chapter 1 lecture note

7/29/2019 Chapter 1 Lecture Note

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Introduction of this Cell Biology course

1. Goals: convey the excitement and challenges of research in contemporary cell

biology

A cell is the basic unit of life. Understanding how cells grow, divide and

respond to environment is the major purpose of biology.

The number of applications of cell biology continues to grow in medicine,

agriculture, biotechnology, and biomedical engineering.

2. Organization of the textbook:

Part I: Introduction

Part II: The flow of genetic information

Part III: Cell structure and function

Part IV: Cell regulation

3. Companion website: www.sinauer.com/cooper5e/

Various information (animation and video clips), Homework

Have to register yourself to solve Online Quizzes (homework)

Online Quizzes Create a new account [email protected]


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Introduction of this Cell Biology course

4. Lecture materials: klms.kaist.ac.kr

5. Your performance and grading

Midterm exam (40%), Final exam (40%),

Other activities (participation/homework/attendance: 20%)

Participation: 6

Homework: 10

Attendance: 4


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Chapter 1. An Overview of Cells and Cell Research

The origin and evolution of cells

Cells and organisms as experimental models

Some of the properties of cells and organisms that make them valuable

experimental model

Tools of cell biology

Progress in cell biology depends on the availability of experimental tools.

Some of important experimental tools are discussed.

Chapter sections


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The Origin and Evolution of Cells

Two types of cells: Prokaryotic cells lack a nuclear envelope. Eukaryotic cells have a nucleus that separates genetic material from

cytoplasm.

The first cell (premordal ancestor) emerged at least 3.8 billion years ago.

Spontaneous synthesis of organic molecules probably provided the basic

materials from which the first living cells arose (Fig. 1.1).


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Macromolecules may have formed by spontaneous polymerization of

monomeric building blocks under plausible prebiotic conditions.

The critical characteristic of the macromolecule from which life evolved must

have been the ability to replicate itself.

Nucleic acids are capable of directing self-replication (Fig.1.2).

Sid Altman and Tom Cech first discovered that RNA is capable of catalyzing

chemical reactions, including the polymerization of nucleotides.

RNA can serve as a template for its own replication, and it is also able to

catalyze its own replication (= self-replicating RNA).

Consequently, RNA is generally believed to have been the initial genetic

system in evolution.

This period is known as the RNA world.


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The first cell probably arose by the enclosure of self-replicating RNA in a

membrane composed ofphospholipids.

Phospholipids are the basic components of all present-day biological

membranes.

Properties of phospholipids Amphipathic When placed in water, they spontaneously aggregate into a bilayer.

Forms a physical barrier against free influx of molecules from outside.


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Cells needed to evolve mechanisms for generating energy and synthesizing

molecules.

The principal pathways of energy generation are highly conserved in

present-day cells; and all cells use ATP as their source of metabolic energy.

The mechanisms of generation of ATP are thought to have evolved in three

stages, corresponding to the evolution of glycolysis, photosynthesis, and

oxidative metabolism (Fig. 1.4).


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Glycolysis evolved when the Earths atmosphere was anaerobic. No involvement of oxygen Breakdown of glucose to lactic acid All present-day cells carry out glycolysis.

Photosynthesis evolved more than 3 billion years ago. It allowed some cells to harness energy from sunlight; and they no longer required

preformed organic molecules. The first photosynthetic bacteria probably used H2S to convert CO2 to organic

molecules: a pathway of photosynthesis still used by some bacteria.

(Analysis of sedimentary rocks: completely anoxic, filamentous microbial tangles) The use of H2O evolved later (~1.5 billion years ago, as H2S diminished); it

changed Earths atmosphere by making free O2 available.

O2 in the atmosphere may have allowed the evolution of oxidative metabolism. It is much more efficient than glycolysis; the complete oxidative breakdown of glucose

yields 36 to 38 ATP molecules.


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Present-day prokaryotes

Archaebacteria: many live in extreme environments (e.g., hot sulfur spring).

Eubacteria: a large group that live in a wide range of environments.

Most bacterial cells are small.

