Graduate Biochemistry Homepage
Earliest signs of life ~3.5 billion years ago
The pre-life atmosphere contained H2O, N2, CO2, CH4, NH3, and SO2.
Lightning or ultraviolet radiation could have provided energy for reactions to form simple organic compounds.
An experimental test of this hypothesis showed that reactions of primitive atmospheric compounds could result in amino acids and other common metabolites found in modern day cells.
These compounds contain many of the functional groups necessary for modern day biochemical reactions.
Simple organic compounds - raw materials for a period of chemical evolution in which simple molecules condensed together to form larger functional units.
At some point, an RNA-like molecule may have arisen, which could direct its own replication.
Next major step: formation of a vesicle around the self-replicating molecules.
Enclosure of self-replicating systems within vesicles would form the first cells.
Allows the cell to maintain high local concentrations of necessary components,
which would otherwise diffuse away.
Early cells acquired ability to catalyze reactions for the synthesis of necessary compounds from simple precursors.
Acquired the ability to harvest energy for these reactions from their environment.
Ability of early cells to synthesize biological molecules, to utilize an energy source, and to direct their own replication, allowed them to propagate.
All organisms appear to be descended from a single common ancestor, based on conserved sequences of key cellular components, such as the ribosome.
Present-day organisms can be divided into three kingdoms of life:
Bacteria and Archaea:
Are commonly grouped together in the classification prokaryotes.
Unicellular and relatively simple internal architecture.
Enclosed in an outer cell membrane, but lack internal membrane compartments.
Internal cytoplasm appears to be organized into different regions, however.
Rendering of contents of E. coli cell. Note the organization of DNA and dense packing of cell contents, particularly ribosomes, the site of protein synthesis.
Typical cell: 70% H2O
Dry weight mostly carbon Ð 98% of dry weight composed of C, N, O, H, Ca, P, K, and S.
Eukaryotes consist of the Eukarya
Distinct from prokaryotes in having a nucleus to encapsulate DNA.
Can be both unicellular and multicellular.
Eukaryotic cells characterized by organellar organization:
Nucleus Ð DNA
Endoplasmic reticulum Ð translation, translocation, modification
Golgi Ð post-translational modification
Mitochondria Ð aerobic metabolism (probably descended from bacterium)
Chloroplastc Ð plant photosynthesis (probably descended from bacterium)
Lysosomes and peroxisomes Ð degradative metabolism
Cytosol- the rest of the cytoplasm
Nitrogenous base Ð planar, aromatic, heterocyclic derivatives of purine or pyrimidine
Nucleosides are comprised of a nitrogenous base linked to a sugar
(usually ribose or deoxyribose)
Nucleotides are nucleosides attached to at least one phosphate group
Base (X= H) |
Nucleoside (X= ribose) |
Nucleotide (X= ribose phosphate) |
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purines |
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Adenine Ade A |
Adenosine Ado A |
Adenylic acid Adenosine monophosphate AMP |
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Guanine Gua G |
Guanosine Guo G |
Guanylic acid Guanosine monophosphate GMP |
pyrimidines |
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Cytosine Cyt C |
Cytidine Cyd C |
Cytidylic acid Cytidine monophosphate CMP |
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Uracil Ura U |
Uridine Urd U |
Uridylic acid Uridine monophosphate UMP |
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Thymine Thy T |
Deoxythymidine dThd dT |
Deoxythymidylic acid Deoxythymidine
monophosphate dTMP |
Nucleotide sugars are commonly ribose or deoxyribose
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5Õ Ribonucleotide Ð sugar=ribose |
5Õ Deoxyribonucleotide Ð sugar=2Õ-deoxyribose |
H3PO4 + |
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+ H2O |
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Adenosine diphosphate (ADP) |
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Adenosine triphosphate (ATP) |
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Adenosine triphosphate (ATP):
Most energy from cellular metabolism passes through ATP as an intermediate
Cellular concentration of ATP ~5 mM
But daily turnover of ATP is Å to total body weight
Energy of hydrolysis is used to drive other reactions.
