Hint: remember that unused glucose is converted to and stored in ...

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14 Δεκ 2012 (πριν από 4 χρόνια και 4 μήνες)

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THE RAW MATERIALS OF
BIOTECHNOLOGY

Learning Outcomes


Identify the levels of biological organization and
explain their relationships


Describe cell structure and its significance in
biotechnology research and product development


Discuss the types of organisms researched and the
types of cells grown and studied in biotechnology
facilities plus the product with which they are
associated

Learning Outcomes


Distinguish between the cellular organization of
prokaryotic and eukaryotic cells


List the 4 main classes of macromolecules and
describe their structure and function


Define genetic engineering and identify products
created with this technology


Explain the Central
D
ogma of Biology and its
importance in genetic engineering

Vocabulary


Fluorometer


Organism


Cell


Escherichia coli


Multicellular


Cytology


Anatomy


Physiology


Respiration


Unicellular


Chlorophyll


Photosynthesis


Steroids


nucleotide


Tissue


Organ


Protein


Eukaryote


Protist


Organelles


Mitochondria


Sugar


Starch


Nucleic acid


Pancreas


Hormone


R group


ribonuicleic

acid


Vocabulary


Chloroplast


Cytoplasm


Lysosome



Ribosome


Cell wall


Cellulose


Plasma membrane


Glucose


ATP


Nucleus


Chromosomes


Enzyme


Cellular respiration


Deoxyribose


Hydrophobic


Triglycerides


Ribose


Phospholipids



Pigments


mRNA


Amino acid


Polypeptide


Chinese hamster ovary cells


Vero cells


Prokaryote


Organic


Carbohydrates


Cytoskeleton


Monomer


Polymer


Monosaccharide


Disaccharide


Polysacccharide


Fructose


Sucrose


Lactose


Hydrophillic


Organisms & Their Components


To manufacture biotechnology products for medical,
industrial, or agricultural applications,
biotechnicians

must work either directly or indirectly with
organisms or their components


Entire
multicellular

organisms


Bacteria


Tissues/cells

Organisms & Their
Cmponents


Working in any area of biotechnology requires a
thorough understanding of the characteristics of life
and the structures that compose organisms


You must have at least a minimal understanding of


Biochemistry: organic molecules structure, function, &
interactions


Cytology: branch of life science, which deals with the
study of cells in terms of structure, function and
chemistry


Anatomy: structure of living things and their components


Physiology: the processes and functions of living systems

The Living Condition


Biologists estimate that there are well in excess of
20million different species, with some estimates
being as high as 150 million


All living things can be classified into one of the
following categories

Prokaryotes

Eukaryotes

Eubacteria

Archaeabacteria


Protists

Plants

Animals

Fungi

The Living Condition


Biologists estimate that there are well in excess of
20million different species, with some estimates
being as high as 150 million


All living things can be classified into one of the
following categories

Prokaryotes

(Unicellular)

Eukaryotes

(mostly
multicellular
)

Eubacteria

Archaeabacteria


Protists


Plants__

Animals__

Fungi

Characteristics of Life


Growth & Development


Reproduction


Organized structure composed of 1 or more cells


Response to stimuli


Conversion of energy


Respiration


Metabolism


Catabolism


Anabolism


Here We Go Again! Cells & Their Parts


The cells of both unicellular and
multicellular

organisms
are tiny microscopic factories that produce thousands of
different molecules


Biotechnology companies exploit the
biologiical

manufacturing capabilities of cells and trick them into
producing particular molecules in large quantities


These become biotechnology products


Organelles: a specialized subunit within a cell that has
a specific function, and is usually separately enclosed
within its own lipid
bilayer


The name
organelle

comes from the idea that these
structures are to cells what an organ is to the body


Organelles involved in Energy
Production


A membrane bound organelle
consisting of an outer
membrane and a highly
folded inner
-
membrane; it is
responsible for generating
large amounts of ATP through
the process of aerobic cellular
respiration


