BIO150 – CELLULAR AND MOLECULAR BIOLOGY 1
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)
THE H1N1 FLU VIRUS
VIRUSES
– Not plants, animals, or bacteria
– Cannot synthesize proteins, because they
lack ribosome
– Cannot generator or store energy in the
form of ATP
– Parasitize the cell for basic building
materials, such as amino acids,
nucleotides, and lipids
– All contain nucleic acid, either DNA or
RNA (but not both), and a protein coat
– Some viruses enclosed by an envelope of
fat and protein molecules
– In its infective form, outside the cell, a
virus particle is called a virion.
– Predominantly two kinds of shapes found
amongst viruses: rods, or filaments, and
spheres
○ Rod shape due to the linear array of
the nucleic acid and the protein
subunits making up the capsid
○ Sphere shape actually a 20-sided
polygon (icosahedrons)
– Influenza virus (an RNA virus)
CAPSID
– The protein shell that encloses the nucleic
acid; with its enclosed nucleic acid, it is
called the nucleocapsid. This shell is
composed of protein organized in
subunits known as capsomers. They are
closely associated with the nucleic acid
and reflect its configuration, either a rod-
shaped helix or a polygon-shaped sphere
– Has three functions:
○ Protects the nucleic acid from
digestion by enzymes
○ Contains special sites on its surface
that allow the virion to attach to a host
cell
○ Provides proteins that enable the
virion to penetrate the host cell
membrane and, in some cases, to
inject the infectious nucleic acid into
the cells’ cytoplasm
ENVELOPE
– Glycoprotein envelope surrounding the
nucleocapsid; composed of two lipid
layers interspersed with protein
molecules (lipoprotein bilayer) and may
contain material from the membrane of a
host cell as well as that of viral origin
– The virus obtains the lipid molecules from
the cell membrane during the viral
budding process. However, the virus
replaces the proteins in the cell
membrane with its own proteins, creating
a hybrid structure of cell-derived lipids
and virus-derived proteins. Many viruses
also develop spikes made of glycoprotein
on their envelopes that help them to
attach to specific cell surfaces.
NUCLEIC ACID
– Encodes the genetic information for the
synthesis of all proteins; only a few
groups of viruses use DNA as most
viruses maintain all their genetic
information with the single-stranded RNA.
– Two types of RNA-based viruses:
○ Plus strand, acts as mRNA for direct
synthesis (translation) of viral protein.
○ Negative strand; virion has an
enzyme, called RNA-dependent RNA
polymerase (transcriptase), which
must first catalyze the production of
complementary mRNA from the virion
genomic RNA before viral protein
synthesis can occur.
TYPES OF INFLUENZA VIRUS
– Three types of influenza viruses: A, B and
C. influenza A and B viruses cause
seasonal epidemics of disease. Influenza
type C infections cause a mild respiratory
illness and are not thought to cause
epidemics.
H1N1 FLU VIRS
– A new influenza virus causing illness in
people
– First detected in people in Mexico in
March 2009.
– Spreading from person-to-person,
probably in much the same way that
regular seasonal influenza viruses spread
– Originally referred to as “swine flu”
because lab testing showed that many of
the genes in this new virus were similar to
influenza viruses that normally occur in
pigs in NA