Sometimes used to form intermediate necessary for further synthesis
Other important nucleotides and their uses:
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Nicotinamide adenine dinucleotide - NAD |
Flavin adenine dinucleotide - FAD |
NAD+ and NADP+ can be reversibly reduced to form NADH or NADPH in oxidation-reduction reactions (nicotinamide is from vitamin niacin).
FAD also is used for oxidation-reduction (riboflavin portion is vitamin B2)
All three act as electron transporters
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Coenzyme A (CoA)- used as acyl group carrier. Pantothenic acid is vitamin B3. |
Phosphodiester bond links each nucleotide unit to form polynucleotide chain
Linkage between nucleotides is typically between 5Õ O and 3ÕO
Terminii named 5Õ and 3Õ ends
Forms polyanion due to negatively charged PO4
Is this RNA or DNA?
DNA base composition:
ChargaffÕs rule: For any organism %A=%T and %G=%C.
But %G+C can vary widely (25 Ð 75%)
Watson and Crick used model building to describe structure of DNA
Explained ChargaffÕs rules
Fit fiber diffraction measurements of DNA
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3-D structure of DNA |
2-D representation showing H-bonds |
DNA Kinemage 1: B-DNA | DNA Kinemage 2: Base pairs. |
Features of the double helix:
Two polynucleotide chains wrapped around a common axis -> double helix
Strands are antiparallel but both are right-handed
The bases are buried in the core of the helix, sugar-phosphate backbone is exposed to solvent
Interior bases partially accessible through major and minor grooves of double helix
Bases form characteristic sets of hydrogen bonds (sharing of proton by dipoles) between complementary bases:
A pairs with T, G pairs with C
termed complementary base pairs
Accounts for ChargaffÕs rules
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Genome sizes:
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Eukarya
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Homo sapiens |
3,127,936,000 |
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Arabidopsis thaliana |
117,950,579 |
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Caenorhabditis elegans |
100,237,364 |
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Schizosaccharomyces pombe |
12,515,113 |
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Saccharomyces cerevisiae |
12,162,624 |
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Bacteria |
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Escherichia coli
K12 |
4,639,221 |
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Haemophilus influenzae
Rd |
1,830,138 |
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Mycoplasma genitalium |
580,074 |
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Methanococcus jannaschii |
1,664,970 |
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Viruses |
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9181 |
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Simian virus 40 |
5243 |
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Bacteriophage l |
48502 |
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168,903 |
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39937 |
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Suggested mechanism for replication of DNA based on complementarity of bases:
One strand can serve as template for synthesis of new copy of its complement
DNA polymers can be enormous.
Total DNA of cell is its genome
Genomes may be divided among several molecules (chromosomes)
Most prokaryotes have only one double strand copy of their genomes per cell Ð they are haploid
Most eukaryotes have not only haploid cells,
but also diploid cells that have 2 copies of each double-strand chromosome
The structure of double-strand DNA is relatively unaffected by the DNA sequence
Single stranded DNA and RNA tend to try to form basepairs with other regions of the molecule that are complementary, so structure can vary for different sequences.
Isolated DNA from pathogenic strain of D. pneumoniae,
mixed it with non-pathogenic strain
recipient strain was transformed (permanently changed) to pathogenic
ð DNA is the source of genetic information
How is the genetic information in the DNA expressed by the cell to affect the way it behaves?
DNA directs transcription of messenger RNAs (mRNAs)
mRNA is translated into protein at the ribosome by base-paring to aminoacyl-tRNAs
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The Central Dogma: DNA->RNA->protein |
Translation of mRNA into protein |
change can affect the mRNA sequence
which can in turn change the protein sequence.
Proteins perform much of the chemical and structural functions of the cell, either directly or indirectly.
Most enzymes (biological catalysts) within the cell are proteins, but a few are made of RNA.
Means of amplifying DNA sequence of interest.
Use to determine DNA sequence
Overproduce gene product
Manipulate gene product (e.g. make mutations for study)
Make reagents for further study (determine fate of gene product in cell or analyze it in other individuals i.e. forensics)
Need some kind of vector to
amplify your DNA:
In vivo:
Plasmids (typically)
Virus (bacteriophage, eukaryotic)
Hybrids
Artificial chromosomes (YACs)
In vitro:
PCR primers
Usually trying to clone into E. coli plasmid vector
Most bacteria can bind and absorb DNA from the environment - transformation
Probably an adaptation to allow individuals to acquire variant genes from others of their species.
