转基因在生物医学中应用的理论与技术
转基因,
在细胞、组织或整体水平上，利用物理、化
学或生物学等手段导入外源基因或外源 DNA，
从而使受体基因组发生改变的一种方式。
生物医学中的应用：
确定基因功能、建立疾病的动物模型、药物
研发、基因治疗、细胞与器官研究、其他等。
教内 容
(一 ） 转基因在生物医学中的应用理论
1,分子生物学概论,( 黄芳, 05/11/12)
2,表观遗传学（ epigenetics） 导论：（傅继梁,05/11/19）
3,模式生物概论：（费俭,05/11/26）
4,病毒学概论：（路阳,05/12/17）
（ 二 ） 转基因在生物医学中的应用技术
1,基因导入； （ 肖啸, 路阳 )
1,1,病毒载体介导的基因导入；
A,腺病毒, 腺相关病毒载体与应用,(05/12/24）
B,逆转录病毒, 慢病毒载体与应用,(05/12/31）
1,2,非病毒载体介导的基因导入,（ 05/12/31）
A,蛋白转导技术；
B,物理或化学方式介导的基因导入 。
2．可调控系统及组织与细胞特异启动子,（ 路阳,05/12/31 )
3,转基因动物：（费俭,05/12/3）
4,基因打靶,(黄芳,05/12/10)
5,基因治疗：（路阳,06/1/7）
基本要求
每次课程的第四节课为学生文献报告时间，每次有四位
学生报告，报告的文献由授课老师提供，并从, 准备、理解、
表达, 几方面给学生一个评价。所有的学生还需在 2006.1.13
号之前交一份读书报告（三千字以内，内容自定）。
何昆燕,kekunyan@21cn.com Tel,54237510
黄 芳, huangf@shmu.edu.cn Tel,54237296
References
Fundamentals of Molecular Biology
? 52th Anniversary of Watson & Crick
? Completion of human genome project
Protein
RNA
DNA
transcription
translation
CCTGAGCCAACTATTGATGAA
PEPTIDE
CCUGAGCCAACUAUUGAUGAA
Central Dogma,DNA -> RNA -> Protein
Central Dogma
? Gene/ Promoter;
? RNA ;
? Homologous recombination；
? Cre-loxP,FLP-FRT；
? Vectors;
Genes
? Protein Coding
? RNA genes
–MicroRNA
–rRNA
–tRNA
–snRNA,snoRNA…
How many genes are there in mammalian cells?
E,coli 4.6 Mb 4,288 genes
S,cerevisiae 13.5 Mb 6,034 genes
C,elegans 97 Mb 19,099 genes
D,melanogaster 165 Mb 12,000 genes
H,Sapiens 3,300 Mb 40,000 genes
Genome project was completed in 2002 (still regions that are unclear)
Gene expression profiling (Exon profiling)
Phenotypes
Studied on the mechanism
of gene expression.
Genomics and proteomics
Transgenic technologies
Distal
>100kb
Proximal
Approx,1kb
Promoter Transcription
Unit
Coding and uncoding sequences
from 1kb to >200kb
Gene Locus:
Includes both the promoter and the transcription unit!
? Cis elements,sequences on DNA that affects
the level of transcription.
? Trans factors,DNA-binding proteins that
change the level of transcription by basal
transcription machinery.
How genes affect each other
Cis elements,sequences on DNA that affects the
level of transcription.
ACTTCATCTAGTATAATACTCATCCCGCGTA…
ACTTCATCTAGTGTACATACTCATCCCGCGTA…
Cis element may be a promoter
e.g,this may be a strong signal,
and the green protein may bind
to it strongly.
This is a weakened version of
the signal,so the protein doesn’t
bind,or maybe binds less strongly.
Examples of Trans Factors
Some genes inhibit other genes; suppose protein Y sticks to DNA at
the precise pattern CAGGCG
genePromoter
The transcription factors just can’t get to where they need to be,so if protein Y
is being produced,then genes with this pattern nearby just won’t be expressed.
CAGGCCG
Prokaryotic Gene Expression
Promoter Cistron1 Cistron2 CistronN Terminator
Transcription RNA Polymerase
mRNA 5’ 3’
Translation Ribosome,tRNAs,Protein Factors
1 2 N
Polypeptides
NC N C N
C
1 2 3
The Prokaryotic Promoter
Determines where transcription begins – what site and
which strand of DNA acts as template
Promoter Structure in Prokaryotes (E.Coli)
Transcription starts
at offset 0.
? Pribnow Box (-10)
? Gilbert Box (-30)
? Ribosomal
Binding Site (+10)
Eukaryotic Gene Expression
Promoter Transcribed Region Terminator
Transcription RNA Polymerase II
Primary transcript 5’ 3’
Translation
Polypeptide
NC
Enhancer
Exon1 Exon2
Intron1
Cap
Splice
Cleave/Polyadenylate
7mG An
7mG An
Transport
Definitions
? Intron (intervening sequence),
– a nucleotide sequence within a gene that is transcribed into
RNA but excised before translation,
– Intron size,50 – 20,000 nts
? Exon:
– A subsegment of a gene that encodes a portion of the final
geneproduct,This subsegment remains after processing and
is ultimately translated into a polypeptide or incorporated
into the structure of an RNA molecule.
– Most exons are < 1000 nts; majority b/n 100 – 200 nts
Primary transcript with introns
Eukaryotic Gene Complexity
? Yeast
– introns rare
– promoters
adjacent
– genome
dense
? ―large‖ Eukaryotes
– introns common,
LONGER than
exons
– Promoter/enhancer
– genome sparse
? Fungi
– introns common,
short relative to
exons
– promoter/enhancer
– genome dense
Intron Prevalence
0
10
20
30
40
50
60
70
80
90
100
0 1 >1
Yeast
Fungi
Mammal
Intron Size
0
10
20
30
40
50
60
70
<100 <200 <1
kbp
1 to
5
>5
Fungi
Verterbrate
Exon Size
0
5
10
15
20
25
30
35
1-
100
100-
200
200-
300
300-
500
>500
Fungi
Verterbrate
0
5
10
15
20
25
30
35
1-
100
100-
200
200-
300
300-
500
>500
Fungi
Verterbrate
Eukaryote Promoter
? Goldberg-Hogness or TATA located at –30
? Additional regions at –100 and at –200
? Possible distant regions acting as enhancers
or silencers (even more than 50 kb),
Basal Promoter Analysis
? ATATAA -30 TBP
? GGCCAATC -75 CTF/NF1
? GCCACACCC -90 SP1
TATACAATGC
+1
DATABASE SEARCH
? BLASTN
– DNA:DNA comparison (ALWAYS!)
