Molecular BiologyIntroductionGenome & Genomics

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)

Department of Biochemistry,

Medical College,

Sher-I-Kashmir Institute of Medical Sciences,

Bemina, Srinagar, Kashmir, 190018. India.

Genome is the genetic material of an organism

Includes both the genes and the non-coding sequences of the DNA/RNA

The term was created by Hans Winkler

Genomics analyzes the function and structure of genomesApplies DNA sequencing methods and bioinformatics for that

Introduction

DISCOVERY……………………

In 1880’s Friedrich Miescher isolated a substance from the pus cells and also from the

sperm cells of salmon fish and

found that it was acidic in nature

He called it nuclein as it was derived from nucleus of these cells

Later on R. Altmann designated it as nucleic acid

Introduction

Introduction

• GRIFFITH’S TRANSFORMATION

• This experiment laid the foundation of the discovery of DNA as the genetic material

• Working at Rockefeller Institute of Medical Research, Frederick Griffith experimented on Diplococcus pneumoniaeand found that the extract from one bacterium could genetically modify other strain

• So that latter exhibited certain traits of the donor strain

Introduction

• GRIFFITH’S TRANSFORMATION

• This bacterium exists in two forms

• Virulent called as S (smooth)

• Avirulent called as R (rough)

{both of which are present in various strains}

• Virulence in this bacterium is due to the presence of a polysaccharide capsule around the cell

Introduction

Griffith’s experimental protocol

Strain Injected in Mice Observation Strain isolated fromblood

Live SIII strain

Live RII strain

Heat Killed SIII strain

Mixture of heat killed SIII &RII strains

Mice died of pneumonia

Mice did not die

Mice did not die

Mice died of pneumonia

Virulent S strain

Avirulent R strain

No strain identified

Virulent S strain identified

Griffith therefore suggested that there was a transforming principle in extract of dead virulent

cells which performed this phenomenon

Introduction

• AVERY, MCLEOD & MCCARTY’S EXPERIMENTATION

• O.T. Avery, C.M. McLeod and M. McCarty repeated the experiments of Griffith

• They found that the transforming principle was DNA

Introduction

Avery, McLeod & McCarty’s experimental protocolStrain in test tube

Chemical from dead SIII extract

Observation

Live RII strain

Live RII strain

Live RII strain

Live RII strain

Proteins

Carbohydrates

Lipids

DNA

No virulent S strain obtained

No virulent S strain obtained

No virulent S strain obtained

Virulent S strain obtained

Introduction• HERSHEY & CHASE

• They utilized the two techniques for their experiments

• One: Osmotic Shock technique for the lysis of the phage particles

• Second: Radiolabeling for the identification of the molecules

Introduction

• HERSHEY & CHASE

• First E. coli cultures were set up which acted as the medium for the growth of T2 phages.

• Later on T2 phages growing on E. coli cultures were divided into two aliquots

• One lot was cultured in a medium containing radioactive phosphorus P32 which labels nucleic acid

• Other half was cultured in the medium containing radioactive sulfur S35- which labels proteins

Introduction

• HERSHEY & CHASE

• After labeling; the phages were used to infect the normal E. coli cultures separately

• The infection of E. coli cells was studied. It was found that during infection the P32 labelled T2 phages transferred the radioactivity to the E. coli cells but S35 labelled failed to do so. As P32 label refers to the presence of nucleic acids thus confirming that it is the DNA which is transmitted from one generation of phage to the other

• The infected E. coli cells were allowed to undergo lytic phase of growth of the phage. The P32 labelled T2 phage infected E. coli cells produced phages with radioactivity while as phages from the S35 labelled T2 phage infected E. coli cells produced non radioactive phages on lysis

Introduction

• HERSHEY & CHASE

Introduction

• FRAENKEL & CONRAT

• They experimented on tobacco mosaic viruses and found that the viruses contain RNA as genetic material

• TMV consists of single-stranded coiled RNA molecule which is covered on the outside by protein units

• There are three strains of TMV A, B & C; each viral strain causes its own characteristic symptoms of infection

