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BIO150 – CELLULAR AND MOLECULAR BIOLOGY 1

1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

THE H1N1 FLU VIRUS

VIRUSES contain material from the membrane of a


– Not plants, animals, or bacteria host cell as well as that of viral origin
– Cannot synthesize proteins, because they – The virus obtains the lipid molecules from
lack ribosome the cell membrane during the viral
– Cannot generator or store energy in the budding process. However, the virus
form of ATP replaces the proteins in the cell
– Parasitize the cell for basic building membrane with its own proteins, creating
materials, such as amino acids, a hybrid structure of cell-derived lipids
nucleotides, and lipids and virus-derived proteins. Many viruses
– All contain nucleic acid, either DNA or also develop spikes made of glycoprotein
RNA (but not both), and a protein coat on their envelopes that help them to
– Some viruses enclosed by an envelope of attach to specific cell surfaces.
fat and protein molecules
– In its infective form, outside the cell, a NUCLEIC ACID
virus particle is called a virion. – Encodes the genetic information for the
– Predominantly two kinds of shapes found synthesis of all proteins; only a few
amongst viruses: rods, or filaments, and groups of viruses use DNA as most
spheres viruses maintain all their genetic
○ Rod shape due to the linear array of information with the single-stranded RNA.
the nucleic acid and the protein – Two types of RNA-based viruses:
subunits making up the capsid ○ Plus strand, acts as mRNA for direct
○ Sphere shape actually a 20-sided synthesis (translation) of viral protein.
polygon (icosahedrons) ○ Negative strand; virion has an
– Influenza virus (an RNA virus) enzyme, called RNA-dependent RNA
polymerase (transcriptase), which
CAPSID must first catalyze the production of
– The protein shell that encloses the nucleic complementary mRNA from the virion
acid; with its enclosed nucleic acid, it is genomic RNA before viral protein
called the nucleocapsid. This shell is synthesis can occur.
composed of protein organized in
subunits known as capsomers. They are TYPES OF INFLUENZA VIRUS
closely associated with the nucleic acid – Three types of influenza viruses: A, B and
and reflect its configuration, either a rod- C. influenza A and B viruses cause
shaped helix or a polygon-shaped sphere seasonal epidemics of disease. Influenza
– Has three functions: type C infections cause a mild respiratory
○ Protects the nucleic acid from illness and are not thought to cause
digestion by enzymes epidemics.
○ Contains special sites on its surface
that allow the virion to attach to a host H1N1 FLU VIRS
cell – A new influenza virus causing illness in
○ Provides proteins that enable the people
virion to penetrate the host cell – First detected in people in Mexico in
membrane and, in some cases, to March 2009.
inject the infectious nucleic acid into – Spreading from person-to-person,
the cells’ cytoplasm probably in much the same way that
regular seasonal influenza viruses spread
ENVELOPE – Originally referred to as “swine flu”
– Glycoprotein envelope surrounding the because lab testing showed that many of
nucleocapsid; composed of two lipid the genes in this new virus were similar to
layers interspersed with protein influenza viruses that normally occur in
molecules (lipoprotein bilayer) and may pigs in NA
BIO150 – CELLULAR AND MOLECULAR BIOLOGY 2
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

– Further has shown that this new virus is – Made in cells rather than grown in eggs
very different from what normally – Unlikely to provide a major boost to the
circulates in NA pigs: it has two genes world’s pandemic vaccine supply
from flu viruses that normally circulate in
pigs in Europe and Asia and avian genes HOW THE VIRUS CAN CHANGE
and human genes. – Antigenic drift:
– Called a “quadruple reassortant” virus ○ Small changes in the virus that
– Binding site of neuraminidase stick happen continually over time
models are anti-influenza virus drugs – Produces new virus strains that may not
(Oseltamivir and Zanamivir) be recognized by the body’s immune
– RNA PB2 and RNA PA 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
ANTIVIRAL DRUGS longer recognize the “newer” virus, and
– Are often nucleoside analogues, (fake reinfection can occur. This is one of the
DNA building blocks), which viruses main reasons why people can get the flu
incorporate into their genomes during more than one time.
replication – Antigenic shift:
– Life cycle of the virus halted since newly ○ An abrupt, major change in the
synthesized DNA is inactive. This is influenza A viruses, resulting in new
because these analogues lack the hemagglutinin and/or new
hydroxyl groups, which, along with hemagglutinin and neuraminidase
phosphorus atoms, link together to form proteins in influenza viruses that infect
the strong “backbone” of the DNA humans. Shift results in a new
molecule influenza A subtype. When shift
– Called DNA chain termination happens, most people have little or no
protection against ht new virus.
NEW NOVARTIS H1N1 VACCINE ○ While influenza viruses are changing
– Produced at a Novartis plant in Marburg, by antigenic drift all the time,
Germany antigenic shift happens only
– Experimental occasionally.
– Has not been tested; cannot be used on
humans yet