– Not sensitive (DNA conservation low)
? BLASTX/TBLASTX
– ?6 frame ORFS:polypeptide database
– 6 frames vs,6 frames of a DNA database
Classes of RNA
? Informational RNA,protein encoding mRNA
– translated into amino acid sequence
? Functional (structural) RNA
– MicroRNA,regulation of transcription and translation
– tRNA,transports amino acid to ribosome;
– rRNA,structural and catalytic component of
ribosomes
– snRNA,structural and catalytic component of
spliceosome snRNPs
– snoRNA,small nucleolar RNA involved in maturation
of rRNA
– scRNA,directs protein traffic in cytoplasm
RNA polymerases
? Prokaryotes,single RNA polymerase
– Transcribes mRNA,rRNA and tRNA
– Transcription and translation are coupled
? Eukaryotes,three RNA polymerases
– RNA polymerase I transcribes rRNA genes
– RNA polymerase II transcribes protein-encoding genes; i.e,
makes mRNA
? primary transcript will be processed
– RNA polymerase III transcribes tRNA genes and 5S rRNA
genes
? Transcription and translation occur in separate
compartments of the eukaryotic cell
– In organelles they occur in the same compartment
Transcription
? RNA polymerase catalyzes RNA synthesis
– uses one DNA strand as template
? always the same strand for a given gene
– locally unwinds DNA
– adds free nucleotides to growing RNA strand at 3’ end
? 5’ to 3’ RNA synthesis
? template read 3’ to 5’
? uses rules of base pairing to synthesize complementary RNA
molecule
? starts RNA chain de novo
? Transcript is identical in sequence to nontemplate
strand,except T’s replaced by U’s
Transcription is asymmetric – only one strand of the DNA is transcribed
into RNA; the template strand
The RNA transcript has the same sequence as the nontemplate strand
RNA is synthesized in a 5’ to 3’ direction only
The template strand is read in the 3’ to 5’ direction
sense
anti-sense
Transcription steps
? Initiation
– at 5’ end of gene
– binding of RNA polymerase to promoter
– unwinding of DNA
? Elongation
– addition of nucleotides to 3’ end of growing chain
– governed by rules of complementary base pairing
– energy from NTP substrates
? Termination
– at 3’ end of gene
– terminator loop (prokaryote) or processing enzyme
coding region5?UTR 3?UTR
Elongation – the addition of ribonucleotides to form the RNA
chain occurs in a 5’ to 3’ direction
Termination is dependent on
a specific sequence of
nucleotides
Elongation & Termination
Eukaryote mRNA processing
? 5’ end,capping
– addition of 7-methylguanosine to the 5’-end
– linked by three phosphates
? 3’ end,poly(A) tail
– addition of 150-200 adenine nucleotides to 3’ end
– downstream of AAUAAA; polyadenylation signal
? Intron removal by spliceosome
– all introns have 5’GU and 3’AG recognition sequence
(GU – AG rule)
– snRNPs of spliceosome provide catalysis
– intron excised as lariat,destroyed Some nonprotein-
encoding genes have
self-splicing introns.
5?- Capping
Capping,Modification of 5’
end of eucaryotic mRNA
– May play a role in
translation initiation
– May provide increase
mRNA stability
5’-cap
– 7-methylguanosine linked to
the 5’-terminal residue of the
mRNA via a 5’ to 5’
triphosphate linkage
Phosphorylase
Guanylyltransferase
Guanine 7-methyl-
transferase
2?-O-methyl-
transferase
PolyA Tail Addition
The endonuclease cleaves the RNA after the AAUAAA signal
The polyA tail is added by polyA polymerase; nontemplated
nucleotide addition
Not all mRNAs have a 3’ polyA tail
Polyadenylate polymerase
? adds 80 – 250 nts to the end of mRNA
? requires the cleaved mRNA as a primer
? does NOT require a template for synthesis
? specific for one nucleotide (ATP)
? increases mRNA stability
RNA+ nATP = RNA-(AMP)n + nPPi
To continue
Removal of Introns
Removal of introns is called splicing
Occurs in mRNA; also occurs in rRNA and tRNA
Introns are removed before the mRNA leaves the nucleus
Splice sites have specific sequences – the GU—AG rule
snRNPs form a functional unit called the spliceosome
Consensus splice sites
snRNAs
snRNA Length (nts) Function
U1 165 Binds 5? splice site,then 3? splice site
U2 185 Binds the branch site and forms part of the catalytic center
U4 116 Masks the catalytic activity of U6
U5 145 Binds the 5? splice site
U6 106 Catalyzes splicing
Splicing mechanism
GU A AGU1 U2
U4 U5
U6
Exon 1 Intron Exon 2
3? splice site5? splice site branch site
U1 U2
ATP
U4 U5
U6
AG
1o transcript
Assembled
spliceosome
Products of
the splicing
reaction
Alternative splicing
? generates protein diversity
? allow generation of different splice variants
– in different tissues
– at various developmental stages
? Estimates,30% of human genes undergo alternative
splicing
Splice variants:
Pre-mRNA
Number of splice variants (SV)
SV = 2 number of internal exons
Structure of tRNA
a,short RNA strand made of 73 to 93 nucleotides
b,some internal complimentarity results in 3D shape
– four short double helix regions with three loops
form a cloverleaf shape (2-D structure)
– five other base-pairs between loops form an,L”
shape (3-D structure))
GAU
CUA
mRNA
3’
5’
tRNA structure
? Single RNA strand
? Internal pairing of bases
? Folding,internal
compliment
– Cloverleaf shape to,L” shape
– Conserved regions
? Modified bases
? CCA sequence –aa
binding
? Anticodon – pairs with
mRNA
– Variable region of tRNA
C
C
A
GC
U A
?
codon
?Degeneracy? of the Genetic Code:
GAU GAU
…..CUA…,….CUG….
leucine leucine
Same tRNA,a.a.
different mRNAs
wobble position
– tRNA matching is less selective in the third
position of the anticodon,the,wobble position”.