1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)
– Further has shown that this new virus is
very different from what normally
circulates in NA pigs: it has two genes
from flu viruses that normally circulate in
pigs in Europe and Asia and avian genes
and human genes.
– Called a “quadruple reassortant” virus
– Binding site of neuraminidase stick
models are anti-influenza virus drugs
(Oseltamivir and Zanamivir)
– RNA PB2 and RNA PA
ANTIVIRAL DRUGS
– Are often nucleoside analogues, (fake
DNA building blocks), which viruses
incorporate into their genomes during
replication
– Life cycle of the virus halted since newly
synthesized DNA is inactive. This is
because these analogues lack the
hydroxyl groups, which, along with
phosphorus atoms, link together to form
the strong “backbone” of the DNA
molecule
– Called DNA chain termination
NEW NOVARTIS H1N1 VACCINE
– Produced at a Novartis plant in Marburg,
Germany
– Experimental
– Has not been tested; cannot be used on
humans yet
INTRODUCTION TO THE STUDY OF THE CELL
THE CELL THEORY
– First two tenets proposed in the 1830s by
Matthias Schleiden, a German botanist,
and Theodor Schwann, a German
zoologist
– Third tenet proposed in 1855 by Rudolf
Virchow, a German pathologist
– All organisms are composed of one or
more cells.
– The cell is the structural unit of life
– Cells arise only by division from a pre-
existing cell
TYPES OF CELLS
– Prokaryotes – all bacteria (eubacteria and
archaebacteria)
BIO150 – CELLULAR AND MOLECULAR BIOLOGY 2
– Made in cells rather than grown in eggs
– Unlikely to provide a major boost to the
world’s pandemic vaccine supply
HOW THE VIRUS CAN CHANGE
– Antigenic drift:
○ Small changes in the virus that
happen continually over time
– Produces new virus strains that may not
be recognized by the body’s immune
system. This process works as follows: a
person infected with a particular flu virus
strain develops antibody against that
virus. As newer virus strains appear, the
antibodies against the older strains no
longer recognize the “newer” virus, and
reinfection can occur. This is one of the
main reasons why people can get the flu
more than one time.
– Antigenic shift:
○ An abrupt, major change in the
influenza A viruses, resulting in new
hemagglutinin and/or new
hemagglutinin and neuraminidase
proteins in influenza viruses that infect
humans. Shift results in a new
influenza A subtype. When shift
happens, most people have little or no
protection against ht new virus.
○ While influenza viruses are changing
by antigenic drift all the time,
antigenic shift happens only
occasionally.
– Eukaryotes – plants, animals, protozoa,
fungi
PRINCIPLES OF LIVING CELLS
– Plasma membrane – selectively
permeable; forms boundary between cell
and environment; structure based on a
phospholipid bilayer permeable to O2,
CO2, water and lipids; grow by expansion
of pre-existing membranes; membrane
proteins allow other molecules to
enter/leave the cell
– Chemical composition (by weight)
○ Water, 70%
○ Salts, lipids, amino acids, nucleotides,
7%
 BIO150 – CELLULAR AND MOLECULAR BIOLOGY 3
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

○ Macromolecules, 23% for templates, e.g. chromatin,

– Genetic information is stored in DNA and
is duplicated is stored in DNA and is
duplicated and passed on to daughter
cells when the cell divides
– Both cell types have similar biochemical
pathways such as DNA replication,
transcription, protein synthesis and
energy production
– Macromolecular structures assemble from
subunits spontaneously without the need
cytoskeletal elements, membranes,
ribosome.
– Cellular constituents move by diffusion,
pumps and motors.
– Receptors and signalling mechanisms
allow cells to adapt to environmental
conditions
– Cells employ molecular feedback
mechanisms to control molecular
– Composition, growth and differentiation.

Feature Prokaryotes Eukaryotes

Genetic Organization No membrane-bound nucleus DNA in membrane-bound nucleus
Less DNA More DNA
DNA without histones, but with a DNA associated with histones
different protein
Presence of plasmids in some Plasmids in yeasts and a few
species plants
Plasma membrane Some bacteria with infoldings No mesosomes but with internal
called mesosomes membranes
No cholesterol With cholesterol
Internal membranes Connected to plasma membrane Extensive, not connected to
(thylakoid vesicles in plasma membrane; with
photosynthetic bacteria may not membrane-bound organelles
connect with plasma membrane)
Ribosomes 70S 80S
Cytoskeleton Absent Present
Cell wall composition Peptidoglycan Cellulose in plants
Reproduction Amitotic Mitotic
Site of electron transport Plasma membrane Mitochondrial and thylakoid
membranes
Endocytosis, phagocytosis, Absent Present in some
cytoplasmic streaming
Intracellular transport Diffusion Transport vesicles