INTRODUCTION TO THE STUDY OF THE CELL

THE CELL THEORY – Eukaryotes – plants, animals, protozoa,


– First two tenets proposed in the 1830s by fungi
Matthias Schleiden, a German botanist,
and Theodor Schwann, a German PRINCIPLES OF LIVING CELLS
zoologist – Plasma membrane – selectively
– Third tenet proposed in 1855 by Rudolf permeable; forms boundary between cell
Virchow, a German pathologist and environment; structure based on a
– All organisms are composed of one or phospholipid bilayer permeable to O2,
more cells. CO2, water and lipids; grow by expansion
– The cell is the structural unit of life of pre-existing membranes; membrane
– Cells arise only by division from a pre- proteins allow other molecules to
existing cell enter/leave the cell
– Chemical composition (by weight)
TYPES OF CELLS ○ Water, 70%
– Prokaryotes – all bacteria (eubacteria and ○ Salts, lipids, amino acids, nucleotides,
archaebacteria) 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 cytoskeletal elements, membranes,
is duplicated is stored in DNA and is ribosome.
duplicated and passed on to daughter – Cellular constituents move by diffusion,
cells when the cell divides pumps and motors.
– Both cell types have similar biochemical – Receptors and signalling mechanisms
pathways such as DNA replication, allow cells to adapt to environmental
transcription, protein synthesis and conditions
energy production – Cells employ molecular feedback
– Macromolecular structures assemble from mechanisms to control molecular
subunits spontaneously without the need – 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 – INTRONS are meaningless sequences of
of mRNA synthesis a few hundred nts long. They are only
– As the “start” end is usually added a removed from mRNA after transcription.
modified, upside down GUANINE CAP – another difference is in the sheer number
– At the other end, goes a string of adenine of genes: 200,000 in a human and 4,000
nucleotides, making a POLY A TAIL up to in E coli
a hundred nucleotides in length. Their – a third peculiarity of Eu genes: they
function is unknown. harbour lots of REPETITIVE DNA,
– A complex protein and RNA grabs the sequences of nts which repeat
mRNA, forming loops. The complex— themselves many times. A possible
called a SPLICEOSOME—then shears off answer is that they consist of “SELFISH
the loop, discards it, splices the remaining DNA” which contributes nothing to the
pieces together, and departs. 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 REMARKS
Nucleolus rRNA synthesis; ribosome With many copies of DNA coding
assembly for rRNA
Nucleoplasm Nonnucleolar regions of the
nucleus
SER Lipid synthesis and metabolism; Network of tubular membranes
detoxification of chemicals
RER Synthesis of certain membrane Extensive network of flattened
and organelle proteins and membranes with ribosomes on
secreted proteins surface
Nucleus DNA replication, transcription Contains the chromosome;
generally the largest organelle;
outer membrane continuous with
RER
Golgi complex Membrane constituent & protein Near the nucleus; a series of
sorting/targeting of; protein and flattened membrane sacs
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 Spherical, no particulate of
membrane debris
Secondary Large, irregular shape, from
fusion of 1o lysosome with other
organelle
Mitochondrion Energy production With 2 membranes; contains DNA
Chloroplast (plant cells) Photosynthesis With outer and inner membrane,
and internal membrane system
(thylakoids); contains DNA
Vacuole (plants and certain Plant cells – storage of nutrients Plant cells – entry of water into
microorganisms) and waste; degradation of cellular the vacuole causes rapid
proteins and other elongation has an acidic pH
macromolecules
Protozoans – osmotic regulation