Found in all eukaryotic cells


ATP: adenosine
triphosphate



a nucleotide that serves as
the main source of usable
energy for ALL living things


A specialized organelle
in plants and some
protists

that uses the
energy from sun light to
convert
inorgaic

molecules (CO
2
& H
2
O)
into energy storing
organic molecules
(glucose) through the
process of
photosynthesis

Mitochondria

Chloroplast

Nucleus: The Organelle that Stores the
Hereditary Material


a double lipid
bilayer

that encloses
the genetic material in eukaryotic
cells.


serves as the physical barrier,
separating the contents of the nucleus
from the
cytosol


nuclear pores are inserted in the
nuclear envelope, which facilitate
and regulate the exchange of
materials (proteins such as
transcription factors, and RNA)
between the nucleus and the
cytoplasm.


The outer membrane is continuous
with the rough endoplasmic reticulum


The outer and inner nuclear
membrane are fused at the site of
nuclear pore complexes.



DNA: a nucleic acid that contains
the genetic instructions used in the
development and functioning of
all known living organisms


main role is the long
-
term
storage of information


Used as a template to make RNA
through the
priocess

of
transcription


Nucleolus: a non
-
membrane
bound structure

composed of
proteins and nucleic acids found
within the nucleus.


Ribosomal RNA (
rRNA
) is
transcribed and assembled within
the nucleolus, forming
ribosomes


Nuclear Envelope

Nuclear Contents

Ribosomes
: involved in Protein Synthesis


Non
-
membrane bound organelles composed of
rRNA

& protein that provide the appropriate
microenvironment to catalyze peptide bond
formation between amino acids based on the
instructions it reads on an mRNA molecule through
the process of translation


Chemistry Connect: Remember, all organic polymers are
formed through condensation reactions, aka
dehydration synthesis

Organelles involved in Trafficking



A highly folded lipid
bilayer

whose surface
ois

studded with
protein
-
manufacturing
ribosomes

giving it a "rough" appearance


Ribosomes

bound to the RER at
any one time are not a stable
part of this organelle's structure
as
ribosomes

are constantly
being bound and released from
the membrane.


A ribosome only binds to the ER
once it begins to synthesize a
protein destined for the
secretory

pathway


The Golgi apparatus
processes and packages
macromolecules, such as
proteins and lipids, after
their synthesis and before
they make their way to
their destination; it is
particularly important in
the processing of proteins
for secretion.

Rough Endoplasmic Reticulum

Golgi Apparatus

Organelles involved in Metabolism


Functions in several metabolic
processes, including synthesis
of lipids and steroids,
metabolism of carbohydrates,
regulation of calcium
concentration, drug
detoxification, attachment of
receptors on cell membrane
proteins, and steroid
metabolism.


It is connected to the nuclear
envelope.


Lysosomes: contain acid enzymes
to break up waste materials and
cellular debris.


found in animal cells, while in
yeast and plants the same roles
are performed by lytic
vacuoles
.


digest excess or worn
-
out
organelles, food particles, and
engulfed viruses or bacteria


The membrane around a
lysosome allows the digestive
enzymes to work at the 4.5 pH
they require.

Smooth Endoplasmic Reticulum

Lysosome

Metabolism: is the set of chemical reactions that
happen in living organisms to maintain life


is the set of pathways
that break down
molecules into smaller
units


These reactions
release energy


the set of metabolic
pathways that
construct molecules
from smaller units.



These reactions require
energy

Catabolism

Anabolism

Critical Thinking: Which
organelles are catabolic and
which are anabolic?

The
Endomembrane

System


Is composed of the different membranes that are
suspended in the cytoplasm within a eukaryotic cell.


These membranes divide the cell into functional and
structural compartments, or organelles.