Within the nucleus, there are modifications
of mRNA synthesis
– As the “start” end is usually added a
modified, upside down GUANINE CAP
– At the other end, goes a string of adenine
nucleotides, making a POLY A TAIL up to
a hundred nucleotides in length. Their
function is unknown.
– A complex protein and RNA grabs the
mRNA, forming loops. The complex—
called a SPLICEOSOME—then shears off
the loop, discards it, splices the remaining
pieces together, and departs.
– INTRONS are meaningless sequences of
a few hundred nts long. They are only
removed from mRNA after transcription.
– another difference is in the sheer number
of genes: 200,000 in a human and 4,000
in E coli
– a third peculiarity of Eu genes: they
harbour lots of REPETITIVE DNA,
sequences of nts which repeat
themselves many times. A possible
answer is that they consist of “SELFISH
DNA” which contributes nothing to the
organism
 BIO150 – CELLULAR AND MOLECULAR BIOLOGY 4
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)
 BIO150 – CELLULAR AND MOLECULAR BIOLOGY 5
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

ORGANELLES OF THE EUKARYOTIC CELL

STRUCTURE FUNCTION/PROCESS
Nucleolus rRNA synthesis; ribosome
assembly
Nucleoplasm
SER Lipid synthesis and metabolism;
detoxification of chemicals
RER Synthesis of certain membrane
and organelle proteins and
secreted proteins
Nucleus DNA replication, transcription
Golgi complex Membrane constituent & protein
sorting/targeting of; protein and
lipid modification
Transport vesicle Transport of proteins from RER to
Golgi and to certain organelles
Secretory vesicle Storage and exocytosis of
secreted proteins
Peroxisome & glyoxisome Degradation of fatty acids and
amino acids, producing H2O2;
degradation of H2O2 by catalase
Lysosome Degradation of cellular proteins,
nucleic acids, lipids, organelles
and of ingested macromolecules
Primary
Secondary
Mitochondrion Energy production
Chloroplast (plant cells) Photosynthesis
Vacuole (plants and certain Plant cells – storage of nutrients
microorganisms) and waste; degradation of cellular
proteins and other
macromolecules
Protozoans – osmotic regulation
REMARKS
With many copies of DNA coding
for rRNA
Nonnucleolar regions of the
nucleus
Network of tubular membranes
Extensive network of flattened
membranes with ribosomes on
surface
Contains the chromosome;
generally the largest organelle;
outer membrane continuous with
RER
Near the nucleus; a series of
flattened membrane sacs
Spherical, no particulate of
membrane debris
Large, irregular shape, from
fusion of 1o lysosome with other
organelle
With 2 membranes; contains DNA
With outer and inner membrane,
and internal membrane system
(thylakoids); contains DNA
Plant cells – entry of water into
the vacuole causes rapid
elongation has an acidic pH

OTHER EUKARYOTIC CELL PARTS ○ Inclusion bodies – granules not

– Cytoplasm – region outside the nucleus
– Cytosol – cytoplasm not contained within
organelles
○ Cytoskeleton – maintains cell shape
and motility; 3 classes of fibrous
proteins (microtubules,
microfilaments, intermediate
filaments)
○ Other proteins, e.g. enzymes in
glycolytic pathway
bounded by a membrane, e.g.
glycogen in liver cells
○ Ribosomes – sites of proteins
synthesis
– Cilia/flagella – hair- or whip like motile
structures that extend form the plasma
membrane; core made up of microtubules
– Centrioles – short cylinders of nine
microtubule triplets in the cell centre
(centrosome) and at the base of cilia and
flagella (basal bodies)

