MOLECULAR GENETICS 5

Criteria for Genetic Material

DNA was found to be the prominent genetic material in most organisms.

Exceptions were some viruses where RNA was the genetic material.

What made DNA eligible to be the genetic material, and not other molecules like proteins, carbohydrates etc.

Important criteria to be fulfilled to be a genetic material are:

 

• Capable of replicating itself
• Chemically & structurally stable
• Provide scope for mutation which can lead to evolution
• Capable of expressing itself in the form of 'Mendelian Characters’

Most of the other molecules like proteins, carbohydrates, lipids failed to fulfill the above mentioned criteria.

However, RNA could also fulfill the criteria; still DNA was a preferred genetic material over RNA because of the following reasons:

• DNA is structurally more stable than RNA
• DNA is chemically more stable than RNA
• DNA has double-stranded structure which provides better ability to rectify errors during replication
• DNA can’t code directly for protein synthesis & thus depends on RNA

DNA was thus used for storage of genetic information due to its structural & chemical stability. RNA, on the other hand was used for expression of genetic information as it could directly code for proteins.

RNA World

The question that was bothering everyone was which molecule (DNA or RNA) started to exist in the world before. Walter Gilbert in 1986 hypothesized that there was once an RNA world. According to this hypothesis, all that we see today has descended from an RNA world.

RNA world was a kind of hypothetical world where RNA performed all the activities which are today performed by DNA & proteins. DNA later evolved from RNA with chemical modifications which made it more stable.

There are many scientists who do not agree to this hypothesis. Research is still going on to validate the RNA world hypothesis.

MOLECULAR GENETICS 4

Biochemical nature of Transforming Principle

Bacteriologists performed a series of experiments to identify the Transforming  Principle.

 

• Transforming principle precipitated with alcohol. This showed it was not Carbohydrate.
• Transforming principle could not be destroyed with Proteases. Thus, it was not Protein.
• Transforming principle could not be destroyed with Lipases. This proved it was neither Lipids.
• Transforming principle could not be inactivated with Ribonuclease, hence not RNA.
• Transforming principle could be inactivated with Deoxyribonuclease

Transforming principle was DNA. Therefore, DNA was the genetic material.

Hershey-Chase Experiment

Hershey-Chase experiment was performed in 1952 to further confirm that DNA was the genetic material. They experimented with Bacteriophages. Bacteriophages are the viruses that infect & replicate within bacteria.

Bacteriophages were grown in two different mediums.

• Some bacteriophages were grown in radioactive phosphorus medium. It was found that these Bacteriophages came up with radioactive DNA
• Some bacteriophages were grown in radioactive sulfur medium. It was found that these Bacteriophages with radioactive protein.

 

1. Bacteriophages with Radioactive DNA were brought in contact with bacteria
2. Bacteria got infected
3. 
   
4. Agitated in a blender to separate phage particles from bacterial cells
5. 
   
6. Centrifugation leaves Phage particles as supernatant
7. 
   
8. Bacterial cells were found to be radioactive
9. No radioactivity was detected in the phage particles

 

It was therefore concluded that it was not the proteins, rather DNA which entered into the bacteria. Therefore, DNA causes the replication of viruses inside the bacteria.

DNA was thus proved to be the genetic material.

MOLECULAR GENETICS 3

Search for the Genetic Material

The study of Genetics started with Gregor Mendel who introduced ‘factors’ for inheritance. 

Boveri-Sutton theory later gave the chromosomal theory of inheritance.

The concept of inheritance was understood at the level of chromosomes further when Morgan came up with the concept of linkage & recombination at chromosomal level.

It was taken a step forward in 1926 when experiments were being performed to understand Inheritance at a molecular level.

The search was for the molecule which acts as a genetic material.

Griffith’s Experiment

Griffith experimented with Streptococcus pneumoniae bacteria. 

This bacteria causes Pneumonia.

Two strains -- R-strain & S-strain.

• S strain
  • Smooth mucous polysaccharide coat
  • Resistant to immune system
  • Virulent
• R strain
• Lacks the coat
• Destroyed by immune system of the host
• Non-virulent

The experiment was performed in multiple steps.

1. S-strain (virulent) was injected into mouse. It was found that the mouse died of pneumonia

R-strain (non-virulent) was injected into mouse. It was found that the mouse remained alive

Heat killed S-strain (S-strain bacteria were killed by heating) was injected into mouse. It was found that the mouse remained alive

Heat killed S-strain & live R-strain were injected into mouse. It was found that the mouse died of pneumonia

Griffith found that Live S-strain bacteria could be recovered from the dead mouse (Step 4).

Griffith thus arrived at the following conclusion:

• Something caused bacteria to change from one type (R) to another type (S)
• Some ‘Transforming principle’, transferred from heat-killed S strain to R strain and transformed it virulent.

However, the biochemical nature of the ‘Transforming principle’ was still unknown.

MOLECULAR GENETICS 2

Detailed structure of DNA

 DNA polynucleotide chain has two free ends:

• 5’ end
  • Free phosphate moiety at 5'-end of deoxyribose sugar
• 3’ end
• Free 3'-OH group of deoxyribose sugar
• 
  

  

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Watson & Crick were the first to propose the Double helix structure of DNA, based on X-ray diffraction technique.