In eukaryotes the organelles of the
endomembrane

system include: the nuclear envelope, the
endoplasmic reticulum, the Golgi apparatus,
lysosomes
, vacuoles (plants), vesicles (all
eukaryotes), and the cell membrane



Cellular Membranes


Acts as a semi
-
permeable barrier that
regulates what enters or leaves the cell or
organelle


They consist of phospholipids which
spontaneously arrange so that the hydrophobic
"tail" regions are shielded from the surrounding
polar fluid, causing the more hydrophilic
"head" regions to associate with the
cytosolic

and extracellular faces of the resulting
bilayer
.

Things in/on Cellular Membrane


Proteins: function in cellular communication, catalyzing
reactions, cell, signaling, cell movement


4 main types


Receptor proteins bind signal molecules and initiate signal
transduction pathways


Enzymes: speed up chemical reactions


Channel proteins: open or close to let target molecules in or out in
response to a signal


Marker proteins: specific for each cell type; also, help the immune
system distinguish self from non
-
self


Cholesterol: establishes proper membrane permeability
& fluidity

Cell Walls


All living things except animals and some
protists

have
cell walls


Cell walls are
semipermeable

boundaries that are more
rigid than, and surround the plasma membrane



provides structural support and protection, and also acts as
a filtering mechanism


Keeps cells from bursting in hypotonic environments


Different organisms have cell walls made of different
organic polymers


Plants: cellulose


Bacteria:
peptidoglycan


Fungi (some): chitin

Cell

Tissue


Organ


Organism


Depending on the type of cell hundreds of different
molecules are being
manuifactured

at any given
moment


Some molecules are unique to a specific type of cell,
others are produced in all cells


Example: Pancreas

organ of the digestive system that
lies in your abdomen, behind your
stomach,with

2 main
functions:


producing digestive enzymes to break down food; and


producing the hormones insulin and glucagon to control
sugar levels in your body.



Case in Point: Pancreas

Cells

Liver cells are called
hepatocytes

α
-
cells: secrete glucagon

β
-
cells secrete insulin

Acinar

cells: secrete enzymes that
allow your body to digest protein, fat
and starch from your food

Tissue

Islets of
langerhans

Secrete hormones

Acinar

tissue

Secrete digestive enzymes

Organ

Pancreas

Organism

People

A closer look at the BIG Picture


When you, the organism, eats a meal, your stomach
(with the help of digestive enzymes from your
pancreas and other organs) break down the
complex carbohydrates into glucose


Glucose is absorbed through the intestines (small I
think) into the blood stream


Rising glucose in the blood stream is detected by
the pancreas as the glucose binds glucose receptors
on pancreatic cells. This stimulates
β
-
cells
to
release insulin

Insulin:
pancreatic homeostasis



Insulin works by improving the uptake of glucose from
the blood across cell membranes and into the cells of
the body by binding to insulin receptors on cells


Specifically, when insulin binds the insulin receptor, the
receptor protein transports phosphate groups from ATP
to other proteins in the cell, initiating signal transduction
pathways that open glucose channels allowing passive
diffusion of glucose into the cell


Once in the cells, the glucose is used as the energy to fuel
the cells doing their different jobs or is stored in the liver or
muscle cells as glycogen.


This results in the glucose level of the blood dropping, which
then triggers the pancreas to switch off the release of insulin

Critical Thinking


Insulin and glucagon are both hormones that work
together to regulate blood glucose levels. Knowing
how insulin works, and that glucagon's effect is
opposite that of insulin, propose a mechanism by
which glucagon may work


Hint: remember that unused glucose is converted to and
stored in the form of glycogen in the pancreas

Glycogenolysis


Glucagon helps maintain the level of glucose in the
blood.


Glucose is stored in the liver in the form of
glycogen, which is a starch
-
like polymer chain made
up of glucose molecules.


Liver cells (
hepatocytes
) have glucagon receptors.


When glucagon binds to the glucagon receptors,
the liver cells convert the glycogen polymer into
individual glucose molecules, and release them into
the bloodstream, in a process known as
glycogenolysis
.





