1. Molecular biology: Tools and techniques
Cloning: clone=a group of genetically identical cells derived by mitosis from a single
ancestral cell.
cDNA=DNA synthesized from an mRNA template, using reverse
transcriptase
hybridization=complementary pairing of an RNA and a DNA strand or
of two different DNA strands.
Cloning
a gene requires a probe with some amount of homology to the gene of
interest. Positional cloning
refers to identification of a gene based only on knowledge of its position in
the genome.
cDNA libraries: large collections of recombinant DNA clones, in
which cDNA fragments have been inserted into a particular vector. *remember---cDNA contains only
the transcribed and processed exons, whereas genomic DNA has exons and introns
*
PCR: (Polymerase Chain Reaction) a
method of amplifying a short sequence of DNA from a complex mixture. This allows you to study a particular
DNA fragment without first having to clone it. It depends on using two flanking oligonucleotide DNA primers
and repeated cycles of primer extension using DNA polymerase. Can also be used to amplify specific
RNA sequences via cDNA.
Steps: (1) DNA is heat
denatured into 2 strands. (2) primers anneal to specific sequence on strand to
be amplified. (3) heat-stable DNA pol replicates the DNA sequence. Steps are repeated multiple times for
amplification.
RFLP: (Restriction Fragment Length Polymorphism) a
variation in DNA sequence that alters the length of a restriction
fragment. These can be point
mutations (which can create or destroy a restriction site) or variable length
regions (called VNTRs). RFLPs are
convenient markers for linkage analysis.
A restriction site is a
sequence which is recognize by a restriction enzyme (like EcoRI), so then the
DNA will be cut at that sequence.
Three
important things to remember: (1)
usually the base change detected isn’t the one responsible for the
disease. (2) the inheritance of
RFLPs follows Mendelian expectations. (3)
If used to follow the inheritance of a chromosome in a family, the most
useful one is the RFLP for which most individuals are heterozygous.
VNTRs
make our “DNA fingerprint” and are used in paternity tests and
forensics.
Restriction Enzymes: (aka restriction endonucleases) enzymes, purified from bacteria, which
have the ability to cut double-stranded DNA at a specific nucleotide
sequence. Some generate sticky
ends (EcoRI, BamHI, PstI), while others have blunt ends (SmaI, HaeIII, AluI).
Sequencing: determining the exact nucleotide sequence of a
PCR product or cloned fragment of DNA.
Most common method=the Sanger method (enzymatic approach).
Southern blot: a DNA sample is electrophoresed on a
gel and then transferred to a filter.
Filter is soaked in denaturant and then exposed to a labeled DNA probe
that recognizes and anneals to the complementary strand. Results in labeled dsDNA that can be
visualized when filter is exposed to film. Key: DNA-DNA hybridization
Northern blot: same technique as above, but use radio-labeled
DNA probe that binds to sample RNA.
Key: DNA-RNA hybridization.
Western blot: sample protein is separated by gel
electrophoresis and transferred to filter. Labeled antibody then binds to the relevant protein. Key: Ab-protein hybridization.
Southwestern blot: protein sample run on a gel, transferred to
filter, exposed to labeled DNA.
This is used to detect DNA-protein interactions (e.g. transcription factors).
2.
Transcriptional regulation:
A. the
operon model of transcription
Lac operon: The
lac operon is an example of negative control because binding of the repressor
block transcription.
gene
responsible for lactose metabolism in E.coli. looked at constitutive mutants (expressed all three metab
genes even when lactose not available) and noninducible mutants (didn’t
express any genes even in the presence of lactose). Found two loci: o = constitutive expression, i = consitutive or
noninducible.
o
is the operator unit of the operon…it controls the
transcription of adjacent genes.
i is the
repressor unit of the operon…it’s a regulatory factor; when
it’s bound to o it blocks transcription (binds in the
absence of lactose/presence of lactose inhibits repressor binding). So the presence of lactose inhibits
transcription.
|
Genotype |
Phenotype
|
|
I+/o+ |
Inducible |
|
i-/o+ |
Constitutive |
|
I+/I- |
Normal |
|
I+/oc |
Constitutive |
Trp operon: The trp
operon is an example of transcriptional attenuation where gene expression is
regulated by controlling the ability of RNA polymerase to continue elongation
past specific sites.