Introduction

• FRAENKEL & CONRAT

• In their experiments they used proteins as well as RNAs obtained from different strains and produced different recombinant strains

• Then these were rubbed (mixed with abrasive) them separately on the leaves of tobacco plants to cause infection and observed the characteristics symptoms developed

• The infection that developed exhibited the symptoms of the disease typical of the strain from which the RNA was taken (and not the protein)

Introduction

Fraenkel & Conrats experimental protocolProtein Part RNA Part Observation

TMV A

TMV A

TMV A

TMV B

TMV C

TMV A

TMV B

TMV C

TMV A

TMV B

Infection Characteristic of TMV A

Infection Characteristic of TMV B

Infection Characteristic of TMV C

Infection Characteristic of TMV A

Infection Characteristic of TMV B

Introduction

• MESSELSON & STAHL

• They provided the experimental proof of semi-conservative method of DNA replication as envisaged by Watson & Crick

• First E. coli cell were grown for several generations in a medium provided with N15 so that E. coli cell produced after many generations contained DNA labeled completely with N15

• Then these N15 labelled E. coli cells were transferred to non-labelled media and allowed to grow & divide

Introduction• MESSELSON & STAHL

• Small samples were removed from the transferred E. coli at regular intervals & their DNA extracted and centrifuged in CsCl tubes and the densities of the DNA at the different times after the transfer from N15 to N14 media were recorded

• They found that DNA molecules exhibited only three densities

• The initial ones were fully heavy (N15); another set was as light as pure DNA (N14); and the third set exhibited an intermediate density (which tallied with DNA molecule having one N15 labelledstrand & other N14 labelled)

• This could be explained in light of semi-conservative model of DNA replication

• Proposed by Francis Crick in 1950s to suggest that there was “the unidirectional flow of genetic information”

• Starts from the DNA and ends in proteins via RNA

• It included three main process:• Replication: For new DNA synthesis

from template DNA

• Transcription: For synthesis of RNA on the information of DNA

• Translation: Synthesis of Protein using information from RNA

Central Dogma

• Structure• Prokaryotes

• Eukaryotes

CHROMATIN

• Prokaryotes have a simple genome in their cells

• The chromatin of an E. coli is a single double-stranded circular DNA molecule, lying usually in the centre of the cell without any surrounding membrane

• This region of the cell known as nucleoid –characterized by a very high density of DNA

• It is made up of 4.5 million bps and remains associated with the proteins

• The DNA consists of 50-100 domains or loops each of about 50-100bp in size.

• The overall E. coli chromatin was found to be highly negatively supercoiled

• Also small basic dimer protein called as HU protein is associated with the DNA domains to further constrain it. This protein is analogous to eukaryotic histone proteins

E. coli

E. coli

• Prokaryotes also have an Extra chromatin material in their cells

• These are self-replicating extra-chromosomal elements, usually small segments of duplex DNA found in some bacterial cells

• They vary in size greatly, are usually covalently-closed-circular (ccc) double-stranded DNA molecules (B form)

• They do not encode essential genes but give bacteria a selective advantage only under certain environmental conditions

• Plasmids can integrate into the host genome and are copied at the same time as the bacterial chromosome. This integrated form of plasmid is called an episome and may pass through many cell divisions before excising itself to exist as a separate plasmid again

• Plasmids vary in size from1 kb to 250 kb in length. Individual plasmids vary according to the host cells in which they occur

Plasmids

• Plasmids may be • Stringent plasmids: that are present as just one or two copies are

said to have a low copy number

• Relaxed plasmids: have high copy number with 10 or more plasmid molecules present

• On the basis of function which they play in the cell they are further classified as:• F plasmids: they are sex factors enabling maleness to the cell termed

as F+. They help in the formation of tube like structures called as sex pili on the surface of the cell which in turn help in conjugation.

• R plasmids: they are capable of conferring antibiotic resistance to the cell containing it.

• Col plasmids: these encode proteins called as colcins that kill other bacteria. There are several types of col plasmids as Col B, Col E, Col I etc, which produce different types of colcins.

• Degradative plasmids: they confer the ability to degrade different types of chemicals to the cell.

Plasmids

Questions?