Why all this matters


The purpose of biotechnology is to increase quality
and/ or quantity of life


Life is dependant on the bodies abilities to


Maintain homeostasis


Transform energy


Reproduce


Grow and develop


Respond to stimuli


Internal: for example a change in levels of blood sugar


External: for example a change in temperature

Biotechnology focuses on
making organisms better able
to accomplish one or more of
these tasks

Central Dogma


If the goal of biotechnology is to increase an
organisms ability to accomplish one or more of the
characteristics of life, then it is important to
understand how the organism naturally carries these
tasks out!



One of the most important aspects of understanding
this is to understand the central dogma of biology

Chromosomes & Genes


We know chromosomes are in the nucleus



We know genes are on chromosomes, and are
segments of DNA that code for protein



What we need to look at more specifically is HOW
the DNA instructs your body to build a protein

Central Dogma: an Overview


DNA is made up of 2 strands of nucleotides that coil
around each other forming a double helix


Each strand is composed of varying arrangements
of 4 nucleotides


Adenine


Cytosine

Thymine

Guanine


When genes are used to instruct the body to build a
protein, they must be
transcribed

into a
complementary sequence of
messenger RNA


The mRNA transcript is made of nucleotides


Adenine


Cytosine

Uracil


Guanine

Central Dogma: an Overview


This mRNA can leave the nucleus and be read by a
ribosome

which actually constructs the protein
through the process of
translation


In translation, the ribosome links amino acids
together in the order specified by the sequence of
nucleotides in the mRNA


These amino acids are held together by special
covalent bonds called
peptide bonds


There are
20 different amino a
cids


A chain of amino acids are called a
polypeptide



Nucleus

Ribosome in cytoplasm or on RER

Central Dogma


The process of Gene Expression is universally found
in all cells


How the DNA code is rewritten into mRNA and then
decoded into a protein


It is called “The Central Dogma of Biology” because
it helps explain how virtually all molecules are
made either


directly protein & nucleic acids


or indirectly carbohydrates and lipids because their
synthesis is controlled by the proteins

Tying it together


DNA is used to make protein


Proteins do most of the work of the cell


The other organic molecules are important too, but
because their production/ destruction is controlled by
proteins we focus on the protein


The cell has specialized organelles, containing
different collections of proteins, giving each
organelle a unique function based on the proteins
(and other organic molecules) in/on it


The organelles work together to help the cell carry
out all of the characteristics of life

Tying it together


Not all cells, even within the same individual, are
the same


Some cells become specialized to perform a
specific function


β
-
cells:
Secrete insulin


Rods: sense light


Cells are able to perform these specialized
functions BECAUSE of the proteins they produce


Different types of cells produce different
types/quantities of proteins at different times

Critical Thinking


If all cells within one organism have the same set of
DNA (in other words, the DNA in your nerve cells is
identical to the DNA in the epithelium of your little
toe), how is it that all cells are not the same?

Cells Commonly Used in Biotechnology


In biotech applications, some cells are used more than
others



Chinese Hamster Ovary (CHO)cells


Vero Cells: African green monkey kidney epithelial cells


HeLa

cells: human epithelial cells


Fungal cells



Aspergillus


yeast


Prokaryotic cells


E. coli


Staphylococcus


Streptococcus

In Other Words

You need to know how
to keep cells alive in
culture, in order to do
most biotechnology
research

NOTE


On page 46 of your text book it claims that



“Due to their lack of mitochondria, bacteria conduct
ONLY anaerobic respiration”


This is WRONG & the author is a MORON


There are SOME strictly anaerobic bacteria but
MOST need or at least can survive in oxygen
environments!

Read section 2.3 on your own!


Things to pay attention to


Carbohydrates


Glycogen: what is it


Structural polysaccharides of cell wall


How polysaccharides
interefere

with purification procedures


The text book reads “Cells break the bonds in glucose, releasing energy in a
form that cells can use” why do I hate that definition


Figure 2.23


lipids




why are lipids referred to as hydrocarbons


The 3 general groups of lipids and their functions


Triglycerides


Phospholipids


Steroids


Structure of a
phospholipid


“DNA is the Flash, Proteins are the Cash!”


