Studying DNA - Chapter 4 Notes

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Dec 12, 2012 (4 years and 11 months ago)

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Giselle Cruz

Period 1

1
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20
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Chapter 4: Studying DNA

1.

4.1
-

DNA Structure and Function

Introduction
:



Industry in which they began to study DNA was
biotechnology



DNA was isolated, studied, and the DNA and RNA were manipulated.



The sequence of nitrogenous bases are based on the sugar

code which would
signify which amino acid is placed on the polypeptide strand



Proteins do the work of cells and they give cells and organisms their unique
characteristics



There are about 40,000 genes in humans. A typical cell synthesizes about 2000
diffe
rent kinds of proteins



The entire sum of DNA is the genome.

o

Gene
-

a section of DNA on a chromosome that contains the genetic
code of a protein

o

Nitrogenous base
-

an important component of nucleic acids (DNA &
RNA), composed
of one or two nitrogen containing

rings

o

Chromatin
-

nuclear DNA and proteins



Figure 4.1
-

Pure salmon DNA isolated from sperm cells



Figure 4.2
-

The Central Dogma of Biology



Table 4.1
-

Sizes of the genomes from some model organisms

A.

Similarities in DNA Molecules Among Organisms

o

DNA molecul
es composed of four nucleotide monomers that contain four
nitrogenous bases



Adenosine deoxynucleotide (A)



Guanosine deoxynucleotide (G)



Cytosine deoxynucleotide (C)



Thymine deoxynucleotide (T)

o

A
ll DNA molecules form a double helix of repeating nucleotides.

o

Nucleotides connect to other in the strand thro
ugh strong phosphodiester
bonds

o

Base pairing in DNA only occurs between adenine and thymine
molecules, or between guanine and cytosine molecules

o

The amount of adenine’s equal the amount of thymine’s, like the amount
of guanine’s equal the amount of cytosine’s in the DNA strand

o

Adenine and guanine are purines (PuAG), and thymine and cytosine are
Pyrimidines

o

The antiparrallel arrangement gives “dir
ectionality” to a DNA strand, so
that the DNA strand is “read” in one direction only

o

The nitrogenous bases are stacked 0.34 nm apart with 10 nitrogen bases
per complete turn of the helix. This ensures that the shape of the DNA is
constant throughout its l
ength

o

During semiconservative replication a strand unzips, and the individual
strands act as templates to build a new side, so that by the time the
replication is complete, two identical DNA strands have been produced.



Base pair
-

two nitrogenous bases th
at are connected by a
hydrogen bond



Phosphodiester bond
-

a bond that is responsible for the
polymerization of nucleic acids by linking sugars and
phosphates of adjacent nucleotides



Hydrogen bond
-

a type of weak bond that involves the
“sandwiching” of a hyd
rogen atom between two fluorine,
nitrogen or oxygen atoms; especially important in the structure
of nucleic acids and proteins



Pyrimidine
-

a nitrogenous base composed of a single carbon
ring; a component of DNA nucleotides

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Purine
-

a nitrogenous base compo
sed of a double carbon ring;
a component of DNA nucleotides



Antiparrallel
-

a reference to the observation that strands on
DNA double helix have their nucleotides oriented in the
opposite direction to one another



Semiconservative replication
-

a form of rep
lication in which
each original strand of DNA acts as a template
, or model, for
building a new side; in this model one of each new copy goes
into a newly forming daughter cell during cell division

1.

Figure 4.3
-

shows the nitrogenous bases in DNA

2.

Figure 4.4
-

shows the DNA strand with the nitrogen
bases, the 5 carbon sugar and the phosphate groups

B.

Variations in DNA Molecules

o

Almost all DNA are very similar from one organism to another, but there
are some variations such as: the number of strands in the
cells of an
organism, the length of the base pairs, the number and type of genes,
the shape of the DNA strands (circular or linear chromosomes).

o

Figure 4.5
-

shows a circular DNA visualized by TEM using
cytochrome C and metal shadowing techniques

o

Figure 4.
6
-

shows DNA replication by the semiconservative
manner. It shows the parent strand and the daughter strand.

Review Questions:

1.

The relationship between genes, mRNA and proteins is
that genes are transcribed into mRNA
molecules, which
are then translated into the proteins of a cell.

2.

The four nitrogen
-
containing bases found in DNA
molecules are adenine, thymine, cytosine, and guanine.
Adenine creates a pair with thymine, and cytosine creates
a pair with guanine.

3.

Antipa
rrallel means that the nucleotides of the DNA are
oriented in the opposite direction to one another.

