I. PostTln processing of proteins:

1

• I. Post-Tln processing of proteins
• -some ptns secreted (a leader sequence called a
  signal sequence might be added at N-terminus.
  hydrophobic 5-25 aa long SEE FIG 10-41
• -some are phosphorylated, acetylated, methylated,
  etc.
• PTN Splicing intervening ptn sequences (IVPS)
  removed or spliced out like RNA autocataltyic.
  Note that all IVPS contain endonuclease activity
• II. Universality of the code Appears common to
  all organisms with minor differences in
  mitochondria (see text)
• Also TLN seems extremely well conserved.
• In vitro Tln can be programmed by foreign
  mRNA.
• In vivo Tln (microinject mRNA into cytoplasm)
  can as well.

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Division of genetic labor in cells and genome
size.Size may (or may not) relate to complexity
yeast 6217 ptn genes (140 rRNA, 40 snRNA, 275
tRNA) COMPLETE SET OF PTN CODING GENES
PROTEOME Distribution of essential core functions
below
3
Recombinant DNA Technology
Chapter 12
4
Goal of Genetics Get at gene function/structure
and details From Mendel to Benzer derivative
information based on mutations, heritable changes
etc. All indirect. No genes had been isolated
and studied in pure form. Rec. DNA changed all
that! Why is gene isolation important?-gets the
sequence to give consensus data, landmarks in
genes, conservation, insights into genetic
evolution, evolutionary relationships-gets at
gene FUNCTION mutate replace in organism, make
transgenic animals -Commercial/pharmaceutical
produce ptn based drugs or valuable gene products
for human medicine. Making Recombinant DNA A
how-to primer on cloning. OVERVIEW note this
is molecular cloning genomes are complex (MANY
genes). Cloning allows for isolation and
amplification of a SINGLE gene. Start with Donor
DNA, cut into clonable fragments, insert into
vector, produce VECTOR PLUS INSERT to give
Recombinant DNA
INSERT
Plasmid vector
5

• Cloning basics
• Cloning works since donor DNA in many fragments
  each forms a recombinant with individual vector
  DNA molecules
• Each individual chimera (foreign DNA vector)
  enter host bacterium which amplifies and
  therefore clones DNA

Recombinant DNA means unnatural union between
species DNA. Process Rec. DNA technolgoy
Individual bacterial clones can be selected as
isolated colonies and DNA plasmid amplified.
6
NOTE resistance markers very important!
Details of the process FIRST VECTOR DNA
(plasmid, phage etc.) -ISOLATE PLASMID DNA (CsCl
gradient shown). Plasmid DNA is extrachromosomal
element in host (E. coli typically). -Must
separate small, circular plasmid from bulk host
DNA in vast excess.
NOTE host DNA denatures plasmid does notwhy??
7
Second Step Cutting DNA relies on RESTRICTION
ENZYMES (RE) -RE a defense in bacteria against
phage invasion (foreign DNA eliminators -Cut
SPECIFIC DNA sites All DNAs have sites (by
chance) but frequency depends on seq.Example of
an EcoRI site (Eco E. coli) 5----------GAATTC--
---------33----------CTTAAG-----------5 Key
issuesPalindrome like the word MADAM or DAD
or sentence MADAM IM ADAM or 2002-after
cutting staggered ends 5----------GAATTC------
-----3 3----------CTTAAG-----------5
5----------G AATTC-----------33----------CTT
AA G-----------5
These are sticky ends
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With the plasmid VECTOR you want a SINGLE site
to clone into
IF vector and donor are cut, this gives
isosticky ends that can easily align and
hybridize prior to ligation with E. coli DNA
ligase
Multiple sites??? That is not going to work.
Here is why
No Sites
Cannot accept insert
1 site
Can accept insert to give selectable recombinant
2 sites
Can accept insert but due to rearrangement, no
recombinants possible.
Sticky ends are ideal for facilitating ligation
and proper (forced) joining, but blunt ends (some
RE blunt end cuts) will still work. Also,
mechanical shearing of Donor DNA is possible.
9
Different RE used in cloning (there are
MANYhundreds of these)
Features Can be 6 base recognition or can be 4
base recognition element If cuts are at symmetry
central site you get blunt end products If cuts
are symmetrically PLACED around the center of
symmetry you get cohesive or sticky ends. of
cuts in a genome is LIMITED and based on
Sequence. One RE will give a specific set and
another will give a different set of
fragments. Frequency of cutting?? 4 base cutter
44 256 6 base cutter 46 4096
Note methylation of bases above. Most RE are
paired with methylases that methylate the same
site. This protects the host genome from
internal degradation (since methylated DNA
resists cutting). BUT if an invading genome
(phage) entersit would be destroyed
. RESTRICTION/METHYLATION system
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Joining/ligating DNA Cohesive termini can bind
but MUST be ligated to seal.
Recall DNA ligase from the DNA replication?
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Cloning and gene amplification
Colonies clones
12
GENE SPECIFIC CLONING GENE SELECTION (goal is to
identify Y.F.G. or your favorite gene)
Cloning Vectors many to select from. Ideal
vectors have following characteristics -Small,
high copy number-Convenient RE sites A
polylinker-Negative or Positive selection
markers (to recover DNAs of interest)-Visible
selection (color selection)
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Plasmids Naturally occurring, small cirucular,
autonomous replicating, high copy number
(gt100/cell!), drug resistance markers
(genes).-DRUG RESIS use to identify
transformants AND clones possessing an insert of
interest. Ampicillin bacterial host cells are
sensitive and dieBacterial hosts having the
plasmid with amp, do not die Tetracyline the
gene is disrupted by insertion of foreign DNA
FIG 12-6 pBR322 an early vector
Tet selection for insert positive plasmids
Note all of the single sites for cloning
Amp selection for transformants
Note Insert inactivates the Tet gene
This is what you want (Tet sensitive) with
insert, but, you still have to further
characterize it to get YFG
14
pUC series of vectors newer with upgrades
(arrows)
Polylinker Many single cloning sites available
Color selection In frame ORF with polylinker,
encoding Beta-Galactosidase If BG is active,
colonies appear BLUE, ie, BG has NOT been
inactivated and these colonies are not
relevant. IF colony is white, BG has been mutated
(insertionally inactivated) and no BG. Color
selection X-gal (colorless substrate for BG).
If BG is active, X-gal converted to a blue
color.select for WHITE/AMPr
Plasmid vectors are size limited typically
inserts gt10 KB unstable and are eliminated.