DNA Double-helix model

• DNA is composed of two polynucleotide chains
• Sugar-phosphate forms the backbone
• Nitrogenous bases form the interior, paired through H- bonds

• Complementary base pairing is an important feature of DNA structure (C-- G, A--T, means, purine with pyrimidine, why not purine and purine OR pyrimidine and pyrimidine?  To maintain equal distance between 2 chains, purine 2ring structure, while pyrimidine 1 ring structure)

• The two polynucleotide chains have anti-parallel polarity (one chain, 5'--3' ,other chain will be 3'--5' )

• Two chains are coiled in a right handed fashion forming a right-handed helix
• Uniform distance is maintained between the two strands of helix
• Base pair distance = 3.4 Angstrom
• Each helix = 10 base pairs = 34 Angstrom

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Packaging of DNA helix

• Length of DNA is found to be far greater than dimension of a typical nucleus

Total number of base pairs in a typical mammalian cell= 6.6 * 109

Distance between two base pairs= 0.34nm

Therefore, Length of DNA= 0.34 *10-9 *6.6 *109 = 2.2m

Size of a nucleus is of the order of 10-6m

So, Length of DNA is greater than the size of nucleus.

DNA is packaged very strategically to fit inside the nucleus.

• In Prokaryotes, DNA is organized into loops held by proteins. The region where DNA is present is termed as ‘Nucleoid’

• In Eukaryotes, there exist positively charged basic proteins called Histones.
• DNA wrap around the histone octamer (group of 8 histone proteins) to form a Nucleosome.

•  Each nucleosome contains 200 base pairs of DNA helix.
•  Nucleosomes in chromatin are seen as ‘beads-on-string’ under Electron microscope.

Based on different types of DNA packaging, there are two forms of Chromatin.

• Euchromatin
  • Less condensed structure with looser DNA packaging
  • Lightly stained when observed under microscope
  • Contains less DNA
  • Transcriptionally active
  • Found in eukaryotes & prokaryotes
• Heterochromatin
  • Highly condensed structure with tighter DNA packaging
  • Dark stained when observed under microscope
  • Contains more DNA
  • Transcriptionally inactive, as those regions of the genes which need to interact with proteins for transcription is inaccessible.
  • Found in eukaryotes

MOLECULAR GENETICS 1

Introduction

Inheritance at a molecular level.

DNA is the genetic material for most of the organisms, 

exceptions - viruses -- RNA

DNA -- synthesis of RNA -- protein synthesis -- control traits of individuals.

In this lesson, we will learn how DNA was discovered to be the genetic material, and how this entire process of protein synthesis takes place.

Structure of Nucleic Acids

Nucleic acids are the biomolecules which play a very important role in the process of Inheritance.

Two types of nucleic acids exist: DNA (Deoxyribo Nucleic Acid) and RNA (Ribo Nucleic Acid).

DNA has a double-stranded structure. It is a polynucleotide whose monomer units are deoxyribonucleotide. 

Length of DNA is determined by number of nucleotides in it.

RNA, on the other hand, has a single-stranded structure. It is also a polymer whose monomer units are ribonucleotide.

A nucleotide has 3 components:                     

• Pentose sugar
• Monosaccharide with 5 Carbon atoms
• Ribose sugar in RNA
• 
  
  
• Deoxyribose sugar in DNA
• 
  

Nitrogenous base

• Nitrogen containing compound with properties of a base
• 2 types: Purines, Pyrimidines
• Purine
• Heterocyclic aromatic organic compound
• 9-membered ring
• Examples: Adenine, Guanine

Pyrimidine

• Heterocyclic aromatic organic compound
• 6-membered ring
• Examples: Cytosine, Uracil, Thymine

Phosphate group

• Inorganic salt of phosphorus
• Forms backbone of polynucleotide chain along with the sugar

Formation of a polynucleotide takes place using the following linkages:

 

1. Nitrogenous base is linked to the pentose sugar through a N-glycosidic bond to form a nucleoside
2. 
   
3. A phosphate group is linked to 5'-OH of a nucleoside through phosphoester bond to form a nucleotide
4. 
   
5. Multiple nucleotides are joined together through 3'-5' phosphodiester bond to form a polynucleotide
6. 
   
   

GENETICS 9

PEDIGREE ANALYSIS

• Used for mendelian disorders.
• Analysis of traits in several generations of family is called pedigree analysis.
• Inheritance of a particular trait is represented in the family tree over generations.
• Symbols used in pedigree analysis

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  .              X linked recessive 
  
  
  
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• Autosomal dominant
• Eg. PKD
• 
  

Autosomal recessive

GENETICS 8

CHROMOSOMAL DISORDERS

• It is caused due to absence or excess or abnormal arrangement of one or more chromosomes.

Change in number-

• Aneuploidy – failure of segregation of chromatids during cell division cycle results in the gain or loss of chromosome.
• Eg. 44A+ XY.  Normal male --- 44+XXY 
• Eg. 46 --- 47.      Trisomy
•        46----- 48.     Tetrasomy
•        46--- 45.     Monosomy
•         46---- 44.   Nullisomy

• Polyploidy – Failure of cytokinesis after telophase stage of cell division -- an increase in a whole set of chromosomes in an organism.

Eg.. normal diploid-- triploid

This condition is usually seen in plants.

• Down’s syndrome – gain of extra copy of chromosome 21 (trisomy 21)
• It was first described by Langdon Down (1866).
• Affected individual is short with small round head, furrowed tongue and partially open mouth.
• Broad palm with characteristic palm crease.
• Physical, psychomotor and mental development is retarded.

• Turner’s syndrome – loss of an X-chromosome in human females i.e. 45 with XO
• Such females are sterile as ovaries are rudimentary

• Klinefelter’s syndrome – presence of an additional copy of X-chromosome in male, resulting into karyotype, 47, XXY.
• Sterile individuals