4.

The original DNA strand is used as a template or model
for the new strand, and they build a new side.

2.

4.2
-

Sources of DNA

Introduction
:
DNA is made in

cells. Sources of DNA can be found in nature or can be grown
in cultures in labs. Cells in cultures can be
collected and broken open by the process of
lysis. Through separation techniques, the DNA can be isolated from the other molecules in
a cell.



Mediu
m
-

a suspension or gel that provides the nutrients (salts, sugars, growth
factors, etc) and the environment needed for cells to survive plural in media



Lysis
-

the breakdown or rupture of cells

A.

Prokaryotic DNA



E. coli

is a prokaryotic bacteria cell, meaning that it does not have a nucleus or
other membrane bound organelles.
E. coli

is a single chromosome containing
approximately



In bacteria, the DNA floats in the cytoplasm. The DNA is normally one long
circular molecu
le (a chromosome). The chromosome is small and contains only
several thousand genes



Some bacteria contain
extra small rings of DNA floating in the cytoplasm, they
are called plasmids. (plasmids contain only a few genes generally 5 to 10 and
they usually c
ode for proteins that offer some other characteristics that may be
needed only for certain situations or conditions.



R plasmids contain antibiotic resistance genes, that can survive exposure to
antibiotics that would have killed them otherwise



Bacteria ca
n transfer plasmids and genetic information. Genes that can be
antibiotic resistance can be transferred between bacteria and lead to deadly
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antibiotic resistant forms of disease
-
causing bacteria. Bacteria can “evolve” by
gaining new characteristics for sur
vival



Genes of interest can be transferr
ed into cells. When they take up the foreign
DNA and start expressing the genes, then they are considered to have being
transformed



Foreign DNA fragments (genes) can be cut and pasted into a plasmid vector.
The cel
l reads the DNA code on the recombinant plasmid and start synthesizing
the proteins coded for on the “foreign” gene(s), which is how
E. coli

was
first
manipulated to make human insulin by Genentech. The
E. coli

cells were tricked
to read human genes that h
ad been inserted into them on recombinant plasmids



The middle of the operon is the section that actually codes for one or more
mRNA molecules, which will later be translated into one or more proteins. For a
gene expression to be expressed in prokaryotes,
the enzyme that synthesizes
and mRNA must attach to a section of DNA at a promoter region of the operon



The RNA polymerase then works its way down the DNA strand to a structural
gene, where it builds and mRNA molecule from free
-
floating nucleotides, using
the
DNA strand as a template. It is then decoded into a peptide at a ribosome



If a regulatory molecule attaché at the operator, the operon is turned off because
the RNA polymerase is blocked from continuing down the strand to the gene(s).
When this happen
s, no proteins are produced.



Genetic engineers utilize the promoter and operator regions to turn on and off the
production of certain genes.

o

R
-
plasmids
-

a type of plasmid that contains a gene for antibiotic
resistance

o

Transformed
-

refers to those cells that have taken
up foreign DNA and
have started expressing the genes on the newly acquired DNA

o

Vector
-

a piece of DNA that carried one or more games into a cell,
usually circular as in plasmid vectors

o

Operon
-

a section of prokaryo
tic DNA consisting of one or more genes
and their controlling elements

o

RNA polymerase
-

an enzyme that catalyzes the synthesis of
complementary RNA strands from a given DNA strand

o

Promoter
-

the region at the beginning of a gene where RNA polymerase
binds;

the promoter “promotes” the recruitment of RNA polymerase and
other factors required for transcription

o

Operator
-

a region on an operon that can either turn on or off expression
of a set of genes depending on the binding
of a regulatory molecule

o

Beta
-
gal
actosidae
-

an enzyme that catalyzes the conversion of lactose
into monosaccharide’s

o

Figure 4.7
-

shows the prokaryotic cell of
E.coli

o

Figure 4.8
-

shows the structure of a bacterium showing the flagella,
cytoplasm, cell wall, chromosome, and plasmid.

o

Figu
re 4.9
-

shows beige and blue
-
black colonies of bacteria transformed

o

Figure 4.10
-

shows an operon containing the controlling elements that
turn genetic expressions On and Off

B.

Bacterial Cell Culture



For bacteria to be grown in laboratories, scientists need

to make the environment
so that the bacteria like it.



Agar mediums are prepared by: a mix powdered agar in water and then heated
until it is completely suspended. Then sterilized at a high temperature (121 C or
higher) and a high pressure of 15 pound per

square inch for a minimum of 15
minutes.