OTHER EUKARYOTIC CELL PARTS ○ Inclusion bodies – granules not


– Cytoplasm – region outside the nucleus bounded by a membrane, e.g.
– Cytosol – cytoplasm not contained within glycogen in liver cells
organelles ○ Ribosomes – sites of proteins
○ Cytoskeleton – maintains cell shape synthesis
and motility; 3 classes of fibrous – Cilia/flagella – hair- or whip like motile
proteins (microtubules, structures that extend form the plasma
microfilaments, intermediate membrane; core made up of microtubules
filaments) – Centrioles – short cylinders of nine
○ Other proteins, e.g. enzymes in microtubule triplets in the cell centre
glycolytic pathway (centrosome) and at the base of cilia and
flagella (basal bodies)
BIO150 – CELLULAR AND MOLECULAR BIOLOGY 6
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

– Microvilli – finger like projections of SUMMARY


plasma membrane in cells specialized for – The cell is the basic unit of life and all
absorption cells come from pre-existing cells
– There are two classes of cells, prokaryotic
THE EXTRACELLULAR MATRIX and eukaryotic
– Collagen – loose connective tissue; – All cells have a plasma membrane, a
contains few cells and many fibers in genetic material and have similar
spaces between cells; most abundant compositions and biochemical pathways
proteins in animal kingdom – The plasma membrane interacts with the
– Proteoglycans – consist mostly of acidic extracellular matrix of the cell wall in
polysaccharides (~95%) such as plant cells
hyaluronic acid, and proteins (5%); form
ground substance in which collagen and THE MEMBRANE SKELETON FENCE
other connective tissue fibers are STRUCTURE
embedded
– Cell wall (plant cells) – cellulose; provide
rigidity

METHODS IN STUDYING THE MOLECULAR BIOLOGY OF THE CELL

LIGHT MICROSCOPY – Cells hydrodynamically focused in a


– Magnification sheath of PBS before intercepting an
– Resolving power optimally focused light source
○ D = 0.61 λ / N sin α – Lasers most often used as a light source
 D – minimum distance between 2 – The sample is injected into the centre of a
distinguishable objects sheath flow. The combined flow is
 Λ – wavelength of incident light reduced in diameter, forcing the cell into
 N – refractive index of medium the centre of the stream. This allows the
between specimen and objective laser to encounter one cell at a time.
lens – As the cells or particles of interest
 α – angular aperture intercept the light source they scatter
○ limit of resolution ~0.2 µm (200 nm) light and fluorochromes are excited to a
○ preparation of samples higher energy state. This energy is
Other Types of Microscopy released as a photon of light with specific
○ Fluorescence spectral properties unique to different
○ Atomic force fluorochromes
○ Confocal scanning and deconvolution – One unique feature of flow cytometry is
○ Phase contrast and Nomarski that it measures fluorescence per cell of
Interference particle. This contrasts with
○ Transmission electron spectrophotometer in which the percent
absorption and transmission of specific
○ Scanning electron
wavelengths of light is measured for a
○ Flow cytometry
bulk volume of sample.
○ Separation of organelles
 Differential centrifugation FLUORESCENCE-ACTIVATED CELL SORTING
 Density-gradient centrifugation – A type of flow cytometry
 Immunological techniques – Cell suspension entrained in the centre of
a narrow, rapidly flowing stream of liquid
FLOW CYTOMETRY – Vibrating mechanism causes the stream
– Uses the principles of light scattering, of cells to break into individual droplets
light excitation, and emission of – Just before the stream breaks into
fluorochrome molecules to generate droplets the flow passes through a
specific multi-parameter data from fluorescence measuring station where the
particles and cells in the size range of 0.5
to 40 µm diameter
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 differences in sedimentation rates
– An electrical charging ring is placed just
at the point where the stream breaks into DENSITY GRADIENT CENTRIFUGATION
droplets. A charge is placed on the ring – Cellular components separated according
based on the immediately prior to density
fluorescence intensity measurement and – Samples placed onto the surface of a
the opposite charge is trapped on the vertical column of liquid, the density of
droplet as it breaks from the stream. which progressively increases from top to
– The charged droplets then fall thought an bottom, and then centrifuges
electrostatic deflection system that – Ex. Sucrose or glycerol
diverts droplets into containers based on
their charge. MONOCLONAL ANTIBODIES
– Clathrin-coated vesicles separated by
SEPARATION OF ORGANELLES using clathrin-specific antibodies bound
– Cells/tissue samples treated with to a bacterial carrier
proteases – Aby-bacterial complex binds to clathrin-
– Cell membrane disrupted coated vesicles and then purified by
(homogenization/sonication/placing in centrifugation
hypotonic solution) – Coat tiny metallic beads with specific
antibodies
– Recover organelles by adhesion with the
use of a small magnet on the side of the
DIFFERENTIAL CENTRIFUGATION 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) – Stamp the imprint onto other plates, one
– Treat bacterial cells with x-rays or a with complete medium, one with minimal
chemical mutagen medium supplemented with all amino
– Grow cells on master plates that contain acids except histidine
a complete medium. Wait for colonies to – After incubation, examine replica plates
grow for differential growth of the colonies.
– Press plates onto blocks with sterile Determine which have mutations in the
velveteen. This results in an imprint of histidine biosynthetic pathway. Start pure
each colony on the cloth. culture of these for further study.
BIO150 – CELLULAR AND MOLECULAR BIOLOGY 8
1st Exam Notes – Daniel Marc G. dela Torre (2007 76293)

