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生物學
Lecture 3:
What is (a) gene?
Central Dogma
生物醫學系
羅時成老師
[email protected]
ext: 3295
What is (a) gene? Central Dogma
• 學習目標:
• To know the history of searching
heredity unit and the definition of gene.
• To understand how genes are regulated
in various organisms (from genotype to
phenotype).
What is (a) gene? Central Dogma
• 1. Discovery of DNA as a genetic material.
• 2. Classic genetics, modern genetics and
molecular genetics.
• 3. Different levels of gene regulation
(transcriptional, translational and posttranslational levels).
• 4. New frontier of small RNA regulation.
決定生物的藍圖
• 龍生龍
• 鳳生鳳
• 老鼠生出來的……..
• 遺傳的觀念
Heredity unit = Gene
Genetics and Evolution
生命怎麼變的如此複雜?
生物演化的三步曲：變異、遺傳與天擇
variation, inheritance and natural selection!
Gregor Mendel (1823-1884)
孟德爾豌豆雜交實驗
Mendel cannot reproduce his
results on peas in hawkweed!
Mendel豌豆實驗結果分析
Important terms in Mendelian genetics.
•
•
•
•
•
Character: color of peas.
Trait: yellow or white.
Gene: unit of heredity.
Allele: version of a gene produces a specific trait.
Homozygous: having two copies of the same
alleles for a given gene.
• Heterozygous: having two different alleles for a
given gene.
Insight of genetics
•
•
•
•
•
•
•
Partial Dominance: dilute concentration.
Codominance: human ABO blood group (AB)
Overdominance: sickle-cell anemia
Dominant: gain-of-function.
Recessive: loss-of-function.
Negative dominant mutation.
Genetic suppression: intra vs intergenic
suppression!
Sex linkage
http://nobelprize.org/nobel_prizes/medicine/articles/lewis/index.html
• Thomas Hunt Morgan in The Fly Room!
(Columbia University 1910)
• Fruit Flies (Drosophila melanogaster)
© 2007 Paul Billiet ODWS
Hypothesis
A cross between the F1 flies should give us: 3 red eye : 1
white eye
F2
Phenotypes
Numbers
So far so good
© 2007 Paul Billiet ODWS
Red eye
White eye
3470
82%
782
18%
An interesting observation
F2
Phenotypes
Redeyed
males
Redeyed
females
Whiteeyed
males
Whiteeyed
females
Numbers
1011
2459
782
0
24%
58%
18%
0%
© 2007 Paul Billiet ODWS
Chromosome linkage
Physical map
Generation of fly mutants by x-ray
radiation
Genetics
• classic genetics古典遺傳學
• modern genetics近代遺傳學
• molecular genetics分子遺傳學
• 尋找遺傳物質及基本單位
The Search for the Genetic Material:
Scientific Inquiry
• When T. H. Morgan’s group showed that genes are
located on chromosomes, the two components of
chromosomes—DNA and protein—became candidates
for the genetic material
• The key factor in determining the genetic material was
choosing appropriate experimental organisms
• The role of DNA in heredity was first discovered by
studying bacteria and the viruses that infect them
© 2011 Pearson Education, Inc.
Milestones in DNA History
• 1869 Johann Friedrich Miescher identifies a weakly
acidic substance of unknown function in the nuclei of
human white blood cells. This substance will later be
called deoxyribonucleic acid, or DNA.
• 1912 Physicist Sir William Henry Bragg, and his son,
Sir William Lawrence Bragg, discover that they can
deduce the atomic structure of crystals from their Xray diffraction patterns. This scientiFic tool will be
key in helping Watson and Crick determine DNA's
structure.
• 1924 Microscope studies using stains for
DNA and protein show that both substances
are present in chromosomes.
• 1928 Franklin Griffith, a British medical
officer, discovers that genetic information
can be transferred from heat-killed bacteria
cells to live ones. This phenomenon, called
transformation, provides the first evidence
that the genetic material is a heat-stable
chemical.
肺炎雙球菌的實驗
• 1944 Oswald Avery, and his colleagues Maclyn
McCarty and Colin MacLeod, identify Griffith's
transforming agent as DNA. However, their
discovery is greeted with skepticism, in part
because many scientists still believe that DNA is
too simple a molecule to be the genetic material.
• 1949 Erwin Chargaff, a biochemist, reports that
DNA composition is speciesspecific; that is, that
the amount of DNA and its nitrogenous bases
varies from one species to another. In addition,
Chargaff finds that the amount of adenine equals
the amount of thymine, and the amount of guanine
equals the amount of cytosine in DNA from every
species.