– e.g,CGU,CGG and CGA all code for arginine
? There are 50 different tRNA genes,50 tRNAs
? Each gene is present in multiple copies in the
genome.
– Located on different chromosomes
– Multiple copies of the genes allow faster production.
- Transcribed from DNA by RNA polymerase III
tRNA Genes
rRNA has complex structure:
? Lots of internal
structure,base – pairing
? Ribosome function is
both structural and
catalytic - including an
apparent ribozyme
function
? Many bases post-
transcriptionally
modified
The purple area is a catalytic site.
Ribosomal Subunits
? The genes responsible for making the rRNAs primary transcript is cut and
chemically modified
? a large 45S rRNA is copied from the DNA,The source of 3 of 4
rRNAs 28S,18S,5.8S
? The 5s gene is outside the nucleolus.
? Ribosome subunit assembly takes place in the nucleolus,bringing together
the genes,the 45S primary transcript and the processing enzymes and the
proteins
– Finished small and large subunits are sent to the cytoplasm separately
to do their work,i.e,translation
? Genes appear in multiple copies,in clusters in nucleolar organizer regions
– multiple copies of the gene are required to supply the ten million
ribosome particles needed by the cell.
– human cells have 200 rRNA gene copies per genome located on five
different chromosomes (E,coli has seven copies)
– Each gene has multiple copies of the 45S gene,all separated by non-
transcribed regions termed,spacer DNA”
– growing rRNA strands make a,christmas tree”appearance
The rRNA Gene
Multiple RNA polymerases
can transcribe a gene
simultaneously creating a
train of RNA polymerases
What is the direction of
transcription of this
rDNA gene?
The rRNA gene continued:
– 3 of the rRNA genes (28S,18S,5.8S) all originate
from one large primary transcript 45S (13,000
nucleotides) rRNA
1,by RNA polymerase I (a amanitin insensitive)
2,10 transcript is cut up after transcription
3,an additional rRNA gene (5S) is made outside the
nucleolus and then imported
– removal of spacers and base modifications are
done by snoRNPs (snorps)
? protein/RNA structures,snRNProtein/snRNA,
TRANSCRIPTION OF rRNA GENES
direction of transcription
18S 5.8S 28S
nontranscribed spacer DNA gene promoter terminator
multiple gene copies,clustered
RNA polymerase 1
transcription unit
Made as a single 45S unit
Growing rRNA
Copy 1 Copy 2 Copy 3
two things happen to pre-rRNA
2) cleavage
18S 5.8S 28S
1) Post transcriptional base
modification:
- 2’ - O - methylation of some riboses
- conversion U to pseudouridine Y
5S rRNA
- 5S rRNAs are not made in the nucleolus
- therefore do not have a nucleolar organizer
- like other rRNA genes,multiple repetitive copies
-transcribed by a different polymerase (RNA pol III)
- requires little or no processing
- shipped to the nucleolus after synthesis to be
incorporated into the ribosome
? Ribosomal genes have nucleolar organizer activity
?recognition by cell and association at one location,the
nucleolus
?site of ribosome assembly
?proteins and 5S rRNA are imported into nucleolus
?large,small subunits are made separately
?associate in cytoplasm,but not until they are actually
being used in the translation process.
Ribosomal Genes and
Nucleolar Organizing Activity
Ribosomes
? Function,protein translation (in cytoplasm)
? Structure,ribosomes are made of rRNA and
proteins.
– eukaryotic ribosome structure.
? Four rRNAs 28S,18S,5.8S,5S
? plus associated proteins
? Prokaryotic structure is very similar,but lacks
5.8S rRNA
Translation
? mRNA is translated at the ribosome using tRNA
as an adaptor molecule
? nucleotide sequence is read three nucleotides at a
time
– each triplet is called a codon
– each amino acid has one or more codons
– 64 possible codons (4 ? 4 ? 4) = genetic code
? used by all organisms with few exceptions
– no punctuation except start and stop
? Genetic code specifies 20 different amino acids
(sometimes selenocysteine)
The Ribosome
Translation at the ribosome
? Ribosome
– large subunit
– small subunit
? 3 ribosomal sites involved in decoding mRNA
– A site (amino site),accepts incoming charged tRNA
– P site (polypeptide site),peptide bond
– E site (exit site),tRNA exits ribosome
? Amino terminus synthesized first,beginning
near but not at the 5’ end of mRNA
– C-terminal end is made last
? UAA,UAG and
UGA correspond to 3
Stop codons that
(together with Start
codon ATG)
? Delineate Open
Reading Frames
Genetic Code and Stop Codons
Codon Usage in Human Genome
Ribosomal Binding Site in E coli.
Translation initiation in prokaryotes
Circular polysomes in Eukaryotes
Protein structure
? Protein is polymer of amino acids (polypeptide)
– each amino acid has an R group conferring unique
chemical properties
– amino acids connected by peptide bond
– each polypeptide has amino end and carboxyl end
? Structure
– primary,amino acid sequence
– secondary,hydrogen bonding,a-helix and ?-sheet
– tertiary,folding of secondary structure
– quaternary,two or more tertiary structures
? Shape and function determined by primary
structure encoded by gene
Peptide Bond Formation
DNA,the genes,template for RNA production,(transcription)
RNA in the cytoplasm.
