Molecular Biology 1: Genome Organization Introduction by Doctor-dr

by Microbiology Doctor-dr

Molecular Biology

Unit 1: Genome Organization

Objectives

• To provide information on the many systems involved in the transmission of genetic information.
• To gain a better understanding of the mechanisms that govern gene expression and regulation.
• To develop a comprehensive grasp of the molecular processes that occur in living organisms.

Introduction: 

• A cell's genome is made up of its genetic complement.
• Organisms can have genomes that are made up of DNA or RNA.
• The genome's size and organisation are extremely complicated.
• Understanding genetic processes necessitates an understanding of the genome's arrangement at the chromosomal level.
• The size and structural features of chromosomes vary.
• The structure and genetic arrangement of eukaryotic and prokaryotic chromosomes differ significantly. 

Genome Size and Evolutionary Complexity: 

• Studies of the nucleic acid content of viruses, bacteria, and eukaryotes have led to the conclusion that genome size grows as evolutionary complexity increases.
• This generalisation is based on the observations that a typical virus genome is smaller than a normal bacteria genome; yeast, a basic unicellular eukaryote, contains more DNA than a typical bacterium; and multicellular eukaryotes have the most DNA per genome.

Genome size and form of some representative viruses, bacteria, and eukaryotes:

Genome	Approximate length in thousands of nucleotides	Form
Virus MS2 SV40 fX174   lamda	3569 nt (4 genes) 5000 nt (5 genes) 5 genes   50 genes	Single-stranded RNA Circular double-stranded DNA Circular single-stranded DNA; double-stranded replicative form Linear double-stranded DNA
Bacteria Mycoplasma hominis Escherichia coli	760 4700	Circular double-stranded DNA Circular double-stranded DNA
Eukaryotes Saccharomyces cerevisiae (yeast) Caenorhabditis elegans (nematode) Arabidopsis thaliana (wall cress) Drosophila melanogaster (fruit fly) Homo sapiens (human being) Zea mays (maize) Amphiuma sp. (salamander)	13,000 100,000 100,000 165,000 3,000,000 4,500,000 76,500,000	Haploid chromosome number 16 6 5 4 23 10 14

The Genetic Material: 

The mid-twentieth century was a period of rapid scientific advancement.

DNA (deoxyribonucleic acid) is the genetic substance of living things, according to geneticists.

Biochemists, on the other hand, were having difficulty describing the structure of DNA.

The genetic material, according to researchers, must be:

1. able to store information that control the development, structure, and metabolic activities of the cell or organism.

2. stable so that it can be replicated with high fidelity during cell division and be transmitted from generation to generation.

3. able to undergo rare changes called mutations that provide the genetic variability required for evolution to occur.

DNA (Deoxyribonucleic Acid)

• DNA (deoxyribonucleic acid) is the cell's hereditary material.
• Friedrich Miescher pioneered the biochemical study of DNA in 1868.
• Miescher extracted a phosphorus-containing compound named "nuclein" from the nuclei of pus cells (leukocytes).
• Miescher discovered that nuclein had an acidic and a basic portion (which we now know as DNA) (proteins).
• Later, he was able to separate a similar acidic material from the heads of Salmon sperm cells.

DNA as a Genetic Material:

• Frederick Griffith carried out a well-known experiment using the Streptococcus pneumoniae bacterium in 1931.
• When these bacteria are cultivated on culture plates, he notices that some, known as S strain bacteria, form glossy, smooth colonies, while others, known as R strain bacteria, develop rough colonies.
• S strain bacteria have a capsule under the microscope, but R strain bacteria do not.
• Griffith found that when he injected mice with the S strain of germs, they died, however when he injected mice with the R strain, they did not.
• In order to see if the capsule alone was responsible for the S strain bacteria's pathogenicity, he injected heat-killed S strain bacteria into mice. The mice were not killed.
• Griffith then injected a mixture of heat-killed S strain and live R strain bacteria into the animals. The mice died unexpectedly, and living S strain bacteria were discovered in their bodies.
• Griffith came to the conclusion that some substance required for the bacteria to create a capsule and be virulent had moved from the dead S strain bacteria to the living R strain bacteria, transforming the R strain bacteria.
• This change in the R strain bacteria's phenotypic must be attributable to a change in their genotype.
• Genetic material must have been the transforming substance that moved from S strain to R strain.
• This encouraged researchers to start seeking for the changing substance in order to figure out what the chemical makeup of the genetic material was.
• Oswald T. Avery, Colin MacLeod, and Maclyn McCarty released a report in 1944 after 16 years of research revealing that DNA is the transforming substance that permits Streptococcus to create a capsule and be virulent.
• They provided the first direct proof that DNA carries genetic information.
• They discovered that extracting DNA from a virulent (disease-causing) strain of Streptococcus pneumoniae genetically changed a nonvirulent strain of the bacteria into a virulent variety.

The Hershey-Chase Experiment:

• The Hershey-Chase Experiment is a study conducted by Hershey and Chase.
• Alfred D. Hershey and Martha Chase did another experiment in 1952 that proved that DNA contains genetic information.
• They demonstrated that when bacteriophage T2 (a bacterial virus) infects Escherichia coli, the virus's sulfur-containing protein remains outside while its phosphorus-containing DNA enters the host cell and provides the genetic information for viral replication using radioactive phosphorus (32P) and sulphur (35S) tracers.

The Chemical Composition of DNA

DNA is a linear polymer of repeating units having a fixed backbone built of sugar-phosphate residues.

The sugar is deoxyribose, from which DNA receives its name.

Each deoxyribose may attach with one of four possible bases:

• Adenine (A)
• Guanine (G)
• Thymine (T)
• Cytosine (C)

• The two bases have a double-ring structure while the other two have a single-ringed structure. 
• The double ringed bases are called purines whereas the single  ringed bases are called pyrimidines.

• In nucleic acids (DNA or RNA) the structure formed by linking of sugar with a base is known as a nucleoside.
• When a phosphate group is also attached to the sugar of nucleoside, the nucleoside becomes a nucleotide.
• the carbon atom to which the base is attached is the 1' carbon and the carbon atom to which phosphate is attached is 5’ carbon.

Polynucleotide

• The nucleotides of DNA and RNA are joined to form a polynucleotide chain, in which the phosphate attached to the 5' carbon of one sugar is linked to the hydroxyl group attached to the 3' carbon of the adjacent sugar.
• Each polynucleotide chain has a 5'-phosphate (5'-P) group at one end and a 3'-hydroxyl (3'-OH) group at the other.
• The nucleotide monomers are joined together through phosphodiester bond indicating a phosphorous atom & diester referring two esters (C-O-P) in each linkage.
• The phosphate group attached to 5’ carbon of one nucleotide is joined to 3’ carbon of next nucleotide.
• The polynucleotide is a linear molecule in which the backbone is made up of a series of sugar phosphate molecules. The bases project to one side of the backbone.

Polynucleotide have distinct Ends

• Two ends off polynucleotides are not same.
• One end is called 5’ or 5-P terminus. The 5C at this end does not participate in phosphodiester bond &
• triphosphate group is attached to it.
• Other end is called 3’ or 3-OH terminus. At this end un reacted group is hydroxyl group.
• This chemical distinction shows that polynucleotide have a direction, which is very important in molecular genetics.

• Because the ends of a DNA strand are asymmetric, each strand has a polarity.
• Along a strand of DNA, the nucleotides can be organised in any order.
• The genetic information that gives instructions for constructing proteins is encoded in this sequence of nucleotides along a DNA strand.

