生物學Lecture 3:

What is (a) gene? Central Dogma

生物醫學系羅時成老師losj@mail.cgu.edu.twext: 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 post-translational 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

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!

Heredity unit = Gene

Genetics and Evolution

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• Thomas Hunt Morgan in The Fly Room!

(Columbia University 1910)• Fruit Flies (Drosophila melanogaster)

http://nobelprize.org/nobel_prizes/medicine/articles/lewis/index.html

© 2007 Paul Billiet ODWS

Hypothesis

A cross between the F1 flies should give us: 3 red eye : 1 white eye

F2 Phenotypes Red eye White eye

Numbers 3470

82%

782

18%

So far so good

© 2007 Paul Billiet ODWS

An interesting observation

F2 Phenotypes Red-eyed males

Red-eyed

females

White-eyed males

White-eyed femal

es

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 X-ray 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.

ＤＮＡ是攜帶遺傳資訊的分子！ How ？

奧斯卡．阿佛來 (Oscar Avery) 1943

Figure 16.4-3

Bacterial cell

Phage

Batch 1:Radioactivesulfur(35S)

Radioactiveprotein

DNA

Batch 2:Radioactivephosphorus(32P)

RadioactiveDNA

Emptyproteinshell

PhageDNA

Centrifuge

Centrifuge

Radioactivity(phage protein)in liquid

Pellet (bacterialcells and contents)

PelletRadioactivity(phage DNA)in pellet

EXPERIMENT

• 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

Fir

st m

RN

A b

ase

(5 e

nd

of

cod

on

)

Th

ird

mR

NA

bas

e (3

en

d o

f co

do

n)

UUU

UUC

UUA

CUU

CUC

CUA

CUG

Phe

Leu

Leu

Ile

UCU

UCC

UCA

UCG

Ser

CCU

CCC

CCA

CCG

UAU

UACTyr

Pro

Thr

UAA Stop

UAG Stop

UGA Stop

UGU

UGCCys

UGG Trp

GC

U

U

C

A

U

U

C

C

CA

U

A

A

A

G

G

His

Gln

Asn

Lys

Asp

CAU CGU

CAC

CAA

CAG

CGC

CGA

CGG

G

AUU

AUC

AUA

ACU

ACC

ACA

AAU

AAC

AAA

AGU

AGC

AGA

Arg

Ser

Arg

Gly

ACGAUG AAG AGG

GUU

GUC

GUA

GUG

GCU

GCC

GCA

GCG

GAU

GAC

GAA

GAG

Val Ala

GGU

GGC

GGA

GGGGlu

Gly

G

U

C

A

Met orstart

UUG

G

• 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 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.)

Class IMutants(mutationin gene A)

Class IIMutants(mutationin gene B)

Class IIIMutants(mutationin gene C)Wild type

Gene A

Gene B

Gene C

Precursor Precursor Precursor Precursor

Ornithine Ornithine Ornithine Ornithine

Citrulline Citrulline Citrulline Citrulline

Arginine Arginine Arginine Arginine

EnzymeA

EnzymeB

EnzymeC

A A A

B B B

C C C

Figure 15.15

Down syndrome

Figure 16.23

5 m

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

Ribozymeribozyme (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

電腦

硬體

2D 資訊在磁碟複製、長久、穩定

軟體硬體

DNA複製、長久、穩定

RNA暫時、不穩定

蛋白質執行工作或通訊

RAM暫時、不穩定

銀幕或其他機器執行工作或通訊

細胞像電腦？電腦像細胞？

（亞瑟．孔伯）Arthur Kornberg（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

Ends of parentalDNA strands

Leading strand

Lagging strand

Last fragment Next-to-last fragment

Lagging strand RNA primer

Parental strand Removal of primers andreplacement with DNAwhere a 3 end is available

3

3

3

5

5

5

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)

To make a unique protein with a specific amino acid sequence

through transcription and translation

Gene expression

(Expression of information)

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 RNA transcript (pre-mRNA)5

Exon 1

Protein

snRNA

snRNPs

Intron Exon 2

Other proteins

Spliceosome

5

Spliceosomecomponents

Cut-outintronmRNA

5Exon 1 Exon 2

Figure 17.11

5 Exon Intron Exon

5CapPre-mRNACodonnumbers

130 31104

mRNA 5Cap

5

Intron Exon

3 UTR

Introns cut out andexons spliced together

3

105 146

Poly-A tail

Codingsegment

Poly-A tail

UTR1146

How many genes do we have ?

