Model Organisms

Star College Workshop for

College Teachers
For Genetics & Genomics I
Semester V
 Model Organism
 Two important feature of all model systems:
first, the availability of powerful tools of and
study the organism genetically. Second,
ideas, methods, tools, and strains could be
shared among scientists investigating the
same organism, facilitating rapid progress.
The choice of a model organism depends on
what question is being asked.
 Here we will describe some of the most
commonly studied experimental organisms
and advantages of each as a model system.
 Bacteriophage
 Bacteriophage (and viruses in general) offer
the simplest system to examine the basic
processes of life. Phage typically consist of
a genome (DNA and RNA, most commonly
the former) packaged in a coat of protein
subunits, some of which form a head
structure (in which the genome is stored)
and some a tail stricture.
Each phage attaches to a specific cell
surface molecule (usually a protein) and
so only cells bearing that “receptor” can be
infected by a given phage.
 Phage come in two basic types-lytic and
temperate. The former, examples of which
include the T phage, grow only lytically.
 FIGURE 21-1 The lytic growth cycle of bacteriophage
 Temperate phage can also replicate
lytically. But they can adopt an alternative
developmental pathway called lysogeny.
(figure 21-2)
 In this integrated, repressed state the
phage is called a prophage.
 The lysogenic state can be maintained in
this way for many generations but is also
poised to switch to lytic growth at any time.
This switch from the lysogenic to lytic
pathway, called induction.
 FIGURE 21-2
 Assays of phage growth
 For bacteriophage to be use ful as an
experimental system, methods are needed
to propagate and quantify phage. To
quantify the numbers of phage particles in a
solution, a plaque assay is used.
 Figure 21-3
Plaques firmed
by phage
infection of
a lawn of
bacterial cells.
 Plaque is the result of multiple round of
infection, a circular clearing in the
otherwise opaque lawn of densely grown
uninfected bacterial cells. Knowing the
number of plaques on a given plate, and
the extent to which the original stock was
diluted before plating, makes it trivial to
calculate the number of phage in that
original stock.
 The single-step growth curve
 This classic experiment revealed the life
cycle of a typical lytic phage and paved the
way for many subsequent experiments that
examined that life cycle in detail. The
essential feature of this procedure is the
synchronous infection of a population of
bacteria and the elimination of any re-
infection by the progeny.
 Figure 21-4 The single-step growth curve
 The time lapse between infection and
release of progeny is called the latent period,
and the number of phage released is called
the burst size.
 Phage crosses an
complementation tests
 Differences in host range and plaque
morphologies of the phage were very often
the result of genetic differences between
otherwise identical phage.
 The ability to perform mixed infection-in
which a single cell is infected with two
phage particles at once-makes genetic
analysis possible in two ways.
 First, it allow one to perform phage
crosses.
 Second, co-infection also allow one to
assign mutations to complementation
groups; that is, one can identify when two
or more mutations are in the sane or in
different genes.
Transduction and recombinant DNA
 The process involves a site-specific
recombination event, and if that event
occurs at slightly the wrong position, phage
DNA is lost and bacterial DNA included is as
known as specialized transduction.
 Because of the ability to promote
specialized transduction, it was natural
that phage λ was chosen as one of the
original cloning vectors.
 Many different λ vectors were developed,
all differing in the restriction sites used and
in how recombinant phage could be
identified.
 Bacteria
The attraction of bacteria such as E. coli or
B. subtilis as experimental systems is that
they are relatively simple cells and can be
grown and manipulated with comparative
ease.
 Molecular biology owes its origin to
experiments with bacterial and phage model
systems.
 Assays of bacterial growth
 Bacterial cells are large enough（about
2µm in length）to scatter light, allowing the
growth of a bacterial culture to be monitored
conveniently in liquid culture by the increase
in optical density.
 Figure 21-5 bacterial growth curve
 The number of bacteria can be determined
by diluting the culture and plating the cells
o solid (agar) medium in a petri dish.
Knowing how many colonies are on the
plate and how much the culture was
diluted makes it possible to calculate the
concentration of cells in the original culture.
 Bacteria exchange DAN by sexual
conjugation, phage-mediated
transduction, and DAN-mediated
transformation

