REASONS OF FEMALE FOETICIDE :-PRINCIPLES OF INHERITANCE AND VARIATION

PRINCIPLES OF INHERITANCE AND VARIATION

PRINCIPLES OF INHERITANCE AND VARIATION

·         Genetics: deals with the inheritance, as well as the variation of characters from parents to offsprings.

·         Inheritance: is the process by which characters are passed on from parent to progeny.

·         Variation: is the degree by which progeny differ from their parents.

MENDEL’S LAWS OF INHERITANCE:

·         Gregor Mendel. Conducted hybridization experiments on garden peas for seven years (1856 – 1863) and proposed laws of inheritance.

·         Mendel conducted artificial pollination/cross pollination experiments using several true-breeding pea lines.

·         A true breeding line is one that, having undergone continuous self-pollination for several generations.

·         Mendel selected 14 true-breeding peas’ plant varieties, as pair’s which were similar except for one character with contrasting traits.

o    True breed selected by Mendel

o    Stem height- Tall / dwarf

o    Flower color- Violet/white

o    Flower position – Axial / terminal

o    Pod shape- Inflated / beaded or constricted

o    Pod color- Green / yellow

o    Seed  color- Yellow/ green

o    Seed shape – round / wrinkled

INHERITANCE OF ONE GENE:

·         Mendel crossed tall and dwarf pea plants to study the inheritance of one gene.

·         He collected the seeds produced as a result of this cross and grew them to generate plants of the first hybrid generation. This generation is called filial progeny or the F₁.

·         Mendel observed that all the F₁ progeny plants ere tall, like one of its parents; none were dwarf.

·         He made similar observations for the other pairs of traits – he found that the F₁ always resembled either one of the parents, and that the trait of the other parent was not seen in them.

·         Mendel then self – pollinated the tall F₁ plants and to his surprise found that in the F₂ generation some of the offsprings were ‘dwarf; the character that was not seen in the F₁ generation was now expressed.

·         The proportion of plants that were dwarf was 1/4^th of the F₂ plants while 3/4th of the F₂ plants were tall.

·         The tall and dwarf traits were identical to their parental type and did not show any blending, that is all the offsprings were either tall or dwarf, none were of in between height.

·         Similar results were obtained with the other traits that he studied: only one of the parental traits was expressed in the F₁ generation while at the F₂ stage both the traits were expressed in the proportion of 3:1.

·         The contrasting traits did not show any blending at either F₁ or F₂ stage.

 Mendel’s proposition:

·         Mendel proposed that something was being stably passed down, unchanged, from parent to offspring through the gametes, over successive generations. He called these things as ‘factors’.

·         Now a day we call them as genes.

·         Gene is therefore are the units of inheritance.

·         Genes which codes of a pair of contrasting traits are known as alleles, i.e. they are slightly different forms of the same gene.

Alphabets used:

·         Capital letters used for the trait expressed at the F₁ stage.

·         Small alphabet for the other trait.

·         ‘T’ is used for Tall and‘t’ is used for dwarf.

·         ‘T’ and‘t’ are alleles of each other.

·         Hence in plants the pair of alleles for height would be TT. Tt. or tt.

·         In a true breeding tall or dwarf pea variety the allelic pair of genes for height are identical or homozygous, TT and tt respectively.

·         TT and tt are called the genotype.

·         Tt plant is heterozygous for genes controlling one character (height).

·         Descriptive terms tall and dwarf are the phenotype.

Test cross:

·         When F₁ hybrid is crossed back with the recessive parent, it is known as test cross.

·         It is used to know the genotype of the given plant/animal.

Law of Dominance:

·         Characters are controlled by discrete units called factors.

·         Factors occur in pairs.

·         In a dissimilar pair of factors one member of the pair dominates (dominant) the other (recessive).

Law of Segregation:

·         The alleles do not show any blending and that both the characters are recovered as such in the F₂ generation though one of these is not seen at the F₁ stage.