Then it is allowed to cool in a temperature of 65 C and then poured
(under sterile conditions) into sterile Petri dishes and cool and solidifies in about
15 to 20 minutes.

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Broth cultures reproduce quickly since th
ey have better access to nutrients than
do colonies growing on the surface of solid media; oxygen and food
diffuse

into
these cells easily



The most important part of all media
preparation

is sterilizing the medium



Sterile technique is
the process of doing

something without contamination by
unwanted microorganisms or their spores. (used in surgery, doctors “scrub up” to
get rid of “germs” before they enter the operating room)



Technicians need a sterilized cell culture because a single unwanted cell could
r
uin an entire experiment or a multimillion dollar production run

o

Therefore, they must use a laminar flow hood, disinfectants, sterile tools,
sterile media and pay attention to detail



Under ideal conditions, cells will reproduce every 20 minutes, and cultur
es will be
seen within a day. The methods to check the concentration of the culture are by
using a spectrophotometer or simply by counting the cells under a microscope.

o

Broth
-

liquid media used for growing cells

o

Agar
-

acid media used for growing bacteria,

fungi, plant, or other cells

o

Media preparation
-

the process of combining and sterilizing ingredients
(salts, sugars, growth factors, pH indicators, etc) of a particular medium

o

Autoclave
-

an instrument that creates high temperature and high
pressure to sterilize equipment and media

o

Figure 4.11
-

shows the Lac Operon (lactose)

o

Figure 4.12
-

shows equipment being sterilized in the autoclave

o

Figure 4.13
-

shows a scientist working under the
hood, in a sterilized
environment

C.

Eukaryotic DNA



Eukaryotic DNA uses the same nucleotides as prokaryotic DNA. They have the
same double helix structure with antiparrallel strand bound to the others by
hydrogen bonds



Eukaryotes generally have several chrom
osomes per cell, and each one is a
single, linear, very long molecule of DNA (one can contain several million or
more nucleotides and up to many thousands of genes)



Prokaryotes only have one chromosome per cell, while eukaryotes can contain
multiple number
s of chromosomes



The total amount of DNA per cell is not directly related to an organism’s
complexity



Much of eukaryotic DNA is noncoding meaning that it does not transcribe into
protein



Eukaryotes have a lack of operators in their DNA, meaning that gen
e
expression is controlled differently; they are usually expressed at a very low
level. Increases occur when enhancer molecules interact with the RNA
polymerase or with other enhancer DNA regions where molecules attach to the
DNA and increase gene transcri
ption



At the structural DNA, the RNA polymerase enzyme builds a complementary
mRNA transcript from one side of the DNA strand; the enzyme transcribes the
entire gene until it reaches a terminal sequence



In eukaryotes, mRNA is often modified before transla
tion



Transcription factors turn genes “on”; these molecules either activate or
repress gene products, such as mRNA



Chromosomes in higher organisms are highly coiled around structural proteins
called histones. When genes are buried this way, RNA polymerase

cannot get
them to transcribe them into mRNA (so it has been essentially turned “off”)

o

Enhancer
-

a section of DNA that increases the expression of a gene

o

Intron
-

the region on a gene that is transcribed into an mRNA
molecule but not expressed in a protein


o

Exon
-

the region of a gene that directly codes for a protein’ it is the
region of the gene that is expressed

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o

Transcription factors
-

molecules that work to either turn on or off the
transcription eukaryotic genes

o

Histones
-

nuclear proteins that bind to th
e chromosomal DNA and
condense it into highly packed coils

o

Figure 4.14
-

bacteria is checked under high
-
powered light microscopes

o

Figure 4.15
-

the
Arabidopsis
plant which contains 10 chromosomes in
each of their cells

o

Figure 4.16
-

shows a chromosome of a m
ale in a human genome

o

Figure 4.17
-

shows the eukaryotic gene that has a promoter but not an
operator region

D.

Mammalian Cells



Mammalian cells are more challenging to grow because they normally grow
within multicellular organisms; they
depend

on other cells
f
or products and
stimuli. So a biotechnologist growing mammalian cells, must provide it with a
substitute but right environment



Typically grown in broth culture, in production facilities, they are grown in
suspension broth cultures in fermenters



They have specific nutrients and special indicators

o

Figure 4.18
-

shows a coiled DNA strand

o

Figure 4.19
-

mammalian cells are being viewed under a light
microscope

o

Figure 4.20
-

a lab technician is checking the mammalian cells growing
in broth cultures in
a carbon dioxide incubator

E.