mRNA rRNA tRNA
-information transfer to cytoplasm - catalytic machinery used to translate mRNA to protein
22,2 28 5 2
i nt e ri or o f nu c l e us
c yt op l a sm
RNA in nucleus
hnRNA,precursors to mRNA
rRNAs,from large precursor
tRNA
small nuclear RNAs
small nucleolar RNAs
Summary
Review of Eukaryotic mRNA
Production
RNA processing
1,any post-transcriptional modifications of the
primary transcript
2,Note,some modifications take place in both
procaryotes and eucaryotes
3,much more extensive in eucaryotes
Purpose of RNA processing
1,Generally and increase in RNA stability
– 5?-capping
– 3?-adenylation
– modifications of individual residues
– structural changes
2,Increase of genetic variability
– The proteome is much larger than the genome
– Splice variants
? Central Dogma was proposed in 1958 by Francis Crick
? Crick had very little supporting evidence in late 1950s
? Before Crick?s seminal paper
all possible information transfers
were considered viable
? Crick postulated that some
of them are not viable
(missing arrows)
? In 1970 Crick published a paper defending the Central Dogma,
Central Dogma,Doubts
From Conversion to Aggregation:
Protofibril Formation of the Prion Protein
PrPC PrPSc
Low pH
M,L,DeMarco and V,Daggett Proc,Natl,Acad,Sci,USA 101,2293-2298,2004.
MD
To continue
Recombination
? Homologous Recombination - Occurs during
prophase of meiosis I and involves exchange
between homologous strands of DNA
? Site Specific Recombination - Short homologous
sections of bacterial and phage DNA serve as a
site for recombination and thus incorporation of
phage DNA into bacterial chromosomes
Recognition site Recombinase
- Cre-LoxP system (phage P1) LoxP Cre
- FRT-Flp systeem (S,cerevisiae) FRT Flp
Site-specific Recombination Systems
Principles of Cre-Mediated Recombination
? Cre is the 38 kDa product of the cre gene of
bacteriophage P1
? It belongs to the Int family of DNA recombinases,but
unlike other family members,Cre requires no
accessory factors
? Cre recognizes a 34 bp site on the bacteriophase
genome called LoxP,catalyzing reciprocal conservative
DNA recombination between LoxP pairs
?LoxP consists of two 13 bp inverted repeats separated by an 8 bp
asymmetric core region
?This core region is responsible for the directionality of the LoxP
site
?Two molecules of Cre bind to each LoxP site,so that [theoretically]
4 molecules of Cre are required for each recombination event
Cre-Mediated Recombination - tandem repeats
Result,Excision of intervening sequence (with retention of 1 LoxP site)
Cre-Mediated Recombination - inverted repeats
Result,Inversion of intervening sequence
Cloning vectors
? Common properties
– origin of DNA replication
– unique restriction sites for insertion of DNA
? multiple cloning sites containing many restriction sites
engineered into many plasmid vectors
– Easy identification and recovery of clones
? Types of vectors
– plasmids containing drug resistance gene
? many commercially available plasmids
– bacteriophage,e.g.,lambda
– cosmids for larger DNA molecules
– BAC,bacterial artificial chromosome
– YAC,yeast artificial chromosome
Other Replication Vectors
? Bacteriophage - Engineered bacterial
viruses
? Used to infect E,coli cells
? Advantage is the insert size can be much
larger than a plasmid
? Can be maintained with at -80 C almost
indefinitely
Other Replication Vectors
? Cosmids - large plasmid-like
autonomously replicating DNA vectors -
rarely used
? BACs - large linear pieces of DNA with
chromosome like attributes
? YACs - Contain yeast centromere and
telemeres as well as yeast replication
features - can clone up to 1 million bp
Examples of two
plasmid vectors
Replication in the
host cell generates
many,many copies
of the DNA
Selection is based on
absence/presence of
antibiotic resistance
Identification of,clones”
is facilitated with these
vectors through
differential antibiotic
resistance or blue/white
screen
Bacteriophage Vectors
Bacteriophage
vectors are
useful for
cloning larger
DNA
fragments
Bacterial artificial
chromosomes (BACs) are
used to clone much larger
DNA fragments (150-300
kb)
These have been extremely
useful in genome sequencing
projects
Use the F plasmid to
provide replication
functions,selection is for
CAM resistance and the
inserts can be large
DNA is easy to recover for
analysis
Bacterial Artificial Chromosomes
Entry into the Cell
Genomic DNA
Chemically synthesized
DNA
cDNA
PCR-amplified DNA
Generating Recombinant DNA (2)
Methodology
? Southern Blot – DNA
? Northern Blot – RNA
? Western Blot – Protein
? EMSA – DNA&protein
? PCR – DNA amplification
? Mutagenesis
Site-directed mutagenesis
PCR mutagenesis
? FISH –labeled probe
? Microarrays (DNA chip,gene chip,biochip)
– allows for thousands of interactions at once
? RNA interference
? Gene mapping
– Functional cloning,Find protein and
work back
– Positional cloning,Uses known sequences
and markers
Microarray gene expression,Two color image overlay
?Red,higher in Cy5
?Green,higher in Cy3
?Yellow,equal in both
channels
?Brightness reflects intensity
of signal
RNA interference - Mechanism
DICER - RNAse III,ds spec,endonuclease
- Dimer,2 catal,domains,helicase
and PAZ motif
- produce 2-3nt 3′overhangs
- ATP-dependent ribonuclease
RISC - RNA-induced silencing complex
- RISC contains siRNA
- precurser activated by ATP
- find and destroy mRNA
of complementary sequence
- contains endo- and exonuclease,