Gene
responsible for tryptophan synthesis in E.coli. Trp operon is regulated by repressor that blocks
transcription when bound to tryptophan.
But there is also an attenuator sequence that mediates expression by
prematurely terminating transcription when tryptophan levels are high. So the presence of tryptophan leads
to repression and transcription attenuation.
B.
Eukaryotic transcription:
TATA box: a conserved sequence 25-30 bp upstream from the
start site of transcription in many, but not all, genes. Apparently involved in the initiation
of transcription.
Enhancers: DNA sequences that act in cis to
increase transcription of a nearby gene.
Can act in either orientation, may be either 5’ or 3’ to the
gene, and may act at considerable distance from the gene.
Transcription factors: proteins that act by
binding to specific DNA sequences within the promoter or enhancer of the target
gene to increase, decrease, or otherwise modulate the level of gene expression.
Steroid hormones: steroids
cross the cell membrane and bind to nuclear receptors, accumulate in the
nucleus and then bind to regulatory DNA sequences (hormone response elements,
HREs). HREs are found in the
enhancer regions and can stimulate or inhibit gene transcription.
3.
Protein synthesis:
Components for translation:
Amino
acids (know which ones are
essential)
tRNA: humans
have 50 types, bac have 30-40
each
tRNA has aa attachment site and
anticodon
mRNA: acts as template
aminoacyl-tRNA
synthetases: to attach aa to tRNA
**
this step requires ATP **
functional ribosomes:
A site binds the incoming aa-tRNA;
the P site binds
the
peptidyl-tRNA.
Protein factors: need initiation, elongation, and
termination factors.
ATP and GTP: need four high energy phosphate bonds to add one aa to
chain.
One ATP for aminoacyl-tRNA synthetase rxn, and
two from GTPs (1 binds to A site, one for
translocation).
Also need ATP and GTP for initiation and termination.
Steps:
1. Initiation: assembly of all the parts. Two mechanisms for ribosome recognizing
the initiation sequence – a. Shine-Dalgarno sequence (in E.coli); b.
initiation codon (AUG), facilitated by eIF-2 in humans. In bac and mito, this initiator tRNA
has a N-formylated methionine. In
eukaryotes the methionine is not formylated. This N-terminal methionine is removed before the protein is
completed.
2. Elongation: ribosome moves from the
5’ end to the 3’ end of the mRNA. Elongation
factors in E.coli: EF-Tu and EF-Ts (need GTP). In eukaryotes, eEF. Translocation requires
Ef-G and GTP in E.coli (similar in eukaryotes).
3.
Termination: when one of the 3 termination codons
moves into the A site. Release
factors recognize these codons.
Inhibitors of these steps:
Streptomycin: binds to 30S subunit and distorts the structure.
Tetracyclines: interact with the small ribosomal subunits, block
access of t-RNA to
ribosomal complex.
Puromycin: becomes incorporated in the chain, stopping
elongation (in pro- &
eukaryotes)
Chloramphenicol: stops prokaryotic peptidyltransferase
Clindamycin/Erythromycin: bind irreversibly to 50S subunit.
Diptheria
Toxin: inactivates the eukaryotic
elongation factor, eEF-2.
Post-translational
Modifications:
1. Trimming (e.g. zymogens becoming activated)
2. Covalent alterations:
Phosphorylation of hydroxyl groups (e.g. regulation of glycolysis,
glycogen syn.)
Glycosylation
(proteins destined for plasma
membrane or secretion)
Hydroxylation
(e.g. proline & lysine of
collagen)
4. Acid-base titration curve of amino acids, proteins
Not really sure what they
want here…see Chapter 1 of Lippincott…
Henderson-Hasselbalch: pH
= pKa + log [A-]/[HA]
Buffer region is about +/- 1
pH unit from the pKa of a weak acid. Maximum buffering at the pKa. If an aa has a side chain with a pK
within 1 of the desired pH, it is an effective buffer for that pH.
Zwitterion = isoelectric
form of an amino acid…has overall charge of zero.
Isoelectric point (pI): the
pH at which an amino acid is electrically neutral.
At physiologic pH, all
amino acids have both a negatively charged group (-COO-) and a
positively charged group (-NH3+). They are dipolar zwitterions.