Cyanobacteria, the group in which photosynthesis evolved, are the largest

and most complex prokaryotes (i.e., large number of genes)

Escherichia coli (E. coli) is a typical prokaryotic cell. It has a rigid cell wall composed of polysaccharides and

peptides (=peptidoglycan).

Beneath the cell wall is the plasma membrane, a

phospholipid bilayer with associated proteins.


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They are much larger and more complex, with a nucleus, other organelles,

and cystoskeleton.

*Organelles= subcellular organelles or compartment

The nucleus is the largest organelle; it contains the linear DNA molecules.

Mitochondria:site of oxidative metabolism.

Chloroplasts: site of photosynthesis.

Lysosomes and peroxisomes: specialized metabolic compartments for the

digestion of macromolecules and for various oxidative reactions.

Present-day eukaryotes


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Vacuoles: in plant cells; perform a variety of functions, including digestion

of macromolecules and storage of waste products and nutrients.

The endoplasmic reticulum is a network of intracellular membranes,

extending from the nuclear membrane throughout the cytoplasm.It functions in the processing and transport of proteins and the synthesis of

lipids.

In the Golgi apparatus, proteins are further processed and sorted for

transport to their final destinations.

It also serves as a site of lipid synthesis, and (in plant cells) the site ofsynthesis of some polysaccharides that compose the cell wall.

The cytoskeleton is a network of protein filaments extending throughout

the cytoplasm.

It provides structural framework, determines cell shape and organization,

and is involved in movement of whole cells, organelles, and chromosomesduring cell division.


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Acquisition of membrane-bound subcellular organelles was a critical step.

These are thought to have arisen by endosymbiosis: prokaryotic cells

living inside the ancestors of eukaryotes. Evidence is especially strong for mitochondria and chloroplasts.

Mitochondria and chloroplasts are similar to bacteria in size Like bacteria, they reproduce by dividing in two. Both contain their own DNA, which encodes some of their components.

The DNA is replicated when the organelle divides; the genes are transcribedwithin the organelle and translated on organelle ribosomes. The ribosomes and ribosomal RNAs are more closely related to those of bacteria

than to those encoded by the nuclear genomes of eukaryotes.

The origin of eukaryotes


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The first eukaryote is thought to derived from the fusion of aerobic

eubacterium with an archaebacterium rather than from one of the two. What

is a supporting evidences? the mosaic nature of eukaryotic genomes consisting of some genes derived

from eubacteria (mostly related to metabolism, e.g., glycolysis) and others from

archebacteria (mostly related to informational processes, e.g., DNA replication).

The DNA sequences of eubacteria

and archaebacteria are as differentas they are from those of present-day eukaryotes.

Mitochondria is contained in bothanimal and plant cells.

But, chloroplast is contained only inplant cells.

Therefore, a scenario would be that anarchaebacterium fused with anaerobic eubacterium first, and laterthe resulting cell fused withphotosynthetic bacterium.

Evolution of cells


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Many eukaryotes are unicellular organisms.

The simplest eukaryotes are the yeasts.

The development of multicelluar organisms

Other unicellular eukaryotes are more complex.

Amoeba proteus:its volume is more than 100,000 times that ofE. coli, and

it can exceed 1 mm in length.

Amoebas use cytoplasmic extensions, called pseudopodia, to move and

to engulf other organisms.

pseudopodia


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Some unicellular eukaryotes form aggregates (=muticellular colonies) that

may represent an evolutionary transition from single cells to multicellular

organisms.

Volvox (a green algae):

Thousands of cells are embedded

in a gelatinous matrix.

Individual cells are connected by

tiny cytoplasmic bridges.

Some division of labor; a small

number of cells are specialized in

reproduction.

Increasing cell specialization might have led to the transition from

aggregates to truly multicellular organisms.


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Cells of plants:

Organized into 3 main systems:

1. Ground tissue

(A) Parenchyma cells: site of metabolic reactions, including

photosynthesis.

(B) Collenchyma and sclerenchyma have thick cell walls and provide

structural support.