cleaves the hybrid in the middle
imm,followed by degradation
- ARO,PAZ domain (assembly)
Figure 1
Zamore,P.D,(2001)
Nat,Struc,Biol,9:746
RNAi Pathway
RNAi = RNA i nterference
siRNA = small interfering RNA
siRNP = small interfering Ribonucleoprotein
RISC = RNA Induced Silencing Complex
Dicer
Quantitative PCR (Q-PCR)
T a q - m a n C Y B R g r e e n
Q-PCR (continued)
24.207
23.099
18.571
18.657
2(23.099- 24.207)
2(18.657-18.571)Regulation = => Regulation = -2.27
Inverse PCR
ligate
Clone into vector
Amplify by PCR
Digest genomic DNA with
restriction endonuclease
Methodology
Restriction Enzymes
Xba I TCTAGA TCTAGmATC
AGATCT AGAT CTAG
INV 110,Dam methylase deficient
转基因,
在细胞、组织或整体水平上，利用物理、化
学或生物学等手段导入外源基因或外源 DNA，
从而使受体基因组发生改变的一种方式。
生物医学中的应用：
确定基因功能、建立疾病的动物模型、药物
研发、基因治疗、细胞与器官研究、其他等。
教内 容
(一 ） 转基因在生物医学中的应用理论
1,分子生物学概论,( 黄芳, 05/11/12)
2,表观遗传学（ epigenetics） 导论：（傅继梁,05/11/19）
3,模式生物概论：（费俭,05/11/26）
4,病毒学概论：（路阳,05/12/17）
（ 二 ） 转基因在生物医学中的应用技术
1,基因导入； （ 肖啸, 路阳 )
1,1,病毒载体介导的基因导入；
A,腺病毒, 腺相关病毒载体与应用,(05/12/24）
B,逆转录病毒, 慢病毒载体与应用,(05/12/31）
1,2,非病毒载体介导的基因导入,（ 05/12/31）
A,蛋白转导技术；
B,物理或化学方式介导的基因导入 。
2．可调控系统及组织与细胞特异启动子,（ 路阳,05/12/31 )
3,转基因动物：（费俭,05/12/3）
4,基因打靶,(黄芳,05/12/10)
5,基因治疗：（路阳,06/1/7）
基本要求
每次课程的第四节课为学生文献报告时间，每次有四位
学生报告，报告的文献由授课老师提供，并从, 准备、理解、
表达, 几方面给学生一个评价。所有的学生还需在 2006.1.13
号之前交一份读书报告（三千字以内，内容自定）。
何昆燕,kekunyan@21cn.com Tel,54237510
黄 芳, huangf@shmu.edu.cn Tel,54237296
References
Fundamentals of Molecular Biology
? 52th Anniversary of Watson & Crick
? Completion of human genome project
Protein
RNA
DNA
transcription
translation
CCTGAGCCAACTATTGATGAA
PEPTIDE
CCUGAGCCAACUAUUGAUGAA
Central Dogma,DNA -> RNA -> Protein
Central Dogma
? Gene/ Promoter;
? RNA ;
? Homologous recombination；
? Cre-loxP,FLP-FRT；
? Vectors;
Genes
? Protein Coding
? RNA genes
–MicroRNA
–rRNA
–tRNA
–snRNA,snoRNA…
How many genes are there in mammalian cells?
E,coli 4.6 Mb 4,288 genes
S,cerevisiae 13.5 Mb 6,034 genes
C,elegans 97 Mb 19,099 genes
D,melanogaster 165 Mb 12,000 genes
H,Sapiens 3,300 Mb 40,000 genes
Genome project was completed in 2002 (still regions that are unclear)
Gene expression profiling (Exon profiling)
Phenotypes
Studied on the mechanism
of gene expression.
Genomics and proteomics
Transgenic technologies
Distal
>100kb
Proximal
Approx,1kb
Promoter Transcription
Unit
Coding and uncoding sequences
from 1kb to >200kb
Gene Locus:
Includes both the promoter and the transcription unit!
? Cis elements,sequences on DNA that affects
the level of transcription.
? Trans factors,DNA-binding proteins that
change the level of transcription by basal
transcription machinery.
How genes affect each other
Cis elements,sequences on DNA that affects the
level of transcription.
ACTTCATCTAGTATAATACTCATCCCGCGTA…
ACTTCATCTAGTGTACATACTCATCCCGCGTA…
Cis element may be a promoter
e.g,this may be a strong signal,
and the green protein may bind
to it strongly.
This is a weakened version of
the signal,so the protein doesn’t
bind,or maybe binds less strongly.
Examples of Trans Factors
Some genes inhibit other genes; suppose protein Y sticks to DNA at
the precise pattern CAGGCG
genePromoter
The transcription factors just can’t get to where they need to be,so if protein Y
is being produced,then genes with this pattern nearby just won’t be expressed.
CAGGCCG
Prokaryotic Gene Expression
Promoter Cistron1 Cistron2 CistronN Terminator
Transcription RNA Polymerase
mRNA 5’ 3’
Translation Ribosome,tRNAs,Protein Factors
1 2 N
Polypeptides
NC N C N
C
1 2 3
The Prokaryotic Promoter
Determines where transcription begins – what site and
which strand of DNA acts as template
Promoter Structure in Prokaryotes (E.Coli)
Transcription starts
at offset 0.
? Pribnow Box (-10)
? Gilbert Box (-30)
? Ribosomal
Binding Site (+10)
Eukaryotic Gene Expression
Promoter Transcribed Region Terminator
Transcription RNA Polymerase II
Primary transcript 5’ 3’
Translation
Polypeptide
NC
Enhancer
Exon1 Exon2
Intron1
Cap
Splice
Cleave/Polyadenylate
7mG An
7mG An
Transport
Definitions
? Intron (intervening sequence),
– a nucleotide sequence within a gene that is transcribed into
RNA but excised before translation,
– Intron size,50 – 20,000 nts
? Exon:
– A subsegment of a gene that encodes a portion of the final
geneproduct,This subsegment remains after processing and
is ultimately translated into a polypeptide or incorporated
into the structure of an RNA molecule.
– Most exons are < 1000 nts; majority b/n 100 – 200 nts
Primary transcript with introns
Eukaryotic Gene Complexity
? Yeast
– introns rare
– promoters
adjacent
– genome
dense
? ―large‖ Eukaryotes
– introns common,
LONGER than
exons
– Promoter/enhancer
– genome sparse
? Fungi
– introns common,
short relative to
exons
– promoter/enhancer
– genome dense
Intron Prevalence
0
10
20
30
40
50
60
70
80
90
100
0 1 >1
Yeast
Fungi
Mammal
Intron Size
0
10
20
30
40
50
60
70
<100 <200 <1
kbp
1 to
5
>5
Fungi
Verterbrate
Exon Size
0
5
10
15
20
25
30
35
1-
100
100-
200
200-
300
300-
500
>500
Fungi
Verterbrate
0
5
10
15
20
25
30
35
1-
100
100-
200
200-
300
300-
500
>500
Fungi
Verterbrate
Eukaryote Promoter
? Goldberg-Hogness or TATA located at –30
? Additional regions at –100 and at –200
? Possible distant regions acting as enhancers
or silencers (even more than 50 kb),
Basal Promoter Analysis
? ATATAA -30 TBP
? GGCCAATC -75 CTF/NF1
? GCCACACCC -90 SP1
TATACAATGC
+1
DATABASE SEARCH
? BLASTN
– DNA:DNA comparison (ALWAYS!)