奧斯卡．阿佛來(Oscar Avery) 1943
ＤＮＡ是攜帶遺傳資訊的分子！ How？
Figure 16.4-3
EXPERIMENT
Phage
Radioactive
protein
Empty
protein
shell
Radioactivity
(phage protein)
in liquid
Bacterial cell
Batch 1:
Radioactive
sulfur
(35S)
DNA
Phage
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Radioactive
DNA
Batch 2:
Radioactive
phosphorus
(32P)
Centrifuge
Radioactivity
Pellet (phage DNA)
in pellet
• 1953 James Watson and Francis
Crick discover the molecular
structure of DNA.
• 1962 Francis Crick, James Watson,
and Maurice Wilkins receive the
Nobel Prize for determining the
molecular structure of DNA.
•
Watson and Crick華生與克立克
Rosalind Franklin 和她的 DNA X-光繞射圖
• 1961 Sidney Brenner and Francis Crick
establish that groups of three nucleotide
bases, or codons, are used to specify
individual amino acids.
• 1966 The genetic code is deciphered when
biochemical analysis reveals which codons
determine which amino acids.
Figure 17.5
Second mRNA base
UUU
U
UUC
First mRNA base (5 end of codon)
UUA
C
Phe
Leu
UAU
UCC
UAC
UCA
Ser
Tyr
UGU
UGC
Cys
U
C
UAA Stop UGA Stop A
UCG
UAG Stop UGG Trp G
CUU
CCU
CAU
CUC
CCC
CAC
Leu
CCA
Pro
CAA
CUG
CCG
CAG
AUU
ACU
AAU
ACC
AAC
AUC
Ile
AUA
AUG
G
UCU
G
UUG
CUA
A
A
C
ACA
Met or
start
Thr
AAA
His
Gln
Asn
Lys
CGU
U
CGC
C
CGA
Arg
CGG
AGU
G
Ser
AGC
AGA
A
Arg
U
C
A
ACG
AAG
AGG
G
GUU
GCU
GAU
GGU
U
GUC
GCC
GAC
GGC
C
GAA
GGA
GUA
GUG
Val
GCA
GCG
Ala
GAG
Asp
Glu
GGG
Gly
A
G
Third mRNA base (3 end of codon)
U
• 1970 Hamilton Smith, at Johns Hopkins Medical
School, isolates the first restriction enzyme, an
enzyme that cuts DNA at a very specific
nucleotide sequence. Over the next few years,
several more restriction enzymes will be isolated.
• 1972 Stanley Cohen and Herbert Boyer combine
their efforts to create recombinant DNA. This
technology will be the beginning of the
biotechnology industry.
•
• 1976 Herbert Boyer cofounds Genentech,
the first firm founded in the United States to
apply recombinant DNA technology
• 1978 Somatostatin, which regulates human
growth hormones, is the first human protein
made using recombinant technology.
尋找基因的實驗
CONCLUSION
Gene A
From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable
to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the
necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded
that each mutated gene must normally dictate the production of one enzyme. Their results
supported the one gene–one enzyme hypothesis and also confirmed the arginine pathway.
(Notice that a mutant can grow only if supplied with a compound made after the defective step.)
Wild type
Class I
Mutants
(mutation
in gene A)
Precursor
Precursor
Precursor
Precursor
A
A
A
Ornithine
Ornithine
Ornithine
B
B
B
Citrulline
Citrulline
Citrulline
C
C
C
Arginine
Arginine
Arginine
Enzyme
A
Ornithine
Gene B
Enzyme
B
Citrulline
Gene C
Enzyme
C
Arginine
Class II
Mutants
(mutation
in gene B)
Class III
Mutants
(mutation
in gene C)
Figure 15.15
Down syndrome
5 m
Figure 16.23
Central dogma of molecular biology : a process of decoding
Genetic code in DNA: A, T, G, C
Genetic code in RNA: A, U, G, C
20 amino acids in protein
The Nobel Prize in Physiology or
Medicine 1975 was awarded jointly
to David Baltimore, Renato
Dulbecco and Howard Martin Temin
"for their discoveries concerning the
interaction between tumour viruses
and the genetic material of the cell".
The Nobel Prize in Physiology or
Medicine 1975
David Baltimore
Renato Dulbecco Howard Martin Temin
Ribozyme
ribozyme (ribonucleic acid enzyme) is an RNA molecule
that is capable of catalyzing specific biochemical reactions,
similar to the action of protein enzymes. The 1982
discovery of ribozymes demonstrated that RNA can be
both genetic material (like DNA) and a biological catalyst
(like protein enzymes), and contributed to the RNA world
hypothesis, which suggests that RNA may have been
important in the evolution of prebiotic self-replicating
systems. Also termed catalytic RNA, ribozymes function
within the ribosome (as part of the large subunit ribosomal
RNA) to link amino acids during protein synthesis, and in
a variety of RNA processing reactions, including RNA
splicing, viral replication, and transfer RNA biosynthesis.
