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HHMI Research Studio
Freshmen Biology Section
Instructor: Michael Lehmann
Mouse Lipin 1 controls adiposity
Control
Lipin 1 mutant
Lipin 1 up
Lipin 1 normal
Peterfy et al., 2001: Nature Genetics 27, 121-124
Phan & Reue, 2005: Cell Metabolism 1, 73-83
Lipin’s domain structure is highly conserved
SWR
SWR
SWR
NLIP domain
mLpin1
hLPIN1
mLpin2
hLPIN2
mLpin3
hLPIN3
D.m.
C.e.
S.c.
S.p.
A.t.
P.f.
CLIP domain
mLpin1
hLPIN1
mLpin2
hLPIN2
mLpin3
hLPIN3
D.m.
C.e.
S.c.
S.p.
A.t.
P.f.
mLpin1
hLPIN1
mLpin2
hLPIN2
mLpin3
hLPIN3
D.m.
C.e.
S.c.
S.p.
A.t.
P.f.
mLpin1
hLPIN1
mLpin2
hLPIN2
mLpin3
hLPIN3
D.m.
C.e.
S.c.
S.p.
A.t.
P.f.
*
dLipin
Lipin1_human
IFSSPNLVVRLNGKYYTWMAACPIVMTMITFQKPLTHDAIEQLMSQTVDGKCLPGDEKQE 656
IIDDPNLVVKIGSKYYNWTTAAPLLLAMQAFQKPLPKATVESIMRDKMP----------- 547
*:..*****::..***.* :*.*::::* :*****.: ::*.:* :.:
dLipin
Lipin1_human
AVAQADNGGQTKRYWWSWRRSQDAAPNHLNNTHGMPLGKDEKDGDQAAVATQTSRPTSPD 716
--------KKGGRWWFSWR-GRNTTIKEESKPEQCLAGKAHSTGEQPPQLSLATR----- 593
: *:*:*** .:::: :. .:..
** .. *:*.. : ::*
Excision of “SWR peptide” from Lipin gene
1. Cut dLipin gene with restriction enzymes on either side of conserved sequence and remove excised
sequence
2. Insert short DNA sequence to restore open reading frame (ORF) and ligate
3. Transform modified gene into bacteria
4. Select clones that harbor correctly modified gene by PCR (Polymerase Chain Reaction)
Chemical composition of DNA
• deoxyribose (sugar)
• 4 organic bases (pyrimidines and purines)
• phosphoric acid
DNA = deoxyribonucleic acid
RNA = ribonucleic acid
DNA contains organic bases, pyrimidines and purines:
9
sugar
Thymine
1
1
Nucleoside (Thymidine)
in DNA:
Thymine
Nucleoside (Thymidine)
in RNA!:
Uracil
Nucleoside (Uridine)
Nucleosides1)
+
Cytosine
+
Adenine
+
Guanine
+
Ribose
Ribose
Ribose
Ribose
Uridine (U)
Cytidine (C)
Uracil
1) in DNA: prefix Deoxy…, e. g.:
Thymine
+
Deoxyribose
Deoxythymidine (dT)
Adenosine (A)
Guanosine (G)
Nucleotides are the basic building blocks of DNA
Phosphate group
1
Nucleotide (2’-Deoxythymidine monophosphate)
DNA = deoxyribonucleic acid
2’-deoxy-
2’-deoxy-
DNA is a polar molecule:
5’
3’
Erwin Chargaff, 1949:
Chargaff’s rules: A=T
C=G
ratio of pyrimidine to purine = 1:1
A+T ≠ C+G
Watson and Crick, 1953: The double helix model of DNA structure
based on:
1. Chargaff’s rules
2. X-ray diffraction analysis of DNA structure
• Rosalind Franklin and Maurice Wilkens, 1953:
• DNA has helical structure
• DNA consists of two polynucleotide strands
Watson and Crick, Nature, 1953:
“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic material”
Denaturation of DNA = separation of DNA strands
Renaturation of DNA = Hybridization:
joining of two complementary DNA strands
The two DNA strands are antiparallel:
(10 base pairs)
Sugar-phosphate
backbone
DNA strands of double helix serve as templates
for synthesis of complementary strands
Reverse transcription
Francis Crick,1957: The “Central Dogma” of molecular biology
Within each cell, genetic information flows from DNA to RNA to protein
Ways to encode sequences of 20 different amino acids…..