1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)
– Microvilli – finger like projections of
plasma membrane in cells specialized for
absorption
THE EXTRACELLULAR MATRIX
– Collagen – loose connective tissue;
contains few cells and many fibers in
spaces between cells; most abundant
proteins in animal kingdom
– Proteoglycans – consist mostly of acidic
polysaccharides (~95%) such as
hyaluronic acid, and proteins (5%); form
ground substance in which collagen and
other connective tissue fibers are
embedded
– Cell wall (plant cells) – cellulose; provide
rigidity
METHODS IN STUDYING THE MOLECULAR BIOLOGY OF THE CELL
LIGHT MICROSCOPY
– Magnification
– Resolving power
○ D = 0.61 λ / N sin α
 D – minimum distance between 2
distinguishable objects
 Λ – wavelength of incident light
 N – refractive index of medium
between specimen and objective
lens
 α – angular aperture
○ limit of resolution ~0.2 µm (200 nm)
○ preparation of samples
Other Types of Microscopy
○ Fluorescence
○ Atomic force
○ Confocal scanning and deconvolution
○ Phase contrast and Nomarski
Interference
○ Transmission electron
○ Scanning electron
○ Flow cytometry
○ Separation of organelles
 Differential centrifugation
 Density-gradient centrifugation
 Immunological techniques
FLOW CYTOMETRY
– Uses the principles of light scattering,
light excitation, and emission of
fluorochrome molecules to generate
specific multi-parameter data from
particles and cells in the size range of 0.5
to 40 µm diameter
BIO150 – CELLULAR AND MOLECULAR BIOLOGY 6
SUMMARY
– The cell is the basic unit of life and all
cells come from pre-existing cells
– There are two classes of cells, prokaryotic
and eukaryotic
– All cells have a plasma membrane, a
genetic material and have similar
compositions and biochemical pathways
– The plasma membrane interacts with the
extracellular matrix of the cell wall in
plant cells
THE MEMBRANE SKELETON FENCE
STRUCTURE
– Cells hydrodynamically focused in a
sheath of PBS before intercepting an
optimally focused light source
– Lasers most often used as a light source
– The sample is injected into the centre of a
sheath flow. The combined flow is
reduced in diameter, forcing the cell into
the centre of the stream. This allows the
laser to encounter one cell at a time.
– As the cells or particles of interest
intercept the light source they scatter
light and fluorochromes are excited to a
higher energy state. This energy is
released as a photon of light with specific
spectral properties unique to different
fluorochromes
– One unique feature of flow cytometry is
that it measures fluorescence per cell of
particle. This contrasts with
spectrophotometer in which the percent
absorption and transmission of specific
wavelengths of light is measured for a
bulk volume of sample.
FLUORESCENCE-ACTIVATED CELL SORTING
– A type of flow cytometry
– Cell suspension entrained in the centre of
a narrow, rapidly flowing stream of liquid
– Vibrating mechanism causes the stream
of cells to break into individual droplets
– Just before the stream breaks into
droplets the flow passes through a
fluorescence measuring station where the
 BIO150 – CELLULAR AND MOLECULAR BIOLOGY 7
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

fluorescence character of interest of each – Cellular components separated based on

cell is measured
– An electrical charging ring is placed just
at the point where the stream breaks into
droplets. A charge is placed on the ring
based on the immediately prior
fluorescence intensity measurement and
the opposite charge is trapped on the
droplet as it breaks from the stream.
– The charged droplets then fall thought an
electrostatic deflection system that
diverts droplets into containers based on
their charge.
SEPARATION OF ORGANELLES
– Cells/tissue samples treated with
proteases
– Cell membrane disrupted
(homogenization/sonication/placing in
hypotonic solution)
DIFFERENTIAL CENTRIFUGATION
differences in sedimentation rates
DENSITY GRADIENT CENTRIFUGATION
– Cellular components separated according
to density
– Samples placed onto the surface of a
vertical column of liquid, the density of
which progressively increases from top to
bottom, and then centrifuges
– Ex. Sucrose or glycerol
MONOCLONAL ANTIBODIES
– Clathrin-coated vesicles separated by
using clathrin-specific antibodies bound
to a bacterial carrier
– Aby-bacterial complex binds to clathrin-
coated vesicles and then purified by
centrifugation
– Coat tiny metallic beads with specific
antibodies
– Recover organelles by adhesion with the
use of a small magnet on the side of the
test tube