– Secondary culture = product of the first


ANIMAL CELL CULTURE subculture
– Require rich media: minimal medium + – Cell line = a population derived from a
essential amino acids (his, ile, leu, lys, primary culture; may be finite specific life
met, phe, thr, trp, val), vitamins, salts, span) or continuous (unlimited
glucose, serum generational potential)
– May require special solid surfaces (glass – Cell strain = lineage of cells originating
or plastic treated with negatively charged from one initial primary culture; used to
groups on the surface) describe a subcultured population based
on specific properties, functional
SOME TERMS TO REMEMBER characteristics or markers
– Primary cell cultures are derived directly – Clonal culture/selection = establishment
from animal tissue of a cultures cell population derived from
– Subculture = to transfer/transplant cells a single cell
of an ongoing culture to a new culture – Transformed cells = derived from animal
vessel to propagate the cell population or tumours or arise spontaneously from
set up replicate cultures primary cells

NORMAL TRANSFORMED
Anchorage-dependent Reduced substrate adhesion
Density-dependent inhibition of proliferation Loss of density-dependent inhibition of proliferation
Finite lifespan Immortal
Greater requirement for serum or growth factors for Reduced requirement for serum or growth factors
optimal growth
More genetically stable Genetically unstable (heteroploidy and aneuploidy)
Longer population doubling time Shorter population doubling time

MONOCLONAL ANTIBODIES cells do not grow, mutant myeloma cells


– Identical, monospecific antibodies cannot make purines via salvage
produced by one type of immune cell that pathway, spleen cells have limited
are all clones of a single parent cell lifespan
– May be used to: – Fused cells survive and proliferate into
○ Label and locate proteins in specific clones called hybridomas. Each
cells or cell fractions hybridoma produced a single antibody
○ Purify proteins by affinity – Hybridomas that produce desired
chromatography antibody are identified and cultured.
○ Diagnose and treat certain diseases
PLAQUE ASSAY
MONOCLONAL ANTIBODY PRODUCTION – Viral plaque – a visible structure (lesion_
– Immortal HGPRT-lacking myeloma cells formed within a cell culture, such as
fused with normal antibody-producing bacterial cultures within some nutrient
spleen cells from an animal immunized medium; develops on the plate wherever
with protein X a single virion initially infects a single cell
– Placed on HAT (contains hypoxanthine, – Assay – used to count viral particles
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 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