2. Dermal tissue covers the surface of the plant (=C); forms a protective coatand allows absorption of nutrients.

3. Vascular tissue (xylem and phloem) (=D):

xylem tissue- transport water mainly

phloem tissue- transport sucrose mainly

The both contains elongated cells.

Stomata


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Cells of animals:

Much more diverse than those of plants; e.g., 200 different kinds of cells in human

body. Components of 5 main types of tissues:

1. Epithelial tissue forms sheets that cover the surface of the body and lineinternal organs.

Functions: protection, nutrient absorption, secretion of molecules.

2. Connective tissueserve a connecting function- binds and support other

tissues. They includes bone, cartilage, and adipose tissue.

Loose connective tissue between organs and tissues is formed by fibroblasts.

4. Nervous tissue is composed of supporting cells and nerve cells, orneurons,

and various types of sensory cells; e.g., olfactory cells

5. Muscle tissue is responsible for the production of force and movement.

*All these complex array of cells differentiate from a single fertilized egg.

3. Blood tissues contains several different types of cells: Red blood cells (erythrocytes) function in oxygen transport. White blood cells (granulocytes, monocytes, macrophages,and

lymphocytes) function in inflammatory reactions and the immune

response.


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Representative animal cells

Epithelial cells

Fibroblasts

Blood cells


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Cells as Experimental Models

Because the fundamental properties of all cells have been conserved during

evolution, the basic principles learned from experiments on one type of cell

are generally applicable to other cells.

Several kinds of cells and organisms are used as experimental models.


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E. co li

The most thoroughly studied species of bacteria.

Our understanding of DNA replication, the genetic code, gene expression,

and protein synthesis derive from studies of this bacterium. E. coliis particularly useful because of its simplicity and ease of culture in

the laboratory.

The genome consists of approximately 4.6 million base pairs and contains

about 4300 genes. (human: 3 billion bps)

The small size of the genome is an advantage in genetic analysis. E. colidivide every 20 minutes. A clonal population can be readily isolated

as a colony grown on agar medium.

Bacterial colonies containing as many as 108

cells can develop overnight.

Selecting genetic variants of an E. colistrain is easy and rapid.

E. colican carry out biosynthetic reactions in simple defined media; thismade them extremely useful in elucidating biochemical pathways.


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Yeasts Yeasts are the simplest eukaryotes, and have been a model for

fundamental studies of eukaryote biology; e.g., RNA processing, protein

sorting and cell division.

The genome ofSaccharomyces cerevisiae consists of 12 million base pairsof DNA and contains about 6000 genes on 16 linear chromosomes.

Contains a nucleus, cytoskeleton, subcellular organelles.

Yeasts can easily be grown in the laboratory as colonies from a single cell.

Yeasts can be used for genetic manipulations similar to those performed

using bacteria.

The unity of molecular cell biology is made clear by the fact that general

principles of cell structure and function revealed by studies of yeasts apply

to all eukaryotic cells.


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Understanding the development of multicellular organisms requires

the experimental analysis of plants and animals.

The nematode C. elegansis one of the most widely used models.

Caenorhabdit is elegans

The genome ofC. elegans contains approximately 19,000 genes: nearly the

same number of genes as in humans.

C. elegans is relatively simple: adult worms consist of only 959 somatic cells.

The embryonic origin and lineage of all the cells has been traced.

Genetic studies have also identified many mutations responsible for

developmental abnormalities.

This led to isolation and characterization of genes that control development

and differentiation.

found that homologous genes in complex animals have similar functions


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The fruit fly Drosophila melanogasterhas been a crucial model

organism in developmental biology.

Drosophila is easy to grow in the laboratory, and the short

reproductive cycle (2 weeks) makes it very useful for genetic

experiments.

Many fundamental genetic concepts were derived from studies

ofDrosophila early in the 20th century.

Drosophi la melanogaster

Studies ofDrosophila have led to advances in understanding the molecular

mechanisms that govern animal development, particularly with respect to

formation of the body plan of complex multicellular organisms.

found that homologous genes and similar mechanisms exist in

vertebrates


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A model for plant molecular biology and development is the small

mouse-ear cress,Arabidopsis thaliana.