– Not sensitive (DNA conservation low)
? BLASTX/TBLASTX
– ?6 frame ORFS:polypeptide database
– 6 frames vs,6 frames of a DNA database
Classes of RNA
? Informational RNA,protein encoding mRNA
– translated into amino acid sequence
? Functional (structural) RNA
– MicroRNA,regulation of transcription and translation
– tRNA,transports amino acid to ribosome;
– rRNA,structural and catalytic component of
ribosomes
– snRNA,structural and catalytic component of
spliceosome snRNPs
– snoRNA,small nucleolar RNA involved in maturation
of rRNA
– scRNA,directs protein traffic in cytoplasm
RNA polymerases
? Prokaryotes,single RNA polymerase
– Transcribes mRNA,rRNA and tRNA
– Transcription and translation are coupled
? Eukaryotes,three RNA polymerases
– RNA polymerase I transcribes rRNA genes
– RNA polymerase II transcribes protein-encoding genes; i.e,
makes mRNA
? primary transcript will be processed
– RNA polymerase III transcribes tRNA genes and 5S rRNA
genes
? Transcription and translation occur in separate
compartments of the eukaryotic cell
– In organelles they occur in the same compartment
Transcription
? RNA polymerase catalyzes RNA synthesis
– uses one DNA strand as template
? always the same strand for a given gene
– locally unwinds DNA
– adds free nucleotides to growing RNA strand at 3’ end
? 5’ to 3’ RNA synthesis
? template read 3’ to 5’
? uses rules of base pairing to synthesize complementary RNA
molecule
? starts RNA chain de novo
? Transcript is identical in sequence to nontemplate
strand,except T’s replaced by U’s
Transcription is asymmetric – only one strand of the DNA is transcribed
into RNA; the template strand
The RNA transcript has the same sequence as the nontemplate strand
RNA is synthesized in a 5’ to 3’ direction only
The template strand is read in the 3’ to 5’ direction
sense
anti-sense
Transcription steps
? Initiation
– at 5’ end of gene
– binding of RNA polymerase to promoter
– unwinding of DNA
? Elongation
– addition of nucleotides to 3’ end of growing chain
– governed by rules of complementary base pairing
– energy from NTP substrates
? Termination
– at 3’ end of gene
– terminator loop (prokaryote) or processing enzyme
coding region5?UTR 3?UTR
Elongation – the addition of ribonucleotides to form the RNA
chain occurs in a 5’ to 3’ direction
Termination is dependent on
a specific sequence of
nucleotides
Elongation & Termination
Eukaryote mRNA processing
? 5’ end,capping
– addition of 7-methylguanosine to the 5’-end
– linked by three phosphates
? 3’ end,poly(A) tail
– addition of 150-200 adenine nucleotides to 3’ end
– downstream of AAUAAA; polyadenylation signal
? Intron removal by spliceosome
– all introns have 5’GU and 3’AG recognition sequence
(GU – AG rule)
– snRNPs of spliceosome provide catalysis
– intron excised as lariat,destroyed Some nonprotein-
encoding genes have
self-splicing introns.
5?- Capping
Capping,Modification of 5’
end of eucaryotic mRNA
– May play a role in
translation initiation
– May provide increase
mRNA stability
5’-cap
– 7-methylguanosine linked to
the 5’-terminal residue of the
mRNA via a 5’ to 5’
triphosphate linkage
Phosphorylase
Guanylyltransferase
Guanine 7-methyl-
transferase
2?-O-methyl-
transferase
PolyA Tail Addition
The endonuclease cleaves the RNA after the AAUAAA signal
The polyA tail is added by polyA polymerase; nontemplated
nucleotide addition
Not all mRNAs have a 3’ polyA tail
Polyadenylate polymerase
? adds 80 – 250 nts to the end of mRNA
? requires the cleaved mRNA as a primer
? does NOT require a template for synthesis
? specific for one nucleotide (ATP)
? increases mRNA stability
RNA+ nATP = RNA-(AMP)n + nPPi
To continue
Removal of Introns
Removal of introns is called splicing
Occurs in mRNA; also occurs in rRNA and tRNA
Introns are removed before the mRNA leaves the nucleus
Splice sites have specific sequences – the GU—AG rule
snRNPs form a functional unit called the spliceosome
Consensus splice sites
snRNAs
snRNA Length (nts) Function
U1 165 Binds 5? splice site,then 3? splice site
U2 185 Binds the branch site and forms part of the catalytic center
U4 116 Masks the catalytic activity of U6
U5 145 Binds the 5? splice site
U6 106 Catalyzes splicing
Splicing mechanism
GU A AGU1 U2
U4 U5
U6
Exon 1 Intron Exon 2
3? splice site5? splice site branch site
U1 U2
ATP
U4 U5
U6
AG
1o transcript
Assembled
spliceosome
Products of
the splicing
reaction
Alternative splicing
? generates protein diversity
? allow generation of different splice variants
– in different tissues
– at various developmental stages
? Estimates,30% of human genes undergo alternative
splicing
Splice variants:
Pre-mRNA
Number of splice variants (SV)
SV = 2 number of internal exons
Structure of tRNA
a,short RNA strand made of 73 to 93 nucleotides
b,some internal complimentarity results in 3D shape
– four short double helix regions with three loops
form a cloverleaf shape (2-D structure)
– five other base-pairs between loops form an,L”
shape (3-D structure))
GAU
CUA
mRNA
3’
5’
tRNA structure
? Single RNA strand
? Internal pairing of bases
? Folding,internal
compliment
– Cloverleaf shape to,L” shape
– Conserved regions
? Modified bases
? CCA sequence –aa
binding
? Anticodon – pairs with
mRNA
– Variable region of tRNA
C
C
A
GC
U A
?
codon
?Degeneracy? of the Genetic Code:
GAU GAU
…..CUA…,….CUG….
leucine leucine
Same tRNA,a.a.
different mRNAs
wobble position
– tRNA matching is less selective in the third
position of the anticodon,the,wobble position”.