 Bacterial often harbor autonomously

replicating DNA elements known as
plasmids.
 Figure 21-6 the three forms of F-plasmid carry cells
 The F-factor can undergo conjugation only
with other E .coli strains, promiscuous
conjugative plasmids provide a convenient
means for introducing DNA into bacterial
strains that are otherwise lacking in their
own systems of genetic exchange.
Yet another powerful tool for genetic
exchange is phage-mediated transduction.
 Generalized transduction is mediated by
phage that occasionally package fragment
of chromosomal DNA during maturation of
the virus rather than viral DNA.
 Figure 21-7 Phage-mediated generalized transduction
 Another kind of phage-mediated
transduction is called specialized
transduction. It involves a lysogenic phage
such as λ that has incorporated a segment
of chromosomal DNA in place of a
segment of phage DNA.
 DNA-mediated transformation: certain
experimentally important bacterial species
possess a natural system of genetic
exchange that enables them to take up
and incorporate linear, naked DNA into
their own chromosome by recombination.
 Often the cells must be in a specialized
state known as “genetic competence” to
take up and incorporate DNA from their
environment.
 Bacterial plasmids can be used as
cloning vectors

 Circular DNA elements in bacterial known as

plasmids can serve as convenient vectors
for bacterial DNA as well as foreign DNA.
 Transposons can be used to
generate insertional mutations and
gene and operon fusion

 Transposons are enormously useful tools for

carrying out molecular genetic
manipulations in bacterial.
 Figure 21-8 Transposon-generated insertional mutagenesis
 It have two important advantages over
traditional mutations induced by chemical
mutagenesis.
 One is that the insertion of a transposon
into a gene is more lifely to result in
complete inactivation of the gene than a
simple nucleotide switch created by a
mutagen.
 The second is that ,having inactivated the
gene, the presence of the inserted DNA
makes it easy to isolate and clone that
gene.
 Transposons can also be used to create
gene and operon fusions on a genome-
wide basis. such a fusion is know as an
operon or transcriptional fusion.
 Figure 21-9 Transposon-generated lacZ fusion
 And a fusion in which the reporter is joined
both transcriptionally and translationally to
the target gene is known as a gene fusion
 Studies on the molecular biology of
bacteria have been enhanced by
recombinant DNA technology, whole-
genome sequencing, and
transcriptional profiling
 With the advent of recombinant DNA
technologies revolutionized molecular
biological studies of higher cells. But they
have had an impact on the study of
bacterial model systems as well.

 The availability of microarrays

representing all of the genes in a
bacterium has made it possible to study
gene expression on a genome basis.
 And the availability of whole-genome
sequences and promiscuous conjugative
plasmids has created opportunities for
carrying out molecular genetic
manipulations in bacterial species that
otherwise lack sophisticated, traditional tools
of genetics.
Biochemical analysis is especially
powerful in simple cells with well-
developed tools of traditional and
molecular genetics
 There are three reasons for this:
 first, large quantities of bacterial cells can
be grown in a defined and homogenous
physiological state.
 Second , the tools of traditional and
molecular genetics.
 Third, the machinery for carrying out DNA
replication, gene transcription, protein
synthesis, and so forth is much simpler
(having far fewer components) in bacterial
than in higher cells.
 Bacterial are accessible to
cytological analysis
 Despite their small size, bacteria are
accessible to the tools of cytology, such as
immunofluoresence microscopy for
localizing proteins in fixed cells with
specific antibodies, fluorescence
microscopy with the Green Fluorescent
Protein for localizing proteins in living cells,
and fluorescence in situ hybridization
(FISH) for localizing chromosomal regions
and plasmids within cells.
 Cytological methods are an important part of
the arsenal for molecular studies on the
bacterial cell.
Phage and bacteria told us most of
the fundamental things about the
gene
 Molecular biology owes its origin to
experiments with bacterial and phage
model systems. Indeed, groundbreaking
work with a pneumococcus bacterium led
to the discovery that the genetic material is
DNA. Since then, experiments with E .coli
and its phage have the way.
 There are countless examples where, by
choosing these simplest of systems,
fundamental processes of life were
understood. An important example comes
from the classic work of Seymor Benzer,
who examined intensely a single genetic
locus in phage T4, called rⅡ.
 BAKER’S YEAST, Saccharomyces
cerevisiae

 Unicellular eukaryotes offer many

advantages as experimental model systems.
And the best studied unicellular eukaryote is
the budding yeast S. cerevisiae.
The existence of haploid and diploid
cells facilitate genetic analysis of S.
cerevisiae

 S. cerevisiae exists in three forms. Two

haploid cell types, a and α, and the diploid
product of mating between these two.
 Figure 21-10 The lifecycle of the budding yeast S.
cerevisiae
These cell types can be manipulate to
perform a variety of genetic assays.
 Genetic complementation can be
performed the two mutations whose
complementation is being tested.
 If the mutations complement each other,
the diploid will be a wild type for mntations
can be made in haploid cells in which
there is only a single copy of that gene.
 Generating precise mutations in
yeast is easy