DNA Molecules Have Distinctive Base Compositions 

In late 1940s Erwin Chargaff and his colleagues found that the four nucleotide bases of DNA occur in different ratios in different organisms 

On the basis of data collected from DNA of many species Chargaff gave following conclusions (called “Chargaff’s rules”): 

• The DNA base composition varies from one species to another 
• The base composition of DNA specimens isolated from different tissues of the same species remains the same 
• The organism’s age, nutritional state, or changing environment does not affect the base composition of DNA of a given organism 
• In all cellular DNAs, A = T, and G = C 
• The sum of the purine residues equals the sum of the pyrimidine residues i.e. (A + G = T + C)

DNA is a Double Helix

• In the early 1950s, Rosalind Franklin and Maurice Wilkins used the powerful process of x-ray diffraction.
• They discovered that a concentrated DNA solution may be split into threads.
• DNA is a double helix, as seen by its X-ray diffraction pattern.
• The crossing (X) pattern in the centre of the shot indicates the helical structure.

Structure of DNA:

• In 1953, Watson and Crick proposed a three-dimensional model of DNA structure, for which they were awarded the Nobel Prize in Chemistry in 1962.
• According to this hypothesis, a right-handed double helix is formed by two helical DNA polynucleotide chains twisted around the same axis.
• On the exterior of the double helix are the hydrophilic sugar phosphate backbones.
• Both strands' purine and pyrimidine bases are packed inside the double helix.
• Hydrogen bonds between pyrimidine and purine bases hold the two strands together.
• On the duplex's surface, a major groove and a minor groove are generated.
• Each nucleotide base on one strand is linked with a base on the other strand so that G makes a hydrogen bond with C and A forms a hydrogen bond with T.
• Inside the double helix, the vertically stacked bases are 3.4 (0.34 nm) apart.
• Per entire turn of the double helix, there were roughly 10.5 base pairs, with a secondary repeat distance of about 34. (3.4 nm).
• Double helical DNA has two antiparallel polynucleotide chains that are complementary to one another.
• There are three hydrogen bonds between Guanine and Cytosine (G≡C), and two hydrogen bonds are present between Adenine and Thymine (A=T).
• Watson and Crick constructed their model of DNA keeping in view the antiparallel arrangement of both strands i.e., one strand of DNA run from 5’→3’ direction and the other strand runs from 3’→5’ direction.

Forms of DNA

DNA can occur in different three-dimensional forms

1. B-form DNA

2. A-form DNA

3. Z-form DNA

1. B-form DNA

• The Watson-Crick structure is also referred to as B-form DNA, or B-DNA.
• Under physiological conditions most DNA occurs as B-form.
• B-DNA is arranged in a right-handed double helix with 10.5 base pairs per  helical turn.
• The bases are stacked almost perpendicular to the main axis.
• The major groove is wide and of moderate depth, while the minor groove is much narrower.
• At conditions of high humidity (95%) and low salt concentration B-DNA occurs within the cell.

2. A-form DNA

• A-form DNA is favored in many solutions that have low water content and high salt concentration.
• The helix of A-DNA is much wider than B-DNA with 11 base pairs per helix turn.
• The DNA is still arranged in a right-handed double helix, but the bases are tilted about 20˚ with respect to the axis.
• Major groove is deep and narrow, while minor groove is shallow and broad.
• The presence of A-DNA in cells is uncertain, however, RNA may adopt A form when it forms double-stranded regions.

3. Z form DNA

• Alexander Rich and associates (1979) discovered a left-handed helix formed by oligonucleotides composed of repeating GC sequences on one strand, and complementary CG sequences on the other strand.
• As the backbone formed a zig-zag structure, they called the structure as Z-DNA.
• Z form DNA is more slender and elongated with 12 base pairs per helical turn.
• The major groove is very shallow while the minor groove is very deep and narrow.
• Z-DNA was first formed under conditions of high salt or in the presence of alcohol. But in the presence of methylated cytosine it can also be stabilized at physiological conditions.
• Z-DNA occur as short stretches in both prokaryotes and eukaryotes and help in regulating the expression of some genes or in genetic recombination.