But if DNA from another species, could wreak havoc with regulation of gene expression.
So, bacteria have a biochemical system that allows them to acquire DNA of their own species, but not from others- The restriction-modification system.
Each species (or strain) of bacteria has a distinct pair of enzymes:
A site-specific DNA methylase (adds a CH3- group to a particular type of nucleotide)
and an endonuclease, which cuts DNA that is not methylated at that site.
(Endonuclease Ð cuts within DNA [or RNA] strand
Exonuclease Ð removes nucleotides from ends)
So if you transform E. coli strain R with DNA from strain R
Select for expression of an acquired gene --> colonies
But if you use DNA from strain K --> no colonies
The DNA from stain R is methylated by the EcoR I methylase
this keeps the EcoRI endonuclease from cutting the DNA
the DNA from strain K is methylated at different sites
so EcoRI can cut --> less efficient transformation
Restriction endonucleases have been purified from 100s of different species
Most recognize palindromic sequences (sequences that looks the same on either strand from either direction)
E.g.
Bacterial
strain |
Endo. |
Site |
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E. coli R |
EcoR I |
5' GAATTC 3' 3' CTTAAG 5' |
cuts 3' of G leaves 5' overhang |
H. influenzae |
HindIII |
5' AAGCTT 3' 3' TTCGAA 5' |
cuts 3' of 1st A leaves 5' overhang |
H. aegyptus |
HaeII |
5' RGCGCY 3' 3' YCGCGR 5' |
cuts 3' of last C leaves 3' overhang |
Used in many ways in DNA
cloning, especially to cut vector DNA and gene of interest for splicing gene
into vector:
"sticky ends" from EcoRI cut of one fragment can base pair with those from another
isolate fragment with insert from gel, anneal sticky ends to plasmid DNA, and ligate with DNA ligase.
DNA ligase - DNA repair enzyme
repairs breaks in DNA backbone
Requires high energy cofactor.
Usually use enzyme from T4 phage, which can ligate blunt or sticky ends (uses ATP).
E. coli enzyme requires NAD+ instead of ATP and only repairs sticky ends.
Plasmids
autonomously replicating circular minichromosomes
most derived from drug-resistant bacteria
have origin of replication for amplifying DNA and marker gene to select for cells carrying it
Modern plasmids have variety
of features to make life easier.
Usually Ampicillin resistance gene, beta lactamase, as selectable marker (only cells with plasmid can grow).
Polylinker sequence - synthetic sequence with many restriction endonuclease cut sites,
Beta galactosidase gene fragment
Plasmids without inserts
produce beta-gal and appear blue on X-gal plates.
Insert DNA disrupts open
reading frame -> no beta gal -> white colonies
Plasmids introduced into
cell by transformation:
Cells permeablized by osmotic shock or electrochemical pulse.
Circular DNA transforms efficiently
linear does not (due to cellular exonucleases)
Larger DNAs not as efficiently taken up as smaller ones for chemically permeablized cells.
Host cell usually defective for restriction modification system, so exogenous DNA not digested.
Other vectors:
Bacteriophage:
Lambda phage has 48.5 kb genome, packages one headful per virus particle.
Can remove middle third of genome to allow insertion of up to 16 kb of insert
Convenient for construction of Genomic and cDNA libraries
Cosmids:
Hybrid of lambda and plasmid, contains only packaging signal from lambda plus usual plasmid replication and marker genes.
Can insert up to 45 kb DNA
Use lambda packaging system to efficiently introduce large plasmid into cell.
Replicates as plasmid thereafter.
P1 phage: allows replication of up to 150 kb of DNA. Great for genomic libraries.
Expression vectors
Include promoter sequences to allow expression of mRNA and protein from insert DNA. Often are expressed as fusion proteins, your gene is fused with coding sequence for another protein. Can make more stable protein than foreign gene by itself. Also provides a handle for purification
ex.