CELL CULTURE
– Microbial, animal, monoclonal antibody production, plaque assays

MICROBIAL CELL CULTURE

MINIMAL MEDIUM RICH MEDIUM
Carbon source: glucose or glycerol Partially hydrolyzed animal or plant tissue (rich in amino acids, short
peptides and lipids)
Nitrogen source: NH4+ Yeast extract (rich in vitamins and enzyme cofactors, nucleic acid
precursors, and amino acids)
Salts: Na+, K+, Mg+, Ca++, SO4+, Cl-, Carbon source, nitrogen source, and salts as in minimal medium
PO43-
Trace elements

– Single cell  colony of cells

– Clone = population of genetically identical cells derived from a single parent cell
– Replica plating – used to detect mutants in bacterial and yeast cultures (e.g. nutritionally
defective mutants)

REPLICA PLATING (HISTIDINE AUXOTRPHS)
– Treat bacterial cells with x-rays or a
chemical mutagen
– Grow cells on master plates that contain
a complete medium. Wait for colonies to
grow
– Press plates onto blocks with sterile
velveteen. This results in an imprint of
each colony on the cloth.
– Stamp the imprint onto other plates, one
with complete medium, one with minimal
medium supplemented with all amino
acids except histidine
– After incubation, examine replica plates
for differential growth of the colonies.
Determine which have mutations in the
histidine biosynthetic pathway. Start pure
culture of these for further study.

ANIMAL CELL CULTURE
– Require rich media: minimal medium +
essential amino acids (his, ile, leu, lys,
met, phe, thr, trp, val), vitamins, salts,
glucose, serum
– May require special solid surfaces (glass
or plastic treated with negatively charged
groups on the surface)
SOME TERMS TO REMEMBER
– Primary cell cultures are derived directly
from animal tissue
– Subculture = to transfer/transplant cells
of an ongoing culture to a new culture
vessel to propagate the cell population or
set up replicate cultures
NORMAL
Anchorage-dependent
Density-dependent inhibition of proliferation
Finite lifespan
Greater requirement for serum or growth factors for
optimal growth
More genetically stable
Longer population doubling time
MONOCLONAL ANTIBODIES
– Identical, monospecific antibodies
produced by one type of immune cell that
are all clones of a single parent cell
– May be used to:
○ Label and locate proteins in specific
cells or cell fractions
○ Purify proteins by affinity
chromatography
○ Diagnose and treat certain diseases
MONOCLONAL ANTIBODY PRODUCTION
– Immortal HGPRT-lacking myeloma cells
fused with normal antibody-producing
spleen cells from an animal immunized
with protein X
– Placed on HAT (contains hypoxanthine,
amonipterin, thymidine) so that unfused
EXPLORING PROTEINS
– Removal of membrane proteins, protein purification, detection of proteins, determination of
amino acid sequence, measurement of mass : time-of-flight spectrometry, determination of
protein conformation
BIO150 – CELLULAR AND MOLECULAR BIOLOGY 8
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)
– Secondary culture = product of the first
subculture
– Cell line = a population derived from a
primary culture; may be finite specific life
span) or continuous (unlimited
generational potential)
– Cell strain = lineage of cells originating
from one initial primary culture; used to
describe a subcultured population based
on specific properties, functional
characteristics or markers
– Clonal culture/selection = establishment
of a cultures cell population derived from
a single cell
– Transformed cells = derived from animal
tumours or arise spontaneously from
primary cells
TRANSFORMED
Reduced substrate adhesion
Loss of density-dependent inhibition of proliferation
Immortal
Reduced requirement for serum or growth factors
Genetically unstable (heteroploidy and aneuploidy)
Shorter population doubling time
cells do not grow, mutant myeloma cells
cannot make purines via salvage
pathway, spleen cells have limited
lifespan
– Fused cells survive and proliferate into
clones called hybridomas. Each
hybridoma produced a single antibody
– Hybridomas that produce desired
antibody are identified and cultured.
PLAQUE ASSAY
– Viral plaque – a visible structure (lesion_
formed within a cell culture, such as
bacterial cultures within some nutrient
medium; develops on the plate wherever
a single virion initially infects a single cell
– Assay – used to count viral particles
 BIO150 – CELLULAR AND MOLECULAR BIOLOGY 9
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