Examples of ribozymes include the hammerhead ribozyme,
the VS ribozyme, Leadzyme and the hairpin ribozyme.
Evolution of enzymes that catalyze
nucleic acids (RNA and DNA)
RNA dependent RNA polymerase
RNA to RNA
RNA dependent DNA polymerase (RT)
RNA to DNA (cDNA)
DNA dependent DNA polymerase
DNA to DNA
DNA dependent RNA polymerase
DNA to RNA (mENA, tRNA, rRNA)
細胞像電腦？電腦像細胞？
硬體
DNA
細
胞
硬體
電 軟體
腦 2D資訊在磁碟
複製、長久、穩定
複製、長久、穩定
RNA
RAM
暫時、不穩定
暫時、不穩定
蛋白質
銀幕或其他機器
執行工作或通訊
執行工作或通訊
（亞瑟．孔伯）
Arthur Kornberg
• 1956 找到複製DNA的酵素
• 1959 Nobel prize winner
• His son, Roger Kornberg
received Nobel Prize in 2006
for his study of structure
basis of gene transcription in
eucaryotes.
Characteristic of DNA synthesis - I
• Primers absolutely necessary
– Usually short stretches of RNA or RNA-DNA
– Some virus use proteins primers.
Characteristic of DNA synthesis - II
• 5’ to 3’ directionality
– Leading strand vs. lagging strand
– End problems for linear DNA molecules
when replication starts internally
Okazaki fragments: discontinue synthesis!
Figure 16.20a
5
Leading strand
Lagging strand
Ends of parental
DNA strands
3
Last fragment
Next-to-last fragment
RNA primer
Lagging strand
5
3
Parental strand
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Proofreading and Repairing DNA
• DNA polymerases proofread newly made DNA,
replacing any incorrect nucleotides
• In mismatch repair of DNA, repair enzymes correct
errors in base pairing
• DNA can be damaged by exposure to harmful chemical
or physical agents such as cigarette smoke and X-rays;
it can also undergo spontaneous changes
• In nucleotide excision repair, a nuclease cuts out and
replaces damaged stretches of DNA
© 2011 Pearson Education, Inc.
Figure 16.21
1 m
DNA (genetic code)
Gene expression
(Expression of information)
To make a unique protein with a
specific amino acid sequence
through transcription and
translation
mRNA
How many polymerase?
• DNA dependent DNA polymerase
– For DNA replication and repair.
– 5 known Prokaryotic DNA polymerases.
– at least 15 Eukaryotic DNA polymerase
• DNA dependent RNA polymerase
– For gene transcription.
• RNA dependent RNA polymerase
– For RNA virus genome replication
• RNA dependent DNA polymerase
– Reverse transcriptase of retrovirus
– Telemerase to make telemere structure
In 1977, when viral mRNA was hybridized with its DNA,
some loops were observed.
Figure 17.12-3
5
RNA transcript (pre-mRNA)
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
Spliceosome
5
Spliceosome
components
5
mRNA
Exon 1
Exon 2
Cut-out
intron
Figure 17.11
5 Exon Intron Exon
Pre-mRNA 5 Cap
Codon
130
31104
numbers
Intron
Exon 3
Poly-A tail
105
146
Introns cut out and
exons spliced together
mRNA 5 Cap
Poly-A tail
1146
5 UTR
Coding
segment
3 UTR
How many genes do we have ?
DNA (genetic code)
what
where
when
how much
Regulation of gene expression at different level!
Transcription factors turn genes on and off.
Transcription factors are proteins that bind
to a specific base sequence in DNA.
…AGCCTACCAAAAAAGGTTCCACG…
…TCGGATGGTTTTTTCCAAGGTGC…
Figure 17.9
Nontemplate
strand of DNA
RNA nucleotides
RNA
polymerase
A
3
T
C
C
A A
5
3 end
C A
U
C
C A
T
A
G
G T
5
5
C
3
T
Direction of transcription
Template
strand of DNA
Newly made
RNA
Promoters, enhancers, silencers etc.