Fusion
product affinity
resin
glutathione-S-transferase glutathione
polyHis Ni++
For bacterial expression, common promoter systems are lac and T7 phage
But many eukaryotic
proteins not properly expressed and modified (e. g. glycosylation) in bacteria,
so eukaryotic expression systems are used. Common promoters are SV40 and CMV in
mammalian cells.
Reporter plasmids
Clone control region of your gene (promoter) upstream of gene encoding enzyme that is easy to monitor.
Can look at effects of changes in cell environment on expression of reporter gene to infer effects on the intact gene of interest
Common reporters.
Chloramphenicol acetyltransferase (CAT) |
modifies chloramphenicol |
Luciferase |
Produces light (ATP-dependent) |
Beta-galactosidase |
hydrolyzes lactose analogs |
The second two can be monitored in situ as well as in vitro
Southern blot used to analyze (genomic) DNA sequences
Total genomic DNA is purified and digested with restriction enzymes
This digest is electrophoresed on an agarose gel to separate the DNA fragments according to size. If you stain the DNA in the gel with ethidium bromide, a smear is usually seen since the digest may contain thousands of different sized fragments.
The gel is treated with Acid, then NaOH. This randomly nicks the DNA then denatures it.
The gel is then placed on moist blotting paper and a nitrocellulose filter is placed on top, followed by a large stack of dry paper towels (or a disposable diaper!).
The dry paper sucks buffer through the gel, carrying the DNA with it. Nicking the DNA (acid treatment above) allows the large fragments to transfer as efficiently as the small ones
Typically one uses a radiolabeled DNA or RNA probe to detect sequence of interest.
The temperature and buffer conditions used for hybridizing and washing the filters have dramatic effects on results.
Factors favoring hybridization
(base-pairing of DNA strands):
Low temperature
High [salt]
Low [denaturant]
probe length
time
%GC content of probe
All can be factored to determine melting temperature of DNA duplex (Tm)
Tm=81+16.6log[Na+] Ð 0.4[%(G+C)] Ð 0.6 (% formamide) Ð 600/n Ð1.5(% mismatch)
where n is length of probe in bases
After autoradiography, a dark band should be seen which corresponds to a DNA restriction fragment homologous to the probe DNA. The size of the fragment can be determined by comparison with known DNA standards run on the same gel.
Southern are useful for optimizing probe conditions for library screening, for mapping genomic sequences flanking cloned regions and in forensic analysis.
Northern blot used to mRNA expression
Instead of DNA total RNA or polyA+ mRNA from tissues of interest is electrophoresed on a denaturing gel (agarose-formaldehyde or acrylamide-urea), separating RNAs by size. The gel is blotted and probed as for a Southern Blot. Very useful for determining whether your DNA sequence is expressed as mRNA and how it is expressed.
Used to monitor regulation
of mRNA levels:
Can isolate mRNA from different tissues
different times in development
cells treated differently in culture.
Once cloned, DNA can be sequenced by either chemical or enzymatic methods. Chemical method is still used for footprinting type experiments, but virtually all sequencing is performed using DNA polymerase and chain terminating nucleotides.
Enzymatic sequencing reaction
Anneal (hybridize) single strand ssDNA to a specific oligonucleotide primer (complementary to flanking vector sequence, or to known sequence within the insert DNA). The oligonucleotide serves as primer for synthesis of DNA by DNA polymerase, using the plasmid as template.
Usually an unlabelled oligonucleotide is used, plus all 4 deoxynucleotide triphosphates (dNTPS)
Plus a mixture of different 2'-3'-dideoxynucleotide triphosphate (ddNTPs), each of which has a characteristic fluorescent label attached.
ddNTPs can be incorporated by the polymerase, but cause synthesis of the chain to stop,
since they have no free 3'-OH to serve as acceptor of 5' PO4 in the polymerization reaction.
Thus, when a ddNTP is
incorporated into the DNA product, the DNA chain cannot be extended and a
specific colored label is incorporated at the same time.
DNA products are denatured (separated from the template) and the products are separated according to size by electrophoresis on a polyacrylamide gel or capillary tube.