Viral DNA



Viruses infect organisms and are often the target of biotechnology therapies



Used as vectors to carry DNA between cells



Do not have cellular structure; collections of protein and nucleic acid
molecules that become
active once they are within a cell. Are tiny, measure
from 25 to 250 nm



Viruses are either bacterial, plant, or animal



Viruses are classified further based on the specific type of cell infected and on
other characteristics, such as genetic material and sha
pe; they all have a thick
protein coat surrounding a nucleic acid core of either DNA or RNA



Lysogenic viruses incorporate their DNA into the host chromosome
while lytic
viruses do not



Viral vaccine molecules recognize specific viral surface proteins and t
arget
them for attack; protease inhibitors destroy proteases made by viruses in their
attempts to take over host cells.



Viral DNA or RNA molecules are short so easy to manipulate, since they do
not create as many proteins as cells do. Viral DNA is sometim
es used as a
vector because they can open to insert genes of interest.



Some companies are exploring the use of gene therapy to treat diabetes by
replacing defective insulin genes in the pancreas



Gene therapy is possible treatment for cystic fibrosis and o
ther genetic
disorders

o

Nonpathogenic
-

not known to cause disease

o

Bacteriophages
-

viruses that infect bacteria

o

Gene therapy
-

the process of treating a disease or disorder by
replacing dysfunctional gene with a functional one

o

Figure 4.21
-

shows a tongue
of a person who has the herpes infection

o

Table 4.2
-

shows examples of viruses and their characteristics

o

Figure 4.22
-

shows
Coronaviruses

that have a crown like appearance

o

Figure 4.23
-

shows a scientist working in the lab, from France working
with mice

Review Questions:

Giselle Cruz

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20
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11

1.

Plasmids differ from chromosomal DNA molecules because plasmids contain only
a few genes, 5 to 10 and they usually code for proteins that offer some additional
characteristics that may be needed only under extreme conditions while
chromosomal DNA molecules

may contain many thousand genes.

2.

An operon is a section of prokaryotic DNA consisting of one or more genes and
their controlling elements, which has a promoter site where the RNA polymerase
binds to make structural DNA, and this is all turned “on” or “of
f” by the operator.

3.

The human genome is made up of the four nucleotides in DNA, adenine, thymine,
guanine and cytosine, has 46 chromosomes in 23 pairs and each chromosome
holds genes for the human.

4.

Gene therapy is the process of treating a disease or dis
order by replacing
dysfunctional gene with a functional one. For example, scientists are using gene
therapy to produce insulin for people who have diabetes to insert into the pancreas.

3.

4.3
-

Isolating and Manipulating DNA

Introduction
: genetic engineering is a term used because the genetic code has been
manipulated to
the interest. Altering a genetic code can lead to the altering of protein
production. The process of genetic engineering is: identify the molecule, isolate the
desired in
terest, manipulate the DNA, and harvest the molecule or product by testing it
and marketing it to the public.

A.

Recombinant DNA Technology
(rDNA)



Creates new DNA molecules by piecing together different DNA molecules.
When a cell accepts the rDNA and begins
expressing it, then it is considered
to be “genetically engineered”.



T
-
PA
-

(tissue plasminogen activator) naturally occurring protein that
decreases the tome it takes to dissolve blood clots

o

Figure 4.24
-

shows a blood clot on blocking a blood vessel

o

Fig
ure 4.25
-

shows a lab technician monitoring the cell growth in a
broth culture

B.

Site
-
Specific Mutagenesis



The process of inducing changes in certain sections of a particular DNA
code. (usually accomplished through the use of chemicals, radiation or
viruse
s)



Bacteria, fungi, plat, or cell cultures may be treated with one or more
mutagen



Exposure of a cell culture to different amount of light, can increase cellular
growth, new pigment productions, and can cause cell DEATH.



To improve the activity of subti
lisin, fungi were treated with chemicals that
cause change



A change in DNA code led to a change in protein structure such that the
protein

would work more effectively in the alkaline (high pH) detergent
solution



Scientists screen large libraries of mutate
d fungi and bacteria while looking
for new or improved ways to produce a product

o

Bioremediation
-

the use of bacteria or other organisms to restore
environmental conditions

o

Site
-
specific mutagenesis
-

a technique that involves changing the
genetic code of
an organism (mutagenesis) in certain sections (site
-
specific

o

Figure 4.26
-

shows Tide ® laundry detergent that contains a
recombinant protease that helps remove gravy, blood and other
protein based stains from clothing

C.