It has a small genome and is easily grown in the lab. Studies ofArabidopsis have led to the identification of genes

involved in aspects of plant development, such as the

development of flowers.

Arabido psis thal iana


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Vertebrates are the most complex animals, and the most difficult to study

due to the huge genome size and long reproductive cycle.

One approach is to use isolated cells in culture. These studies haveelucidated the mechanisms of DNA replication, gene expression, protein

synthesis, and cell division.

Vertebrates

The ability to culture cells in chemically defined media has allowed studies

of signaling mechanisms that normally control cell growth and differentiation

within the intact organism.

Highly differentiated cells are important models for studying particular

aspects of cell biology.

e.g.,

Muscle cells: a model for studying cell movement at the molecular level.

Giant neurons in squid: a model studying ion transport across the plasma

membrane and the transport of subcellular organelles


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The frogXenopus laevisis an important model for

studies of early vertebrate development.

Xenopus produces large eggs in large numbers,

facilitating laboratory study and biochemical analysis.

Zebrafish are small and reproduce rapidly.

Embryos develop outside of the mother and are transparent; early stages of

development can be easily observed. Zebrafish bridge the gap between humans and simpler invertebrate systems,

like C. elegans and Drosophila.

embryo


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The mouse is the most common mammal model.

Many mutations affecting mouse development or behavior have been

identified.

Genetically engineered mice with specific mutant genes are now used to study

the functions of these genes in the context of the whole animal.

The mouse and human genes are very similar with each other.

Not surprising that mutations in homologous genes result in similar

developmental defects in both species.

f C


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Research in cell biology depends on available laboratory methods and

experimental tools.

Many important advances have directly followed the development of new

methods that have opened novel avenues of investigation.

Tools of Cell Biology

The discovery of cells arose from the development of the light

microscope. Robert Hooke coined the term cell following his observations

of a piece of cork in 1665.

In the 1670s Antony van Leeuwenhoek was able to observe a

variety of cells, including sperm, red blood cells, and bacteria.

The cell theory proposed by Matthias Schleiden and Theodor Schwann in

1838 resulted from their studies of plant and animal cells using microscopes.

It was soon recognized that cells are not formed de novo but arise only from

division of pre-existing cells.

Light Microscopy


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Contemporary light microscopes can magnify objects up to about 1000x.

Most cells are between 1100 m, so they can be observed by light

microscopy, as can some organelles.

Resolution: the ability to distinguish objects separated by small distances;

is even more important than magnification.

The limit of resolution of the light microscope is approximately 0.2 m.

Objects separated by less than that distance appear as one object.

This limit is determined by the wavelength of visible light (), and the

numerical aperture (NA): the light-gathering power of the lens.

is fixed at approximately 0.5 mm.

NA can be envisioned as the size of the cone of light that enters the lens.

NA

0.61Resolution

sinNA

= refractive index of the medium between the objective lens and specimen.

=

vin a vacuum

vin a medium


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Numerical aperture ( sin):

= ~1.0 for air, but can be increased to a maximum of 1.4 by using an oil-

immersion lens.

Maximum foris 90, at which sin = 1, so maximum possible for NA = 1.4.

The theoretical limit of resolution is thus:

m22.04.1

5.061.0Re m

solution

Larger or :

Object lens is more closer to specimen


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Light passes directly through the cell.

Cells are often preserved with fixatives (e.g., formaldehyde) and stained withdyes to enhance the contrast.

This technique cant be used to study living cells.

Types of light microscopy

(a) formaldehye-Asn adduct

(b) cross-linking of Lys and Asn by formaldehyde Fixed and stained kidney tumor

Bright-field microscopy:


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Both convert variations in density or thickness to differences in contrast that

can be seen in the final image.

(by modification of optics, e.g., different angles of incident light)

(A) Bright-field (B) Phase-contrast (C) differential interference-contrast

Modification of optics + computer-assisted image analysis and processing. It

allows visualization of protein filaments with a diameter of only 0.025 m.

Single microtuble can be observed

Phase-contrast microscopy and differential interference-contrast microscopy:

Video-enhanced differential interference-contrast microscopy:

Shadow at this side

Fl i


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Used for molecular analysis (e.g., location of a protein).