REMOVAL OF constants of – 2-D gel according to

MEMBRANE molecules electrophoresis: mass
PROTEINS – More dense to separate
– Transmembran molecules proteins with CHROMATOGRAPH
e proteins sediment faster similar masses Y
○ Precipitate – Differential – Gas, liquid, ion
from centrifugation: 2-D GEL exchange,
aqueous insoluble ELECTROPHORESIS affinity
solutions materials – Useful in
when sediment into a studying DETECTION OF
separated pellet; soluble expression of PROTEINS
from proteins remain various genes – Chromogenic
membranes in supernatant in differentiated and light-
○ Solubilised – Rate-zonal cells because emitting
by centrifugation; as many as enzyme
detergents, separate 1000 proteins reactions
whether particles based can be resolved ○ A color
ionic (SDS) on differences simultaneously change is
or non-ionic in mass; use – Steps: observed
(Triton X- sucrose ○ Isoelectric when a
100) gradient; focusing chromogenic
– Peripheral proteins (IEF): substrate is
proteins migrate down separates added.
○ Bound to the tube at a proteins Alternatively
membranes rate determined based on , substrate
by ionic or by their mass charge; run can be
other weak and shape sample in linked to a
interactions gel with pH chromogenic
○ Removed by ELECTROPHORESIS gradient; molecule
solutions of – Separates charged – Western
high ionic molecules in a proteins will Blotting
strength mixture under migrate (Immunoblottin
(high salt the influence of thought the g)
concentratio an applied gradient ○ Separate
ns) or electric filed until they proteins
chemical – Molecules reach their then identify
that bind separated isoelectric a specific
divalent based on point (pI) protein of
cations differences in ○ SDS interest
charge:mass electrophore
PROTEINS rations sis: place IEF WESTERN BLOT
PRUTIFICATION: – SDS-PAGE: SDS gel on – SDS-PAGE
CENTRIFUGATIONS dissociates second PAG, – Transfer
– To separate subunits in when proteins onto a
particles multimeric electric field membrane
– To measure proteins and is applied, – Flood
physical forces in a proteins membrane with
properties such linear migrate antibody
as MW, density, conformation from IEF gel specific for
shape, and with similar into SDS gel protein of
equilibrium charge:mass and interest
binding ratios separate – Wash
 BIO150 – CELLULAR AND MOLECULAR BIOLOGY 10
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

– Incubate with RECOMBINANT cryogenic – Limited to

second DNA TECHNIQUES temperatures proteins smaller
antibody, which – Deduce protein – Allows the than ~20kD
is linked to from mRNA or observation of ○ Concentrate
alkaline DNA sequence specimens that d protein
phosphatise have not been solution in
– Add substrate MEASUREMENT OF stained or fixed magnetic
for alkaline MASS – TIME OF in any way field 
phosphatise FLIGHT ○ Sample exposed to
– Observe for SPECTROMETRY rapidly different
formation of – Proteins mixed frozen in radio
deep purple with organic liquid helium frequencies
precipitate acid  examined  observe
– Dried on metal in frozen, effects on
USE OF target hydrated resonances
RADIOISOTOPES – Laser light state under of different
– For labelling ionizes cryoelectron atoms 
and tracing proteins, which microscope determine
molecules “fly” down to a  computer distances
– Examples: met detector programs between
and cys with – Time of flight analyze residues 
35
S; NA inversely images elucidate 3D
precursors with proportional to recorded on structure of
3
H; NAs with 32P charge film  protein
– Autoradiograph protein’s 3D
y DETERMINATION structure DNA CLONING
– Specialized OF PROTEIN determined – Cloning –
instruments: CONFORMATION: process of
Geiger and X-RAY NUCLEAR preparing large
scintillation CRYSTALLOGRAPH MAGNETIC numbers of
counters Y RESONANCE (NMR) identical DNA
– X-rays passed SPECTROSCOPY molecules
DETERMINATION through crystal – Exploits – Plasmid –
OF AMINO ACID of protein  magnetic circular,
SEQUENCE- scattered  properties of extrachormoso
EDMAN diffraction certain nuclei mal double-
DEGRADATION pattern on – Study mixtures stranded DNA
– Amino group at photographic of analytes; molecules
N-terminus film  complex understand – Restriction
allowed to react calculations and dynamic effects enzymes –
with protein such as change bacterial
phenylisothiocy modifications in temperature enzymes that
anate (PTC) needed to solve and reaction recognize
– N-terminal AA structure of mechanisms; specific 4- to 8-
cleaved by acid protein invaluable tool bp usually
hydrolysis; in palindromic
identified by CRYOELECTRON understanding sequences,
HPLC MICROSCOPY protein and then cleave
– Repeat until all – Also called nucleic acid both strands at
residues are cryo-EM structure and this site
identified – Sample is function – DNA library – a
studied at collection of
clones that
 BIO150 – CELLULAR AND MOLECULAR BIOLOGY 11
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