Water makes up 75% of a cells mass



Of 25% of dry mass, 75% of it is protein



In biotechnology, proteins are often the
manufactured product



Often, 50
-
75% of a companies staff is devoted to
protein research (proteomics)

Proteins fall into 9 different categories
depending on their function


Structural
:
proteins with the primary purpose of
producing the essential structural components of the cell


Enzyme
:
speed up chemical reactions by lowering activation energy


Transport
:
involved in the movement of substances across
biological membranes; or carries specific molecules (example
oxygen is transported by hemoglobin)


Contractile
:
involved in muscle contraction, also in the functioning
of the mitotic spindle


Hormone
:

a chemical released by a cell or a gland in one part
of the body that sends out messages to affect cells in other parts of
the organism

Proteins fall into 9 different categories
depending on their function


Antibody
:
used by the immune system to identify and
neutralize foreign objects, such as bacteria and viruses


Pigment
:
a material that changes the color of reflected or
transmitted light as the result of wavelength
-
selective absorption


Marker/ Recognition
:
proteins usually found on the
extracellular membrane that allows other molecules to identify the
cell type


Toxins:

small molecules, peptides, or proteins that are capable of
causing disease on contact with or absorption by body tissues
interacting with biological macromolecules such as enzymes or
cellular receptors


Proteins


Grouping is based on functions, and often proteins
within the same group are more similar to each other
than to proteins in different groups


Note: some proteins can have different functions, even
within the same organism, depending on which cell they
are produced in


A typical cell produces about 2,000 different proteins


Proteins are the work horses of the cell, each able to
accomplish its task due to its specific structure and
properties

Proteins


The structure of a protein is determined by the amino
acid sequence


Chains of amino acids are called polypeptides


Polypeptides are not functional until they fold into their
3
-
dimensional shape


Folded polypeptide chains are called proteins


The way a polypeptide folds into a functional protein is
determined by the sequence of the amino acids and
their properties


The sequence of amino acids is determined by the
sequence of mRNA which is determined by the
sequence of DNA


There are 20
different amino
acids



All amino acids
have the same
basic structure


R
-
Groups


R
-

groups are the chemical side groups on an amino
acid that varies between different amino acids


It is the R
-
group that is responsible for the unique
characteristics of each amino acid


The chemical nature of each R
-
group results in
attractions and repulsions between certain amino acids


Example: the negatively charged R
-
group of
glutamic

acid
is attracted to the positively charged R
-
group of
arginine


The various folding patterns of each protein are a result of
these interactions

R
-
Groups


The R
-
groups of protein chains can interact between
proteins as well


Many proteins function by attracting or repelling
other protein chains


Many recognition proteins, antibodies, enzymes, and
protein hormones function because the R
-
group on
one of their amino acids is strategically placed
(active site) to interact with its target molecule
(
ligand
)

Nucleic Acids


Information carrying molecules that direct the
synthesis of all cellular molecules, including
themselves



Ultimately, each protein, carbohydrate, and lipid
molecules production can be traced back to genetic
information stored in the sequence of DNA, which is
packaged into chromatin or chromosomes,
depending on what stage of the cell cycle the cell is
in

Quick Comparison of Prokaryotic Chromosomes
to Eukaryotic Chromosomes

Chromosome located in the
cytoplasm in a region known
as the
nucleoid

1 chromosome (may have
additional plasmids)