 The genetic analysis of S. cerevisiae is

further enhanced by the availability of
techniques used to precisely and rapidly
modify individual genes.
 Figure 21-11 recombinational transformation in yeast
 The ability to make such precise changes in
the genome allows very detailed questions
concerning the function of particular genes
or their regulatory sequences to be pursued
with relative ease.
 S. cerevisiae has a small, well-
characterized genome

 Because of its rich history of genetic studies and

its relatively small genome, S. cerevisiae was
chosen as the first eukaryotic ( nonviral ) organism
to have its genome entirely sequenced. This
landmark was accomplished in 1996.
 The availability of the complete genome
sequence of S. cerevisiae has allowed
“genome-wide” approaches to studies of this
organisn.
S. cerevisiae cells change shape as
they grow

 As S. cerevisiae cells progress through the

cell cycle. They undergo characteristic
changes in shape.
 Figure 21-12 The mitotic cell cycle in yeast
 Simple microscopic observation of S.
cerevisiae cell shape can provide a lit of
information about the events occurring
inside the cell.
 A cell that lacks a bud has yet to start
replicating its genome. A cell with a very
large bud is almost always in the process
of executing chromosome segregation.
 THE NEMATODE WORM,
caenorhabditis elegans
 In 1965 Sydney Brenner settled on the
small nematode worm caenorhabditis
elegans to study the important questions
of development and the molecular basis of
behavior, because it contained a variety of
suitable characteristics.
 And due to its simplicity and experimental
accessibility, it is now one of the most
completely understood metazoan.
C. elegans has a very rapid life cycle
 At 25℃ fertilized embryos of C. elegans
complete development in 12 hours and
hatch into free-living animals capable of
complex behaviors.
 The first stage juvenile(L1) passes through
four juvenile stages(L1-L4) over the course
of 40 hours to become a sexually mature
adult.
 Figure 21-13 The life cycle of the worm, C.elegans
Under stressful conditions, the L1 stage
animal can enter an alternative
developmental stage in which it forms what
is called a dauer.
 Dauers are resistant to environmental
stresses and can live many months while
waiting for environmental conditions to
imptove.
C. elegans is composed of relatively
few, well studied cell lineages

 C. elegans has a simple body plan. Its

lineages is relatively few and well studied.
 Figure 21-14 The body plan of the worm
 Among genes that function to control the
generation, specification, and
differentiation of the vulva cells are
components of a highly conserves
receptor tyrosine kinase signaling pathway
that controls cell proliferation.
 Many of the mammalian homologs of
these genes are oncogenes and tumor-
supressorgenes that when altered canlead
to cancer. But in C. elegans, mutations
that inactivate this pathway eliminate vulva
development.
 The cell death pathway was
discovered in C. elegans
 The most notable achievement to date in
C. elegans research has been the
elucidation of the molecular pathway that
regulates apoptosis or cell death.
 Analysis of the ced mutants showed that,
in all but one case, developmentally
programmed cell death is cell autonomous,
that is, the cell commits suicide.
 Cell death is as important as cell
proliferation in development and disease
and is the focus of intense research to
develop therapeutics for the control of
cancer and neurodegenerative diseases.
RNAi was discovered in C. elegans

 In 1998 a remarkable discovery was

announced. The introduction of dsRNA into
C. elegans silenced the gene homologous to
the dsRNA. It significant in two respects.
One is that RNAi appears to be universal
since introduction of dsRNA into nearly all
animal, fungal, or plant cells leads to
homology-directed mRNA degradation.

 The second was the rapidity with which

experimental investigation of this
mysterious process revealed the
molecular mechanisms.
 THE FRUIT FLY, Drosophila
melanogaster

Drosophila has a raid life cycle

 The salient features of the Drosophila life
cycle are a very rapid period of
embryogenesis, followed by three period of
larval growth prior to metamorphosis.
 Figure 21-15 The Drosophila life cycle
 One of the key processes that occurs
during larval development is the growth of
the imaginal disks, which arise from
invaginations of the epidermis in mid-stage
embryos.

 Imaginal disks differentiate into their

appropriate adult structures during
metamorphosis (or pupation).
 Figure 21-16 Imaginal disks in Drosophila
 The first genome maps were
produced in Drosophila
 Morgan labs studied on Drosophila in 1910
led to two major discoveries: genes are
located on chromosomes, and each gene is
composed of two alleles that assort
independently during meiosis; genes located
on separate chromosomes segregate
independently, whereas those linked on the
same chromosome do not.
 Hermann J. Muller provided the first
evidence that environmental factors can
cause chromosome rearrangements and
genetic mutations.