A fluorescent dye is used to label the molecule of interest (called staining) in

fixed or living cells.

The fluorescent dye molecules absorb light at one wavelength and emit lightat a different wavelength.

This fluorescence is detected by illuminating the specimen with a

wavelength of light that excites the fluorescent dye, then using filters to

detect the specific wavelength of light that the dye emits.

Fluorescence microscopy:


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The green fluorescent protein (GFP) of jellyfish: widely used to visualize

proteins in living cells, by fusing it to a protein of interest. (no need for staining)

Fluorescence recovery after photobleaching (FRAP):

This is used to study rate of protein movement in living cells.

Methods using GFP:

Fluorescence resonance energy transfer (FRET):

This is used to study interactions between proteins in a cell.

The light emitted by one GFP variant excites the second.

High-intensity light

destroying the

chromophore


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Increases contrast and detail by analyzing fluorescence from a single point.

A small point of light from a laser is focused on the specimen at a particular

depth. The emitted fluorescent light is collected by detector such as a videocamera.

Confocal microscopy:

The emitted light must pass through a pin-

hole aperture (confocal aperture). Thus

only light emitted from the plane of focus is

able to reach the detector. Scanning across the specimen generates

a two-dimensional image of the plane of

focus.

A series of images can be used to

reconstruct a three-dimensional image.

*In fluorescence microscopy, out-of-focus

emitted lights give a blurred image.


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Excitation of a fluorescent dye is achieved by 2 or more photons.

Therefore, excitation occurs only at the point in the specimen where the

laser beams are focused.

No need for passing the emitted light through a pinhole aperture. The localization of excitation minimizes damage to the specimen, allowing

three-dimensional imaging of living cells.

Multi-photon excitation microscopy:

Fluorophore can absorb multiple low-

energy photons simultaneously and be

excited. The total energy equals its one-

photon excitation energy.

El t i


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Transmission electron microscopy Specimens are fixed and stained with salts of heavy metals, which provide contrast

by scattering electrons. A beam of electrons is passed through the specimen and forms an image on a

fluorescent screen.

Electron beams deflected by heavy metal

ions do not contribute to the final image,so that the stained area appears dark.

Specimens can be prepared by either

positive or negative staining.

Electron microscopy can achieve much greater resolution (0.2 nm) than

light microscopy because of the short wavelength of electrons (0.004 nm;

105

times shorter than the visible light).

The aperture angle of the electromagnetic lens is ~0.5

o

. The max. resolutionis about 0.2 nm (~0.61x0.004/1xsin0.5).

Resolution for biological samples is about 1 to 2 nm because of their

inherent lack of contrast. But, it is still 100x better resolution than light

microscopy.

Electron microscopy:

Negative staining of actin filament


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Electron tomography generates three-dimensional images by computer analysis

of multiple two-dimensional images obtained over a range of viewing directions.

Metal shadowing is another technique used to visualize the surface of

subcellular structures or macromolecules.

The specimen is sprayed with evaporated metal, such as platinum.

Surfaces facing the evaporated metal are coated more heavily than other

surfaces, which results in a shadowing effect.

Actin/myosine filaments


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Freeze fracturing:specimens are frozen in liquid nitrogen and then

fractured with a knife blade. This often splits the lipid bilayer, revealing the interior faces of a cell membrane. The specimen is then shadowed with platinum.

Membranes of two

adjacent cells; membrane

proteins are observed as

particles


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Scanning electron microscopy provides a three-dimensional image of cells. The surface of the cell is coated with a heavy metal, and a beam of electrons is used to scan

across the specimen.

The electron beam does not pass through the specimen.

The electrons that are scattered from the sample surface are collected to generate a3D image.

The resolution of scanning is ~10 nm, it is restricted to studying whole cell

rather than subcellular organelles or macromolecules.


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In order to determine the function of organelles, they were isolated from the

cell.

Differential centrifugation was developed in the 1940s and 1950s to

separate cell components on the basis of size and density.