DNA (genetic code)

whatwherewhen

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…

Nontemplatestrand of DNA

RNA nucleotides

RNApolymerase

Templatestrand of DNA

3

35

5

5

3

Newly madeRNA

Direction of transcription

A

A A A

AA

A

T

TT

T

TTT G

GG

C

C C

CC

G

C CC A AA

U

U

U

end

Figure 17.9

Promoters, enhancers, silencers etc.

Figure 17.26TRANSCRIPTION

DNA

RNApolymerase

ExonRNAtranscript

RNAPROCESSING

NUCLEUS

Intron

RNA transcript(pre-mRNA)

Poly-A

Poly-A

Aminoacyl-tRNA synthetase

AMINO ACIDACTIVATION

Aminoacid

tRNA

5 C

ap

Poly-A

3

GrowingpolypeptidemRNA

Aminoacyl(charged)tRNA

Anticodon

Ribosomalsubunits

A

AE

TRANSLATION

5 Cap

CYTOPLASM

P

E

Codon

Ribosome

5

3

Figure 16.22a

DNA double helix(2 nm in diameter)

DNA, the double helix

Nucleosome(10 nm in diameter)

Histones

Histones

Histonetail

H1

Nucleosomes, or “beads ona string” (10-nm fiber)

Figure 16.22b

30-nm fiber

30-nm fiber

Loops Scaffold

300-nm fiber

Chromatid(700 nm)

Replicatedchromosome(1,400 nm)

Looped domains(300-nm fiber) Metaphase

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

Brandeis, M., Ariel, M. & Cedar, H. (1993) Bioessays 15, 709-713.

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 EXPANDING THE GENE CONCEPT BEYOND THE PROTEIN ENCODING BEYOND THE PROTEIN ENCODING

SEQUENCESES of DNASEQUENCESES of DNA::

TRANSCRIPTION OF SOME GENES PRODUCES NONCODING RNAs

Non-Coding RNA: Formerly known as “JUNK” A Key to Eukaryotic Complexity?

Types of RNATypes of RNA

CODINGCODINGIn translationIn translation (mRNA) (mRNA)

NON-CODINGNON-CODINGIn translation (tRNAs and rRNAs)In translation (tRNAs and rRNAs)

IIn RNA processingn RNA processingRegulatory RNAs: lincRNA, Riboswitch Regulatory RNAs: lincRNA, Riboswitch

and microRNAand microRNA

A decade of riboswitch Cell January 17, 2013 page 17

What are lincRNAs?What are lincRNAs?LLarge arge iintergenic ntergenic nnononccoding RNAs (lincRNAs) oding RNAs (lincRNAs) are emerging as key regulators of diverse are emerging as key regulators of diverse cellular processes. cellular processes.

Determining the function of individual Determining the function of individual lincRNAs remains a challenge. lincRNAs remains a challenge.

Recent advances in RNA sequencing (RNA-seq) Recent advances in RNA sequencing (RNA-seq) and computational methods allow for an and computational methods allow for an unprecedented analysis of such transcripts.unprecedented analysis of such transcripts.

Cell July 3, 2013. Page 26

Cell nucleus is a highly organized structure just like a Rome city!

24 MAY 2013 VOL 340 SCIENCE page 910

Long Noncoding RNAs May Alter Chromosome’s 3D Structure

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 OrganizationGenomic 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*

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*

MiRs AS PROGNOSTIC FACTORS: GASTRIC CANCER SURVIVAL*

0

1

2

3

4

5

StagesI-II

StagesIII-IV

3.2

high low high low low high01

234

567

89

10

Let-7g

miR-199

miR-495

HAZ

ARD

RAT

IO(d

isea

se fr

ee s

urvi

val)

HAZ

ARD

RAT

IO(d

isea

se fr

ee s

urvi

val)

Intestinal-Type Gastric Cancer

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).

ANTIMETASTATIC ACTIVITY OF AHR AGONISTS IN ER BREAST CANCER (Mol Cancer Therap. 11, 108-118,

2012).

Normal cells Preneoplastic cells

Cancer cells(Invasive carcinoma)

Metastasis

SOX4

SOX4 and other miR-335 regulated proteins

miR-335

miR-335

SOX4

SOX4 and other miR-335 regulated metastatic

mRNAs

arntAhRLigand activated Ahr

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)