Chapter 2

Genes and Chromosomes

2.1 DNA & DNA Structure

1. Nucleoside and Nucleotide

(1) Components ： pentose, base, phosphate group

Base

Pentose

In DNA or RNA, a pentose is associated with only one phosphate group, but a cellular free nucleotide (such as ATP) may contain more than one phosphate group.  If all phosphate groups are removed, a nucleotide becomes a nucleoside.

Adenine (A)

Cytosine (C)

Guanine (G)

Thymine (T)

Uracil (U).

(2) Bases in DNA or RNA

Albrecht Kossel

A, C, G and T  exist in DNA;

A, C, G and U  exist in RNA.

condensation reaction, phosphodiester bond, DNA polymerase

2. Nucleic Acid Chain

连接反应(ligation reaction)

Synthesis of a nucleic acid chain

always proceeds from 5’ to 3’.

(1) DNA Primary Structure    The primary structure of DNA is the sequence itself - the order of nucleotides in the deoxyribonucleic acid polymer.

3. DNA Structure

     Why is DNA sequence important?

Question 3

(2) DNA Secondary Structure

Right-handed or left-handed

Helix Diameter          

Base Inclination     

Bp / helical turn  

Helix pitch  

Major groove

Minor groove

10 bp/turn B-form

A B Z

Handedness

Pitch

Base Inclination

Animation

(3) DNA Supercoil

4. DNA denaturation and renaturation

Denaturation: dsDNA → ssDNA

Renaturation: ssDNA → dsDNA

2.2 Gene & Genome

1. Concepts of Gene

(1) Gene = In molecular terms, it is the entire D

NA sequence including exons, introns, and nonc

oding transcription-control regions necessary f

or production of a functional protein or RNA.

exons introns

noncoding transcription-control regions

(2) Regulatory Gene

A DNA sequence or a gene that functions

to control the expression of other genes.

(3) Structural Gene

A DNA sequence or a gene that codes the

production of RNA, a specific protein or peptide.

(4) Gene Cluster

It is a group of adjacent genes that are identical

or related.

(5) Gene Family A group of related genes having similar DNA

sequence evolved from a single ancestral. These

genes make similar products and may or may not

be located in the same region of a chromosome.

Question:

What is different between gene cluster or gene

family?

2. Genome

(1) Genome Complete collection of a cell or an organism’s genetic information as linked genes in a long strand of DNA.

(2) Genomics The branch of genetics that studies organisms in terms of their genomes (their full DNA sequences).

(3) Functional genomics

The branch of genomics that determines the

biological function of the genes and their resulting

proteins, and the role played by the proteins in the

organisms’ biochemical processes.

(4) Structural genomics

The branch of genomics that determines the three-

dimensional structures of proteins.

3. Size of genome

(1) Prokaryotic cell vs Eukaryotic cell

(2) Size ↔ Evolution ?

4. Model Organisms

Bacteria (E. coli, several others) Yeast (Saccharomyces cerevisiae) Plant (Arabidopsis thaliana) Caenorhabditis elegans Fruit fly Zebrafish Mouse Human………

Question

Why do we choose these organisms as models?

Animation

2.3 Features of Genomes

1. Features of Prokaryotic Genomes

(1) Relatively small -- size, content

(2) Simple structure

(3) Transcription unit

concept…..

(4) Overlapping gene

concept…..

Now, it is also found in eukaryotic genomes…..

2. Genomes of Prokaryotes

Most of them: double-helix , circular

Small number: single strand, DNA

Scaffold: loops, supercoiled

(1) E.coli Genome

4.6 million bp 90% of genome encodes protein 4288 genes. almost no repeated DNA

λ phage genome

Regulatory genes

Replication genes – 2 genes

Lysis genes – 3 genes

Recombination genes – 10 genes

Head – 10 genes

Tail – 12 genes

(2) Phage genome

3. Features of Eukaryotic Genomes

(1) Big size, big content

(2) Repeat sequence

(3) Single cistron

(4) Gene uncontinuous (intron and exon)

(5) Pseudogene

(6) mtDNA & ctDNA

4. Genome of Eukaryotes

(1) Human Genome

nuclear genome + mitochondrial genome

Information derived from the HGP

► less than 2% of the human genome codes for proteins

► almost all (99.9%) nucleotide bases are exactly the

same in all people

► ("junk DNA") make up at least 50%

► 24% important for translation

► Repetitive elements Satellites (regular, mini-, micro-)

Transposons

Retrotransposons

Parasites

Question

Is the junk DNA real junk?

HGP Goals Now

To identify the function of the human genome

To understand how and why genes can cause diseases

To speed up the use of genetic information in biomedical research and put it to work

(2) Other Genomes of Eukaryotes

Yeast genome

Drosophila genome

C. elegans genome

Mouse genome

Arabidopsis thaliana genome genome

2.4 Nucleosome and Chromosomes

2. Nucleosome

(1) concept

~200 bp of DNA Histone contains 5 kinds : H1, H2A, H2B, H3, H4

(2) packing

1. Histones and Non-histones

(1) Packing ratio

(2) Packing levels

solenoid

chromatin fiber

chromatid

chromosome

3. DNA Coiling into a chromosome

2.5 DNA Replication

1. Semiconservative Replication

(1) Models of DNA Replication

What is semiconservative replication?

Each strand acts as a template for a new double hel

ix. The established model of DNA replication in which

each double-stranded molecule is composed of one par

ental strand and one newly polymerized strand.