Figure 17.26
DNA
TRANSCRIPTION
3
5
RNA
polymerase
RNA
transcript
Exon
RNA
PROCESSING
RNA transcript
(pre-mRNA)
AminoacyltRNA synthetase
Intron
NUCLEUS
Amino
acid
AMINO ACID
ACTIVATION
tRNA
CYTOPLASM
mRNA
Growing
polypeptide
3
A
Aminoacyl
(charged)
tRNA
P
E
Ribosomal
subunits
TRANSLATION
E
A
Anticodon
Codon
Ribosome
Figure 16.22a
Nucleosome
(10 nm in diameter)
DNA double helix
(2 nm in diameter)
H1
Histones
DNA, the double helix
Histones
Histone
tail
Nucleosomes, or “beads on
a string” (10-nm fiber)
Figure 16.22b
Chromatid
(700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber
30-nm fiber
Replicated
chromosome
(1,400 nm)
Looped domains
Metaphase
(300-nm fiber)
chromosome
DNA
Nucleosome
Epigenetic chromatin regulation
A. Modification at the DNA level
1. cytosine methylation
B. Histone modification - the histone code
1. Histone acetylation
2. Histone methylation
3. Histone phosphorylation
4. Histone ubiquitilation
5. Different types of histones
The five nucleotides that make up the DNA
Maintenance of methylation
Brand eis, M., Ariel, M. & Cedar, H. ( 1 99 3 ) Bioessays 1 5 , 70 9-71 3.
Imprinting is maintained by DNA methylation
Genomic imprinting
Some genes are expressed only from the
maternal genome and some only from the
paternal genome
It is estimated that about 40 genes are
imprinted and they can be found on
several different chromosomes
For example - igf2, h19, igf2r and genes involved
in the Angelman and Prader Willi syndromes
Histone modification
EXPANDING THE GENE CONCEPT
BEYOND THE PROTEIN ENCODING
SEQUENCESES of DNA:
TRANSCRIPTION OF SOME GENES
PRODUCES NONCODING RNAs
Non-Coding RNA: Formerly known as “JUNK”
A Key to Eukaryotic Complexity?
Types of RNA
CODING
In translation (mRNA)
NON-CODING
In translation (tRNAs and rRNAs)
In RNA processing
Regulatory RNAs: lincRNA, Riboswitch
and microRNA
A decade of riboswitch
Cell January 17, 2013 page 17
What are lincRNAs?
Large intergenic noncoding RNAs (lincRNAs)
are emerging as key regulators of diverse cellular
processes.
Determining the function of individual lincRNAs
remains a challenge.
Recent advances in RNA sequencing (RNA-seq)
and computational methods allow for an
unprecedented analysis of such transcripts.
Cell July 3, 2013. Page 26
Cell nucleus is a highly organized structure just like a Rome city!
Long Noncoding RNAs May Alter Chromosome’s 3D Structure
24 MAY 2013 VOL 340 SCIENCE page 910
What are miRNAs?
•
•
•
•
•
•
Small non-coding double stranded RNAs
Approximately 19-22 nt long
Repress activity of complementary mRNAs
Regulate 30% of mammalian gene products
1 miRNA = hundreds of mRNAs
Many are conserved between vertebrates and
invertebrates
Genomic Organization
miRNA processing
Microprocessor
Complex
Differences in miRNA Mode of
Action
Extended thinking
•miRNA and Cancer
CORRELATION OF MIR EXPRESSION WITH
PROGRESSION AND PROGNOSIS OF GASTRIC CANCER*
PATIENTS: 181 patients from 2 cohorts (Japan)
CLASSIFICATION: Stages I-IV
Diffuse vs. Intestinal type
ANALYSIS:
• Custom miR microarray chip (Ohio State Univ.)
• miR expression in 160 paired samples
(tumor vs. non-tumor)
• Correlations of miR expression vs. stage,
type and prognosis (survival)
* Lancet Oncol. 11,136, 2010
MiRs AS PROGNOSTIC FACTORS:
GASTRIC CANCER SURVIVAL*
Intestinal-Type Gastric Cancer
miR-495
HAZARD RATIO
(disease free survival)
5
4
3.2
3
2
1
0
Stages
I-II
Stages
III-IV
HAZARD RATIO
(disease free survival)
10
9
8
7
6
5
4
3
2
1
0
miR-199
Let-7g
high
low
high
low
low
high
I-II
III-IV
I-II
III-IV
I-II
III-IV
ANTIMETASTATIC ACTIVITY OF AHR AGONISTS IN ER
BREAST CANCER (Mol Cancer Therap. 11, 108-118, 2012).
Ligand
activated
Ahr
AhR
arnt
miR-335
Normal cells
Preneoplastic
miR-335cells
Cancer cells
(Invasive carcinoma)
Metastasis
SOX4
SOX4 and other miR-335
regulated metastatic
mRNAs
SOX4
SOX4 and other miR-335
regulated proteins
RNA world
• Carry information (DNA)
• Catalyze chemical reaction (protein enzyme)
• Nutrient sensor to control gene expression
(protein receptor)
• Broadly control gene expression through
mRNA stability, translational efficiency etc.
(protein activator or repressor)
• Global control nuclear and chromosome
structure.(Histone code)