Chromosome is circular

No introns, exons only

DNA is NOT associated with
histone

proteins



Chromosomes found in the
nucleus

# of chromosomes varies
with species

Chromosomes are linear

DNA contains both introns &
exons

DNA is associated with
histone

proteins

-
Genetic
information

-
Contains
A,C,T,G

-
Composed
of
nucleotides

Prokaryotes

Eukaryotes

Overview of Gene Expression


DNA is composed of long stretches of nucleotides
covalently linked together


Genetic information lies in the sequence of
nitrogenous bases


There are 5 nitrogenous bases


DNA: adenine, cytosine, guanine, thymine


RNA: adenine, cytosine, guanine,
uracil



Cytosine on 1 strand always pairs with guanine on the other
strand


Adenine pairs with either thymine (if you’re copying DNA) or
Uracil
, (if your transcribing RNA



DNA is the template to build RNA

A
A
T
C
C
G
T
C
T
A
T
G

T
T
A
G
G
C
A
G
A
T
A
C

REPLICATION

A
A
U
C
C
G
U
C
U
A
U
G

TRANSCRIPTION

TRANSLATION

The sequence of bases is read in groups
of 3


Each group of 3 bases is called a
codon

and specifies 1 amino acid

Asparagine

Proline

Serine

Methionine

Occurs on a ribosome in the
cytoplasm or attached to the
RER

Occurs in the nucleus

Peptide bonds are a type of covalent bond that hold
amino acids together

What type of reaction forms peptide bonds?

Genetic Code


This is how a molecule with only 4 nitrogenous bases
can code for over 25,000 different proteins!
Because RNA and so the gene is “read” in groups of
3


We can use this knowledge to perhaps not just treat
but actually fix genetic mistakes and or give
organisms new characteristics that make them more
fit, substantially improving the quality and quantity
of life


Genetic Engineering


Companies employ genetic engineers to isolate and
alter the DNA codes for a particular protein or
group of proteins



Sometimes the protein, like insulin, is the product
itself



The goal, then, is to produce it in sufficient quantities
to sell in the marketplace and make a profit

The “New” Biotechnology


Organisms & their products have been harvested
and improved for centuries


The most significant breakthrough in the
manipulation of plant and animal cells occurred
when scientists learned how to move pieces of DNA
within and between organisms


The key to this discovery was restriction enzymes
and DNA
ligase
, which allow us to cut and paste
DNA to make
rDNA

Recombinant DNA


rDNA

usually contains fragments from different
organisms


They are usually novel molecules not seen in
existence anywhere else, and thus are referred to
as being “engineered”


DNA fragments containing genes of interest can be
pasted into vectors and carried back into cells


Once in cells they are transcribed and translated
into protein molecules that the recipient cell has
never produced


This organism has been “genetically engineered”

The 1
st

Genetic Engineering Experiment


Occurred in 1973 when 3 scientists


Stanley Cohen (Stanford)


Herb Boyer (University of California)


Paul Berg (Stanford)


Excised (cut out) a segment of amphibian DNA from
the African Clawed Toad,
Xenopus
, and pasted it
into a small ring of bacterial DNA called a plasmid


The new recombinant plasmid (r
-
plasmid) was
placed in an
E. coli
cell which then transcribed the
DNA into ribosomal RNA

Human Insulin


The 1
st

genitically

engineered product to reach the
market place was human insulin


Scientists used similar methods as before to excise a
healthy human insulin gene using restriction enzymes,
paste it into a bacterial plasmid using DNA
ligase
, and
then transferred the plasmid to E. coli cells which then
transcribed the DNA into RNA, and then translated the
mRNA into the insulin protein


The cells were grown in large volumes, and then the
insulin was purified out of the cell culture


The FDA approved r
-
human insulin (
rhinsulin
)in 1982

Genentech


In 1976 Robert Swanson & Herb Boyer founded the 1
st

biotechnology company called Genentech in San
Francisco


Their 1
st

product was
rhInsulin


Genentech has grown into one of the largest
pharmaceutical companies in the world


It currently markets or is developing


t
-
PA: treatment of
heartattachs


Nutropin
: a human growth hormone


Rituxin
: an antibody that targets cancer
cellsin

B
-
cell non
-
Hodgkins

lymphoma


Pulmozyme
: for cystic fibrosis treatment