new species as closely
related processes
• From studies made years after Darwin’s
voyage, biologists have concluded that this is
what happened to the Galápagos finches
• In 1844, Darwin wrote an essay on natural
selection as the mechanism of descent with
modification, but did not introduce his theory
publicly
• Natural selection is a process in which
individuals with favorable inherited traits are
more likely to survive and reproduce
• In June 1858, Darwin received a manuscript
from Alfred Russell Wallace, who had
developed a theory of natural selection
similar to Darwin’s
• Darwin quickly finished The Origin of Species
and published it the next year
The Origin of Species
Darwin explained three broad observations:
 The unity of life
 The diversity of life
 The match between organisms and their
environment
Descent with Modification
• Darwin never used the word evolution in the
first edition of The Origin of Species
• The phrase descent with modification
summarized Darwin’s perception of the unity
of life
• The phrase refers to the view that all
organisms are related through descent from
an ancestor that lived in the remote past
• In the Darwinian view, the history of life is like
a tree with branches representing life’s
diversity
• Darwin’s theory meshed well with the
hierarchy of Linnaeus
Artificial Selection, Natural Selection, and
Adaptation
• Darwin noted that humans have modified
other species by selecting and breeding
individuals with desired traits, a process
called artificial selection
• Darwin drew two inferences from two
observations
Observation #1: Members of a population often vary
in their inherited traits
Observation #2: All species can produce more
offspring than the environment can support, and
many of these offspring fail to survive and reproduce
Inference #1: Individuals whose inherited traits give
them a higher probability of surviving and
reproducing in a given environment tend to leave
more offspring than other individuals
Inference #2: This unequal ability of individuals to
survive and reproduce will lead to the accumulation
of favorable traits in the population over generations
• Darwin was influenced by Thomas Malthus,
who noted the potential for human population
to increase faster than food supplies and
other resources
• If some heritable traits are advantageous,
these will accumulate in a population over
time, and this will increase the frequency of
individuals with these traits
• This process explains the match between
organisms and their environment
Natural Selection: A Summary
• Individuals with certain heritable
characteristics survive and reproduce at a
higher rate than other individuals
• Natural selection increases the adaptation of
organisms to their environment over time
• If an environment changes over time, natural
selection may result in adaptation to these
new conditions and may give rise to new
species
• Note that individuals do not evolve;
populations evolve over time
• Natural selection can only increase or
decrease heritable traits that vary in a
population
• Adaptations vary with different environments
Evolution is supported by an overwhelming
amount of scientific evidence
New discoveries continue to fill the gaps
identified by Darwin in The Origin of Species
Direct Observations of Evolutionary Change
Two examples provide evidence for natural
selection: natural selection in response to introduced
plant species, and the evolution of drug-resistant
bacteria
Natural Selection in Response to Introduced
Plant Species
• Soapberry bugs use their “beak” to feed on
seeds within fruits
• In southern Florida soapberry bugs feed on
balloon vine with larger fruit; they have longer
beaks
• In central Florida they feed on goldenrain
tree with smaller fruit; they have shorter
beaks
• Correlation between fruit size and beak size
has also been observed in Louisiana,
Oklahoma, and Australia
• In all cases, beak size has evolved in
populations that feed on introduced plants
with fruits that are smaller or larger than the
native fruits
• These cases are examples of evolution by
natural selection
• In Florida this evolution in beak size occurred
in less than 35 years
The Evolution of Drug-Resistant Bacteria
• The bacterium Staphylococcus aureus is
commonly found on people
• One strain, methicillin-resistant S. aureus
(MRSA) is a dangerous pathogen
• S. aureus became resistant to penicillin in
1945, two years after it was first widely used
• S. aureus became resistant to methicillin in
1961, two years after it was first widely used
• Methicillin works by inhibiting a protein used
by bacteria in their cell walls
• MRSA bacteria use a different protein in their
cell walls
• When exposed to methicillin, MRSA strains
are more likely to survive and reproduce than
nonresistant S. aureus strains
• MRSA strains are now resistant to many
antibiotics
Natural selection does not create new traits, but
edits or selects for traits already present in the
population
The local environment determines which traits
will be selected for or selected against in any specific
population
Homology
• Homology is similarity resulting from
common ancestry
Anatomical and Molecular Homologies
• Homologous structures are anatomical
resemblances that represent variations on a
structural theme present in a common
ancestor
• Comparative embryology reveals anatomical
homologies not visible in adult organisms
 • Vestigial structures are remnants of • Endemic species are species that are not
features that served important functions in found anywhere else in the world
the organism’s ancestors
• Islands have many endemic species that are
• Examples of homologies at the molecular often closely related to species on the
level are genes shared among organisms nearest mainland or island
inherited from a common ancestor
• Darwin explained that species on islands
Homologies and “Tree Thinking” gave rise to new species as they adapted to
new environments
• Evolutionary trees are hypotheses about
the relationships among different groups What Is Theoretical About Darwin’s View of Life?
• Homologies form nested patterns in • In science, a theory accounts for many
evolutionary trees observations and data and attempts to
explain and integrate a great variety of
• Evolutionary trees can be made using
phenomena
different types of data, for example,
anatomical and DNA sequence data • Darwin’s theory of evolution by natural
selection integrates diverse areas of
A Different Cause of Resemblance: Convergent
biological study and stimulates many new
Evolution
research questions
• Convergent evolution is the evolution of
• Ongoing research adds to our understanding
similar, or analogous, features in distantly
of evolution
related groups
• Analogous traits arise when groups
independently adapt to similar environments
in similar ways
• Convergent evolution does not provide
information about ancestry
The Fossil Record
• The fossil record provides evidence of the
extinction of species, the origin of new
groups, and changes within groups over time
• Fossils can document important transitions
• For example, the transition from land
to sea in the ancestors of cetaceans
Biogeography
• Biogeography, the geographic distribution
of species, provides evidence of evolution
• Earth’s continents were formerly united in a
single large continent called Pangaea, but
have since separated by continental drift
• An understanding of continent movement
and modern distribution of species allows us
to predict when and where different groups
evolved