– e.g,CGU,CGG and CGA all code for arginine
? There are 50 different tRNA genes,50 tRNAs
? Each gene is present in multiple copies in the
genome.
– Located on different chromosomes
– Multiple copies of the genes allow faster production.
- Transcribed from DNA by RNA polymerase III
tRNA Genes
rRNA has complex structure:
? Lots of internal
structure,base – pairing
? Ribosome function is
both structural and
catalytic - including an
apparent ribozyme
function
? Many bases post-
transcriptionally
modified
The purple area is a catalytic site.
Ribosomal Subunits
? The genes responsible for making the rRNAs primary transcript is cut and
chemically modified
? a large 45S rRNA is copied from the DNA,The source of 3 of 4
rRNAs 28S,18S,5.8S
? The 5s gene is outside the nucleolus.
? Ribosome subunit assembly takes place in the nucleolus,bringing together
the genes,the 45S primary transcript and the processing enzymes and the
proteins
– Finished small and large subunits are sent to the cytoplasm separately
to do their work,i.e,translation
? Genes appear in multiple copies,in clusters in nucleolar organizer regions
– multiple copies of the gene are required to supply the ten million
ribosome particles needed by the cell.
– human cells have 200 rRNA gene copies per genome located on five
different chromosomes (E,coli has seven copies)
– Each gene has multiple copies of the 45S gene,all separated by non-
transcribed regions termed,spacer DNA”
– growing rRNA strands make a,christmas tree”appearance
The rRNA Gene
Multiple RNA polymerases
can transcribe a gene
simultaneously creating a
train of RNA polymerases
What is the direction of
transcription of this
rDNA gene?
The rRNA gene continued:
– 3 of the rRNA genes (28S,18S,5.8S) all originate
from one large primary transcript 45S (13,000
nucleotides) rRNA
1,by RNA polymerase I (a amanitin insensitive)
2,10 transcript is cut up after transcription
3,an additional rRNA gene (5S) is made outside the
nucleolus and then imported
– removal of spacers and base modifications are
done by snoRNPs (snorps)
? protein/RNA structures,snRNProtein/snRNA,
TRANSCRIPTION OF rRNA GENES
direction of transcription
18S 5.8S 28S
nontranscribed spacer DNA gene promoter terminator
multiple gene copies,clustered
RNA polymerase 1
transcription unit
Made as a single 45S unit
Growing rRNA
Copy 1 Copy 2 Copy 3
two things happen to pre-rRNA
2) cleavage
18S 5.8S 28S
1) Post transcriptional base
modification:
- 2’ - O - methylation of some riboses
- conversion U to pseudouridine Y
5S rRNA
- 5S rRNAs are not made in the nucleolus
- therefore do not have a nucleolar organizer
- like other rRNA genes,multiple repetitive copies
-transcribed by a different polymerase (RNA pol III)
- requires little or no processing
- shipped to the nucleolus after synthesis to be
incorporated into the ribosome
? Ribosomal genes have nucleolar organizer activity
?recognition by cell and association at one location,the
nucleolus
?site of ribosome assembly
?proteins and 5S rRNA are imported into nucleolus
?large,small subunits are made separately
?associate in cytoplasm,but not until they are actually
being used in the translation process.
Ribosomal Genes and
Nucleolar Organizing Activity
Ribosomes
? Function,protein translation (in cytoplasm)
? Structure,ribosomes are made of rRNA and
proteins.
– eukaryotic ribosome structure.
? Four rRNAs 28S,18S,5.8S,5S
? plus associated proteins
? Prokaryotic structure is very similar,but lacks
5.8S rRNA
Translation
? mRNA is translated at the ribosome using tRNA
as an adaptor molecule
? nucleotide sequence is read three nucleotides at a
time
– each triplet is called a codon
– each amino acid has one or more codons
– 64 possible codons (4 ? 4 ? 4) = genetic code
? used by all organisms with few exceptions
– no punctuation except start and stop
? Genetic code specifies 20 different amino acids
(sometimes selenocysteine)
The Ribosome
Translation at the ribosome
? Ribosome
– large subunit
– small subunit
? 3 ribosomal sites involved in decoding mRNA
– A site (amino site),accepts incoming charged tRNA
– P site (polypeptide site),peptide bond
– E site (exit site),tRNA exits ribosome
? Amino terminus synthesized first,beginning
near but not at the 5’ end of mRNA
– C-terminal end is made last
? UAA,UAG and
UGA correspond to 3
Stop codons that
(together with Start
codon ATG)
? Delineate Open
Reading Frames
Genetic Code and Stop Codons
Codon Usage in Human Genome
Ribosomal Binding Site in E coli.
Translation initiation in prokaryotes
Circular polysomes in Eukaryotes
Protein structure
? Protein is polymer of amino acids (polypeptide)
– each amino acid has an R group conferring unique
chemical properties
– amino acids connected by peptide bond
– each polypeptide has amino end and carboxyl end
? Structure
– primary,amino acid sequence
– secondary,hydrogen bonding,a-helix and ?-sheet
– tertiary,folding of secondary structure
– quaternary,two or more tertiary structures
? Shape and function determined by primary
structure encoded by gene
Peptide Bond Formation
DNA,the genes,template for RNA production,(transcription)
RNA in the cytoplasm.