Gene therapy



The process of correcting faulty DNA codes that cause genetic disease and
disorders



The most common way to conduct gene therapy is to use a virus to carry a
normal gene into cells containing defective genes. It is a replacement gene

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Companies are working
on correcting genes that are responsible for
Parkinson’s disease, diabetes, cystic fibrosis (CF), and some cancers



Gene therapy has been used to correct the genetic defect of cystic fibrosis
(but still in clinical trials)

Review Questions:

1.

-

identificati
on of the molecule to be produced

-

isolation of the instructions (DNA sequence/gene)

-

manipulation of the DNA instructions

-

harvest of the molecule or product; then marketing

2.

the “naming” designation used with recombinant products made through
genetic

engineering is that they have an
r

in front of their names.

3.

The smallest change in DNA molecule that can occur is that they can have
new pigment production, cell death, or cell growth. The effect that the change
can have is that is makes the cells grow f
aster due to the UV radiation.

4.

The gene that has been the target of CF gene therapy is the Tran membrane
conductance regulator. It is related to the common cold virus.

4.

4.4
-

Using Gel Electrophoresis to Study Molecules

Introduction
: gel electrophores
is uses electricity to separate molecules on a gel slab.
Researchers can easily separate and visualize charged molecules such as DNA
fragments, RNA and proteins.

A.

Components of Gel Electrophoresis



When the power is turned on, an electric current runs into the gel box, and an
electric field is established between the positive and the negative electrodes.



If the molecules have a net negative charge, they

move toward the positive
end of the gel; if th
ey have a net positive charge, they move toward the
negative end of the gel



DNA and RNA both are negatively charged so they move toward the positive
pole. RNA molecules are smaller than most DNA samples and, because they
are single
-
stranded, they move quic
kly through the gel; since DNA molecules
are usually much larger than RNA molecules, it is more difficult for them to
actually penetrate the gel and move through it

o

Gel electrophoresis
-

a process that uses electricity to separate
charged molecules, such as

DNA fragments, RNA and proteins on a
gel slab

o

Agarose
-

a carbohydrate from seaweed that is widely used as a
medium for horizontal gel electrophoresis

o

Polyacrylamide
-

a polymer used as a gel material in vertical gel
electrophoresis; used to separate small
er molecules, like proteins
and very small pieces of DNA or RNA

o

Figure 4.27
-

shows a model of an Agarose gel tray

o

Figure 4.28
-

shows a gel box with a buffer and all the parts to it are
pointed out

o

Figure 4.29
-

gels are mixed with loading dye because most
molecules are colorless

o

Figure 4.30
-

Molecules in a Gel Box

o

Table 4.3
-

represents the behavior of molecules in Agarose gel
electrophoresis

B.

Agarose Gel Concentrations



Most commonly used when separating pieces of DNA no smaller than 500 bp
and no larger t
han 25,000 bp.



The buffer usually contains the chemical TRIS, a buffering salt that stabilizes
the pH, maintains the shapes of the molecules being analyzed, and conducts
electricity



Experimentation is the most effective way to determine which gel materia
l
and concentration to use for the best separation

o

Figure 4.31
-

shows a horizontal gel box setup.

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o

Figure 4.32
-

shows three different Agarose concentrations

C.

Gel Stains



The voltage of the gel box (electric potential) is set to about 110 volts (V);
produce
s (current) kinetic energy of about 45 mAmps



The gel is run until molecules of different sizes are thought to have
completely separated



Since nucleic acids are colorless, technicians must “stain” the gels to see the
bands of separated molecules



Only mol
ecules of negative charge would run on the gel; each dark band
represents a large number of molecules of similar size

o

Ethidium bromide
-

a DNA stain (indicator), glows orange when it is
mixed with DNA and exposed to UV light; abbreviated EtBr

o

Methylene blu
e
-

a staining dye/indicator that interacts with nucleic
acid molecules and proteins, turning them to a very dark blue color

o

High through
-
put screening
-

the process of examining hundreds or
thousands of samples for a particular activity

o

Figure 4.33
-

shows

an Agarose gel machine running

o

Figure 4.34
-

this picture shows an EtBr
-
strained gel

o

Figure 4.35
-

shows how DNA samples may appear on a gel

o

Figure 4.36
-

shows how technicians run large
-
sized gels with many
samples at a time

Review Questions:

1.

T
hey can be

use to study small size of DNA fragments
.


2.

The 1% refers to concentration in the buffer.

3.

What cause molecules to be separated on an Agarose gel is the different
sizes of them.

4.

Ethidium bromide and methylene blue are two common DNA stains used to
visua
lize stains.