includes all the into the – DNA molecules e filter or

DNA sequences bacterium and cloned using nylon
of a given is allowed to expression membrane
species replicate vectors, then ○ Incubated
– If proper allowed to be under
RECOMBINANT regulatory transcribed and hybridization
DNA regions were translated to conditions
– Combination of included, proteins with specific
human and E substantial – Proteins from radiolabeled
coli DNA amounts of individual probe
– The tails snap protein may be clones bound to ○ Position of
together and extracted nitrocellulose fragment of
after treatment filter interest
with LIGASE, IDENTIFYING – Replica filter revealed by
an enzyme that CLONED DNA: screened using autoradiogra
seals nicks in MEMBRANE- a monoclonal phy
the sugar HYBRIDIZATION antibody
phosphate ASSAY specific for NORTHERN
chain, the – Recombinant protein of BLOTTING
recombinant gamma virions interest – For detecting
DNA is in plaques on – Position of specific RNA
complete lawn of E. coli clone detected fragments in a
What are the transferred to by incubation mixture
applications of nylon with second ○ Digest RNA
the membrane radioactively 
Recombinant – Incubate in labelled electrophore
DNA? alkaline solution antibody, then se
– First, choose a (disrupts autoradiograph ○ Transfer
human gene virions, y fragments to
encoding some releasing and nitrocellulos
useful protein. denaturing GEL e filter 
– For your DNA) ELECTROPHORESIS expose to
bacterial DNA, – Dry membrane labelled DNA
you need – Incubate with SOUTHERN probe 
something that radiolabeled BLOTTING autoradioagr
will be probe, which – For detecting aphy
replicated once hybridizes to specific DNA
it’s returned to bound nucleic fragments in a DNA SEQUENCING:
the cell – a acid mixture of MAXAM-GILBERT
VECTOR – Unhybridized restriction METHOD
– Luckily, E coli probe washed fragments – Radiolabel 5’
has small rings away; ○ RE digestion end of molecule
of DNA called recombinant  to be
PLASMIDS clone detected electrophore sequenced
separate from by sis – Generate
the autoradiograph ○ Fragments breaks by
chromosome y in gel chemical
– The human denatured treatment at
gene is spliced IDENTIFICATION with alkali; small
into the plasmid BASED ON transferred proportion of
containing PROPERTIES OF by blotting one or two of
GAATTC and is ENCODED to nucleotide
placed back PROTEINS nitrocellulos bases in each of
 BIO150 – CELLULAR AND MOLECULAR BIOLOGY 12
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

four reactions – DB
G, G+A, C+T, C (immunoglobulins,
– Electrophorese T-cell receptors,
– Perform MHC molecules of
autoradiograph all vertebrate
y species
OMIM – Online
DNA MICROARRAY Mendelian
Inheritance in Men
DATABASES (catalog of human
GenBank genes and genetic
EMBL disorders)
DDBJ BLAST = Basic
PDB Local Alignment
SwissProt Search Tool
IMGT = FASTA = Fast
International Alignments/Fast All
Immunogenetics