4 nucleotides (A, T, C, and G)
two-letter code:
42 = 16
three-letter code: 43 = 64
triplet = codon = 3-lettered code word
Nirenberg & Leder, 1964: The triplet binding assay
Nirenberg & Leder, 1964: The triplet binding assay
The genetic code:
Stop
Stop
Stop
Met
Stop codon = nonsense codon (UAG = amber; UAA = ocher; UGA = opal)
Start codon = initiation codon = AUG (for N-formylmethionine in bacteria)
The genetic code…….
• is a triplet code; a codon consists of 3 nucleotides
• is nonoverlapping: each nucleotide is part of only one codon
• is commaless: no punctuation is used to separate the code words
• includes start and stop codons that set the (open) reading frame (ORF)
• is degenerate: an amino acid can be specified by more than one codon
• is unambiguous: each codon specifies only one amino acid (one exception)
• is universal: is used by all organisms and viruses (with minor exceptions)
non-overlapping:5’-AGTTAGTTCCAGTAAGGTTAACC-3’
overlapping: 5’-AGTTAGTTCCAGTAAGGTTAACC-3’
The genetic code is nearly universal
1979: deviations in mitochondria
Triplet
normal
code
altered
code
UGA
Stop
trp
mitochondria (human/yeast)
Mycoplasma
CUA
leu
thr
mitochondria (yeast)
AUA
ile
met
mitochondria (human)
AGA
AGG
arg
stop
mitochondria (human)
UAA
stop
gln
Paramecium
Tetrahymena
UAG
stop
gln
Paramecium
Where?
Frameshift mutations
THE FAT CAT ATE THE BIG RAT
Insertion:
Deletion:
Insertion
+
Deletion:
THE FAT CAA TAT ETH EBI GRA T
T
THE FAT CAT AET HEB IGR AT
T
THE FAT CAA TAE THE BIG RAT
The genetic code is a triplet code
THE FAT CAT ATE THE BIG RAT
3 Insertions:
THE FAT TCA AGT ATE THE BIG RAT
Cloning of DNA
Restriction enzyme digest
linearized vector
Ligation
recombinant DNA
Transformation of E. coli
Plasmid
E. coli chromosome
Ampicillin-resistant clones
further selection
Restriction and modification in bacteria
progeny grows unrestricted on K12
axis of symmetry
5’
5’
sticky ends
• restriction sites are often palindromic
Palindrome: MADAM I’ M ADAM
OT TO
Restriction fragments can have different ends
Frequency of restriction sites
4n
n = # of bp in site
bp = base pairs
1000 bp = 1kb (or kbp)
Restriction mapping
cloned DNA fragment
separate restriction fragments
by gel electrophoresis
_ (cathode)
agarose gel
+ (anode)
construct restriction map
DNA ligase closes the remaining open phosphodiester bond
nick
Kary Mullis, 1986
PCR: Polymerase Chain Reaction
• only minute amounts (as little as 1 DNA molecule) of starting material required!
denaturation
PCR
cycle
extension
• Taq from thermophilic bacterium, Thermus aquaticus
annealing
PCR: Polymerase Chain Reaction
22 cycles
> 4 million copies from a single DNA molecule
Structure of a eukaryotic gene
gene: nucleotide sequence that encodes a protein or a functional RNA; it
includes regions that are required for the regulated expression of the gene
upstream
5’
3’
promoter
-20
nontemplate (RNA-like) strand
downstream
+1
5’
5’-UTR
+20
ATG
intron
exon
AUG
template strand
3’-UTR: 3’ untranslated region (trailer)
3’-UTR
3’
3’
5’
hnRNA (pre-mRNA)
transcription unit
5’-UTR: 5’ untranslated region (leader)
UGA
Each of the two strands of a DNA double helix can be the template strand
Gene 1
5’
3’
5’
RNA
3’
3’
RNA
5’
3’
5’
Gene 2
1)
> 80 introns!
< 1% of hnRNA (not drawn to scale!)
1) defect in genetic disorder called Duchenne muscular dystrophy
Construction of a cDNA library
Purify mRNA from specific cell type,
e.g.
cDNA = copy or complementary DNA
Transform E. coli
Insert cDNA in vector
Life cycle of a retrovirus
(provirus)