mRNA rRNA tRNA
-information transfer to cytoplasm - catalytic machinery used to translate mRNA to protein
22,2 28 5 2
i nt e ri or o f nu c l e us
c yt op l a sm
RNA in nucleus
hnRNA,precursors to mRNA
rRNAs,from large precursor
tRNA
small nuclear RNAs
small nucleolar RNAs
Summary
Review of Eukaryotic mRNA
Production
RNA processing
1,any post-transcriptional modifications of the
primary transcript
2,Note,some modifications take place in both
procaryotes and eucaryotes
3,much more extensive in eucaryotes
Purpose of RNA processing
1,Generally and increase in RNA stability
– 5?-capping
– 3?-adenylation
– modifications of individual residues
– structural changes
2,Increase of genetic variability
– The proteome is much larger than the genome
– Splice variants
? Central Dogma was proposed in 1958 by Francis Crick
? Crick had very little supporting evidence in late 1950s
? Before Crick?s seminal paper
all possible information transfers
were considered viable
? Crick postulated that some
of them are not viable
(missing arrows)
? In 1970 Crick published a paper defending the Central Dogma,
Central Dogma,Doubts
From Conversion to Aggregation:
Protofibril Formation of the Prion Protein
PrPC PrPSc
Low pH
M,L,DeMarco and V,Daggett Proc,Natl,Acad,Sci,USA 101,2293-2298,2004.
MD
To continue
Recombination
? Homologous Recombination - Occurs during
prophase of meiosis I and involves exchange
between homologous strands of DNA
? Site Specific Recombination - Short homologous
sections of bacterial and phage DNA serve as a
site for recombination and thus incorporation of
phage DNA into bacterial chromosomes
Recognition site Recombinase
- Cre-LoxP system (phage P1) LoxP Cre
- FRT-Flp systeem (S,cerevisiae) FRT Flp
Site-specific Recombination Systems
Principles of Cre-Mediated Recombination
? Cre is the 38 kDa product of the cre gene of
bacteriophage P1
? It belongs to the Int family of DNA recombinases,but
unlike other family members,Cre requires no
accessory factors
? Cre recognizes a 34 bp site on the bacteriophase
genome called LoxP,catalyzing reciprocal conservative
DNA recombination between LoxP pairs
?LoxP consists of two 13 bp inverted repeats separated by an 8 bp
asymmetric core region
?This core region is responsible for the directionality of the LoxP
site
?Two molecules of Cre bind to each LoxP site,so that [theoretically]
4 molecules of Cre are required for each recombination event
Cre-Mediated Recombination - tandem repeats
Result,Excision of intervening sequence (with retention of 1 LoxP site)
Cre-Mediated Recombination - inverted repeats
Result,Inversion of intervening sequence
Cloning vectors
? Common properties
– origin of DNA replication
– unique restriction sites for insertion of DNA
? multiple cloning sites containing many restriction sites
engineered into many plasmid vectors
– Easy identification and recovery of clones
? Types of vectors
– plasmids containing drug resistance gene
? many commercially available plasmids
– bacteriophage,e.g.,lambda
– cosmids for larger DNA molecules
– BAC,bacterial artificial chromosome
– YAC,yeast artificial chromosome
Other Replication Vectors
? Bacteriophage - Engineered bacterial
viruses
? Used to infect E,coli cells
? Advantage is the insert size can be much
larger than a plasmid
? Can be maintained with at -80 C almost
indefinitely
Other Replication Vectors
? Cosmids - large plasmid-like
autonomously replicating DNA vectors -
rarely used
? BACs - large linear pieces of DNA with
chromosome like attributes
? YACs - Contain yeast centromere and
telemeres as well as yeast replication
features - can clone up to 1 million bp
Examples of two
plasmid vectors
Replication in the
host cell generates
many,many copies
of the DNA
Selection is based on
absence/presence of
antibiotic resistance
Identification of,clones”
is facilitated with these
vectors through
differential antibiotic
resistance or blue/white
screen
Bacteriophage Vectors
Bacteriophage
vectors are
useful for
cloning larger
DNA
fragments
Bacterial artificial
chromosomes (BACs) are
used to clone much larger
DNA fragments (150-300
kb)
These have been extremely
useful in genome sequencing
projects
Use the F plasmid to
provide replication
functions,selection is for
CAM resistance and the
inserts can be large
DNA is easy to recover for
analysis
Bacterial Artificial Chromosomes
Entry into the Cell
Genomic DNA
Chemically synthesized
DNA
cDNA
PCR-amplified DNA
Generating Recombinant DNA (2)
Methodology
? Southern Blot – DNA
? Northern Blot – RNA
? Western Blot – Protein
? EMSA – DNA&protein
? PCR – DNA amplification
? Mutagenesis
Site-directed mutagenesis
PCR mutagenesis
? FISH –labeled probe
? Microarrays (DNA chip,gene chip,biochip)
– allows for thousands of interactions at once
? RNA interference
? Gene mapping
– Functional cloning,Find protein and
work back
– Positional cloning,Uses known sequences
and markers
Microarray gene expression,Two color image overlay
?Red,higher in Cy5
?Green,higher in Cy3
?Yellow,equal in both
channels
?Brightness reflects intensity
of signal
RNA interference - Mechanism
DICER - RNAse III,ds spec,endonuclease
- Dimer,2 catal,domains,helicase
and PAZ motif
- produce 2-3nt 3′overhangs
- ATP-dependent ribonuclease
RISC - RNA-induced silencing complex
- RISC contains siRNA
- precurser activated by ATP
- find and destroy mRNA
of complementary sequence
- contains endo- and exonuclease,
cleaves the hybrid in the middle
imm,followed by degradation
- ARO,PAZ domain (assembly)
Figure 1
Zamore,P.D,(2001)
Nat,Struc,Biol,9:746
RNAi Pathway
RNAi = RNA i nterference
siRNA = small interfering RNA
siRNP = small interfering Ribonucleoprotein
RISC = RNA Induced Silencing Complex
Dicer
Quantitative PCR (Q-PCR)
T a q - m a n C Y B R g r e e n
Q-PCR (continued)
24.207
23.099
18.571
18.657
2(23.099- 24.207)
2(18.657-18.571)Regulation = => Regulation = -2.27
Inverse PCR
ligate
Clone into vector
Amplify by PCR
Digest genomic DNA with
restriction endonuclease
Methodology
Restriction Enzymes
Xba I TCTAGA TCTAGmATC
AGATCT AGAT CTAG
INV 110,Dam methylase deficient