As the samples run off the bottom of the sequencing gel, they are detected by the laser fluorimetry and automatically recorded.
polymerase: usually thermostable polymerases and Òcycle sequencingÓ similar to PCR.
Automated sequencing of genomes of humans and other model organisms is revolutionizing the way molecular biology is done. The other player in this revolution is the Polymerase Chain Reaction
Polymerase Chain Reaction
(PCR)
PCR is the amplification of a specific DNA fragment in vitro, directed by using a pair of oligonucleotides to prime DNA synthesis of the region between the primers using DNA polymerase.
Successive rounds of DNA synthesis are accomplished by first heating the template DNA and primers to 94¡ to denature the template (separate strands),
cooling to approximately 50¡ to allow the primers to anneal (base pair) to the template,
followed by a polymerization reaction using DNA polymerase
The cycle of DNA denaturation, annealing and synthesis and polymerization is repeated many times.
Each time the reaction is repeated, the products of the previous cycle are used as template, as well as the original template, causing an exponential synthesis of the fragment of DNA between the primers.
This process was greatly facilitated by the use of thermostable DNA polymerases and automatic temperature cylers.
Using this procedure, a single DNA molecule can be specially amplified and detected.
Used in a myriad of ways in molecular biology.
Need not know sequence of DNA between the primers,
so can amplify DNA of related organism based on primers of regions known to be conserved.
Or use sequence identified from genome sequencing projects
Sensitive method to detect small amounts of contaminating bacteria, mutants cells, etc.
Can be used in combination with reverse transcriptase to amplify specific mRNA sequences.
Choice of polymerase is again important. Thermus aquaticus (Taq) most commonly used, but it is error-prone, lacks proofreading 3'->5' exonuclease and loses activity after about 35 cycles at high temperature.
Other organisms, like Pyrococcus furiousus, produce polymerases that are more thermostable and less error prone and have proofreading function. Allowing more faithful replication of longer genes (up to 40 kb)
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Many different strategies, all require: synthetic oligonucleotide(s) with desired mutation extending oligonucleotide by DNA polymerase to reconstruct entire sequence |
strategy for eliminating starting Òwild-typeÓ template
e.g. Òquick changeÓ strategy:
Cloning gene of interest:
Usually have to isolate gene of interest from a library of clones.
Library - representative collection of all (hopefully) DNA encoded in genome or cDNA of all mRNAs expressed in cells.
Genomic DNA - usually partially digested with restriction endo or mechanically sheared and ligated into vector.
--> overlapping clone sequences which span the genome, only a few of which contain your gene.
cDNA - copy of mRNA sequence made by reverse transcription of mRNA using retroviral reverse transcriptase. --> only exon sequences, much more convenient than genomic sequence for most genes (but may lose important regulatory sequences). Can make library of expressed genes for specific tissue.
For either type of library, you need a probe to detect the gene you want:
homologous DNA from related organism
cDNA for obtaining genomic clone (and vice versa)
antibody (need to use expression library)
oligonucleotide probe based on protein sequence
PCR product obtained from oligos based on shorter regions of homology
RFLP marker that maps near the gene you are interested in.
Need to know how many colonies (or phage plaques, which are usually easier to screen) are necessary to screen and be reasonably sure to get your clone.
to screen genomic DNA:
P= 1-(1-f)N or N=ln(1-P)/ln(1-f)
where f is size of average insert/size of genome, P is probability, and N= number of clones to screen
for 10kb insert you would need 2200 clones to screen the E. coli genome of 4640kb. This could be done on a single small petri plate
You would need 1.4 million of these inserts to screen the human genome
this would require nearly 30 large petri plates.
(expression libraries require even more: gene has to be in proper orientation and reading frame for detection with antibody)
The library is plated out
and after plaques are seen, a nitrocellulose or nylon filter is overlaid onto
the plate and marked to allow realignment of filter and plate later on. Filter
is treated and probed just like a Southern blot
Once the filters are probed, washed and dried, they are exposed to film. Black spots indicated putative clones of interest.
Once isolated, clones must be tested to determine whether they represent the gene of interest. One common test is the Northern blot to determine whether the DNA is homologous to a mRNA in the cell type of interest.
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