 Bridges used the polytene chromosomes

to determine a physical map of the
Drosophila genome (the first produced for
any organism).
 Figure 21-17 Genetic maps, polytene chromosome, and
deficiency mapping
 A variety of additional genetic methods
were create to establish the fruit fly as the
premiere model organism for studies in
animal inheritance.

 For example, balancer chromosomes were

created that contain a series of inversions
relative to the organization of the native
chromosome.
 Figure 21-18 Balancer chromosome
 Embryos that contain two copies of the
balancer chromosome die because some
of the inversions produce recessive
disruptions in critical genes.

 In addition, embryos that contain two

copies of the normal chromosome die
because they are homozygous for the eve
null mutation.
Genetic mosaics permit the analysis
of lethal genes in adult flies
 Mosaics are animals that contain small
patches of mutant tissue in a generally
“normal” genetic background.
 The analysis of genetic mosaics provided
the first evidence that Engrailed is required
for subdividing the appendages and
segments of flies into anterior and
posterior compartments.
 The most spectacular genetic mosaics
are gynandromorphs.
 Figure 21-19 Gyandromorphs
 These are flies that are literally half male
female. The X instability occurs only at the
first division.

 And the “line” separating the male and

female tissues is random. Its exact
position depends on the orientation of the
two daughter nuclei after the first cleavage.
The yeast FLP recombinase permits
the efficient production of genetic
mosaics
 Drosophila possesses several favorable
attributes for molecular studies and whole-
genome analysis. Most notably, the
genome is relatively small.

 The frequency of mitotic recombination

was greatly enhanced by the use of the
FLP recombinase from yeast.
 Figure 21-20 FLP-FRT
 This method is quite efficient. In fact, short
pulse of heat shock are often sufficient to
produce enough FLP recombinase to
produce large patches of zˉ/zˉ tissue in
different regions of an adult fly.
It is easy to create transgenic fruit
flies that carry foreign DNA

 P-elements are transposable DNA

segments that are the causal agent of a
genetic phenomenon called hybrid
dysgenesis.
 Figure 21-21 Hybrid dysgenesis
 P-element excision and insertion is limited
to the pole cells, the progenitors of the
gametes (sperm in males and eggs in
females).

 P-elements are used as transformation

vectors to introduce recombinant DNAs
into otherwise normal strains of flies.
 Figure 21-22 P-element transformation
 This method of P-element transformation
is routinely uses to identify regulatory
sequences such as those governing eve
stripe 2 expression.

 In addition, this strategy is used to

examine protein coding genes in various
genetic backgrounds.
The House Mouse, Mus musculus
 The mouse enjoys a special status due to its
exalted position on the evolutionary tree: it is a
mammal and, therefore, related to humans.

 The mouse provides the link between the basic

principles, discovered in simpler creatures like
worms and flies, and human disease.
 Mouse Embryonic Development
Depends on Stem Cells
 Their small size prohibits grafting experiments of
the sort done in zebrafish and frogs, but
microinjection methods have been developed for
introducing.

 Figure 21-23 shows an overview of mouse

embryogenesis.
 Figure 21-23 Overview of mouse embryogenesis
 It Is Easy to Introduce Foreign DNA
into the Mouse Embryo

 DNA is injected into the egg pronucleus, and

the embryos are places into the oviduct of a
female mouse and allowed to implant and
develop.
 The injected DNA integrates at random
positions in the genome
 Figure 21-24 Creation of transgenic mice by
microinjection of DNA into the egg pronucleus
 Figure 21-25 In situ expression patterns of embryos obtained from
transgenic mice
Homologous Recombination Permits
the Selective Ablation of Individual
 The single most Genes
powerful method of
mouse transgenesis is the ability to disrupt,
or “knock out，” single genetic loci. This
permits the creation of mouse models for
human disease.
 Gene disruption experiments are done
with embryonic stem (ES) cells
 Figure 21-26 Gene knockout via homologous
recombination
Mice Exhibit Epigenetic Inheritance
 Studies on manipulated mouse embryos led
to the discovery of a very peculiar
mechanism of non-Mendelian, or epigenetic,
inheritance.

 This phenomenon is known as parental

imprinting.
 Figure 21-27 Imprinting in the mouse
The basic idea is that only one of the two
alleles for certain genes is active.
 It has been suggested that imprinting has
evolved to protect the mother from her own
fetus.