Two requirements:

● DNA template

● Free 3' -OH group

(2) Replication elements

Origin, Ori

Replicon

Replication fork

Bidirection

2. The Enzymology of DNA Replication

substrate,

polymerase,

DNA template,

primer,

enzymes,

proteins

……

(1) Topoisomerase

(2) Helicase

(4) Primase

(5) DNA Polymerase III

(6) DNA polymerase I

(7) DNA ligase

3. Features of DNA Replication in Prokaryores

(1) Replication Ori in E.coli

(2) Initiation of DNA replication in E.coli

SynthesisDNA repairDNA repair

1011subunit

--+5 exdonuclease

+++ 5 exdonuclease

+++5 polymerase

pol IIIpol IIpol I

(3) DNA polymerase in E.coli

Question

What is Klenow fragment?

Animation

General Model for DNA Replication Process

(4) Semicontinuous replication

(5) Termination of Replication

Forks meet opposite originForks meet opposite origin Two termination regions Two termination regions Regions act as “traps” Regions act as “traps”

(6) Rolling Circle

Replication

(1) Multiple replicons

(2) DNA Polymerase

(3) Replication of telomere

4. Features of DNA Replication in Eukaryores

(4) Replication of mtDNA

Replicating DNA of Drosophila melanogaster

(1) Multiple replicons

(2) DNA Polymerase in Eukaryotes

Pol α: acts as a primase

Pol β: repairing DNA

Pol γ: replicates mt DNA

Pol δ: proofreading

Pol ε: proofreading

Others: η, ι, κ, θ, λ, φ, σ, μ, Rev1

Telomerase

It is an enzyme made of protein and RNA

subunits. Its role is to elongate chromosomes

by adding telomeric sequences to the end of e

xisting chromosomes.

(3) Replication of telomere

Replication of telomere

(4) mtDNA Replication

D-loop replication

mtDNA & ctDNA

Progress

Discovery of a Major D-Loop Replication Origin

Reveals Two Modes of Human mtDNA Synthesis.

Science , 2004, 306: 2098 - 2101

Schematic representation of H-strand replication initiation in human mtDNA D-loop region.

Proofreading

4. Proofreading

2.6 DNA Mutagenesis

1. Mutation types

(1) Base Substitution

(2) Frameshift

Deletion

Insertion

(3) Exon skipping

2. Mutagens

(1) Base analog

(2) Base modifier

(3) Intercalation dye

(4) Ultraviolet, ionizing radiation

3. Meanings of Mutation

2.7 DNA Repair

Errors in DNA replication

Endogenous DNA damage and mutated

Environmental insults to DNA

Why repair DNA?

Un-repaired damage leads to:

Mistakes in RNA/protein synthesis

Inherited as genetic alteration

Mutation

Death

1. Excision Repair - Dar Repair

(1) Mismatch Repair

(2) Base Excision Repair – AP Repair

(3) Nucleotide Excision Repair

3. Recombinational Repair

2. Direct Repair – Light Repair

(1) Concept (2) Involved Genes and Proteins Repressor: LexA Inducer: RecA Repair proteins: UvrABC SOS genes SOS box

4. SOS response—Error prone repair4. SOS response—Error prone repair

(3) Mechanism

ProgressPrecise temporal modulation in the response of the SO

S DNA repair network in individual bacteria.

PLoS Biol. 2005. 3:e238.

(A) 8 min and (B) 70 min after irradiation with a UV

filamentation

2.8 DNA Recombination

1. Overview

(1) Important Contributions

(2) Types of Recombination

homologous recombination

site-specific recombination

transpositional recombinatio

n

2. Homologous recombination

(1) Concept

(2) Holiday model

Nicking of two strandsNicking of two strands

Crossing over: Holliday junctionCrossing over: Holliday junction

Branch migrationBranch migration

ResolvingResolving

Isomerization and resolution of Holliday junctions

3. Site-specific

Recombination

4. Transposition and Retrotransposition

(1) Concepts

Transposition is a unique form of recombination wh

ere mobile genetic elements can virtually move from one

region to another within one chromosome or to another c

hromosome entirely.

Retrotransposon is genetic elements and mobilizes vi

a an RNA form. It is transcribed into RNA, and then rev

erse-transcribed into DNA, which is inserted at a new sit

e in the genome.

(2) Types and Features

Insertion Sequences (IS elements)

Transposon

Composite Transposons

Retrotransposon

long terminal repeat (LTR)

non-LTR retrotransposons

(3) Transposition mechanism (3) Transposition mechanism 

conservative transposition

replicative transposition

retrotransposition

(4) The difference between Transposition and Retrotransposon

Summary

Concepts

Genomes of prokaryotes and eukaryotes

Genomic structure of prokaryotes and eukaryotes

DNA polymerase of prokaryotes and eukaryotes

Mechanism of DNA replication in prokaryotes and eukaryotes

Mechanism of DNA mutation, repair and recombination

Mechanism of DNA transposition and retrotransposition

Gene Genomics Excision Repair

Regulatory

Gene

Functional Genomics Directed Repair

Structural Gene Structural Genomics Recombination Repair

Gene Cluster DNA Primary Structure Error-prone Repair (SOS

response)

Gene Family DNA Secondary Structure Homologous Recombination

Genome DNA Supercoiling Site-specific Recombination

Exon & Intron Semi-conservative

Replication

Transposition

Pseudogene Semi-discontinous Replicatio

n

Transposon

C value Substitution Retrotransposon

C value paradox Mismatch Repair

Concepts