Subcellular fractionation

ER at 200,000g

ribosomes at > 200,000gBreaks the plasma membranes and ER

into small fragments without breaking

up other cell compartments

Larger and more dense

ones at lower speed


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Greater purification can be achieved by density-gradient, in which organelles

are separated by sedimentation through a gradient of a dense substance,

such as sucrose. In velocity centrifugation in density-gradient, the starting material is layered on top

of the sucrose gradient. Particles of different sizes sediment through the gradient atdifferent rates.

Sample at the top

Sedimentation velocity


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Equilibrium centrifugation in density gradient is used to separate

subcellular components on the basis of their buoyant density. Sample is mixed together with sucrose or cesium chloride in a centrifuge tube;

centrifugation forms a concentration gradient of the solutes.

The sample particles are centrifuged until they reach an equilibrium position atwhich their buoyant density is equal to that of the surrounding sucrose or cesium

chloride solution. Example: separation of

14N or

15N labeled DNA molecules

Centrifugal force

G th f i l ll i lt


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In vitro culture systems of plant and animal cells enable scientists to study

cell growth and differentiation, and perform genetic manipulations.

Most animal cell types attach and grow on the plastic surface of dishes used

for cell culture.

Growth of animal cells in culture

The culture media for animal cells are complex and must include salts and

glucose, and various amino acids and vitamins that cells cant make for

themselves.

Serum provides polypeptide growth factors. The identification of individualgrowth factors makes it possible to culture cells in serum-free media.

Harry Eagle was the first researcher to describe a defined medium for


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Harry Eagle was the first researcher to describe a defined medium for

animal cells, in 1955.

This has enabled scientists to grow a wide variety of cells under defined

experimental conditions, which is critical to studies of animal cell growth and

differentiation.


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An initial cell culture from tissue is a primary

culture.

They can be replated at a lower density to form

secondary cultures many times.

Most normal cells such as fibroblasts cannot bereplated and grown indefinitely. They stop

growing and die.

Embryonic stem cells and tumor cells can proliferate

indefinitely in culture and are referred to as permanent orimmortal cell lines.

Permanent cell lines have been particularly useful for many

types of experiments because they provide a continuous and

uniform source of cells.

*Doubling time of most actively growing animal cells is ~20 hrs. That of E. coli

is 20 min.


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Plant cells can also be cultured.

In contrast to polypeptide growth factors of

animal cells, the growth factors of plant cells are

small molecules. Given appropriate growth factors, plant cells

produce a mass of undifferentiated cells called a

callus.

Many plant cells are capable of differentiation into

many different cell types. (Pluripotency)

Sometimes an entire plant can be propagated from a single cell.

This makes it easy to introduce genetic alterations, opening possibilities for

agricultural genetic engineering.

Plant cells:


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Viruses reproduce by infecting host cells and usurping the cellular machinery

to produce more virus particles.

Viruses consist of DNA or RNA surrounded by a protein coat.

Viruses:

Viruses provide simple systems that can be used to investigate the functions

of cells.

Bacterial viruses (bacteriophages) have simplified the study of bacterial

genetics.

Bacteriophage T4 infects E. coli.

In a culture of bacteria on agar, the replication of T4

leads to the formation of clear areas of lysed cells

(plaques).

Viral mutants (e.g. that will grow in one strain ofE. coli

but not another) are easy to isolate. Thus, T4 is

manipulated even more readily than E. colifor studies

of molecular genetics.


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The genome of T4 is 23 times smaller than that ofE. coli, further facilitating

genetic analysis.

Bacterial viruses have provided extremely useful experimental systems for

molecular genetics and have led to understanding many fundamental

principles.

Viruses are also important in studies of animal cells.

There are many diverse animal viruses.

The retroviruses have RNA genomes but synthesize a DNA copy of their

genome in infected cells. These viruses first demonstrated the synthesis ofDNA from RNA templates.

Some animal viruses convert normal cells to cancer cells.

This was first described by Peyton Rous in 1911.

Studies of these viruses have contributed to our current understanding ofcancer, and many of the molecular mechanisms that control animal cell

growth and differentiation.

chapter 1 lecture note

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