·         The parents contain two alleles during gamete formation; the factors or alleles of a pair segregate or separate from each other such that a gamete receives only one of the two factors.

·         Homozygous parent produces all gametes that are similar i.e. contain same type of allele.

·         Heterozygous parents’ produces two kinds of gametes each having one allele with equal proportion.

Incomplete dominance:

·         When a cross between two pure breed is done for one contrasting character, the F₁ hybrid phenotype dose not resemble either of the two parents and was in between the two, called incomplete dominance.

·         Inheritance of flower color in the dog flower (snapdragon or Antirrhinum sp.) is a good example of incomplete dominance.

·         F₂ generation phenotypic ratio is 1:2:1 instead of 3:1 as Mendelian monohybrid cross.

·         Genotypic ratio of F₂ generation is 1:2:1.

Co – dominance:

·         F₁ resembled either of the two parents (complete dominance).

·         F₁ offspring was in-between of two parents (incomplete dominance).

·         F₁ generation resembles both parents side by side is called (co-dominance).

·         Best example of co-dominance is the ABO blood grouping in human.

·         ABO blood group is controlled by the gene I.

·         The plasma membrane of the RBC has sugar polymers (antigen) that protrude from its surface and the kind of sugar is controlled by the gene-I.

·         The gene I has three alleles I ^A, I ^B and i.

·         The alleles I ^A and I ^B produce a slightly different form of sugar while allele i doesn’t produce any sugar.

·         Each person possesses any two of the three I gene alleles.

·         I ^A and I ^B are completely dominant over i.

·         When I ^A, and I ^B present together they both express their own types of sugar; this because of co-dominance. Hence red blood cells have both A and B type sugars.

Multiple Alleles:

·         Example of ABO blood grouping produces a good example of multiple alleles.

·         There are more than two i.e. three allele, governing the same character.

A single gene product may produce more than one effect:

·         Starch synthesis in pea seeds is controlled by one gene.

·         It has two alleles B and b.

·         Starch is synthesized effectively by BB homozygote and therefore, large starch grains are produced.

·         The ‘bb’ homozygous has less efficiency hence produce smaller grains.

·         After maturation of the seeds, BB seeds are round and the bb seeds are wrinkle.

·         Heterozygous (Bb) produce round seed and so B seems to be dominant allele, but the starch grains produced are of intermediate size.

·         If starch grain size is considered as the phenotype, then from this angle the alleles show incomplete dominance.

INHERITANCE OF TWO GENES:

Law of independent Assortment:

·         When two characters (dihybrid) are combined in a hybrid, segregation of one pair of traits is independent of the other pair of traits.

CHROMOSOMAL THEORY OF INHERITANCE:

Why Mendel’s theory was remained unrecognized?

·         Firstly communication was not easy in those days and his work could not be widely publicized.

·         Secondly his concept of genes (or factors, in Mendel’s word) as stable and discrete units that controlled the expression of traits and of the pair of alleles which did not’ blend’ with each other, was not accepted by his contemporaries as an explanation for the apparently continuous variation seen in nature.

·         Thirdly Mendel’s approach of using mathematics to explain biological phenomena was totally new and unacceptable to many of the biologists of his time.

·         Finally he could not provide any physical proof for the existence of factors.

Rediscovery of Mendel’s result:

·         1990 three scientists (deVries, Correns and von Tschermak) independently rediscovered Mendel’s result on the inheritance of character.

Chromosomal theory of inheritance:

·         Proposed by Walter Sutton and Theodore Bovery in 1902.

·         They work out the chromosome movement during meiosis.

·         The behavior of chromosomes was parallel to the behavior of genes and used chromosome movement to explain Mendel’s laws.

·         Sutton united the knowledge of chromosomal segregation with Mendelian principles and called it the chromosomal theory of inheritance.

o    Chromosome and genes are present in pairs in diploid cells.

o    Homologous chromosomes separate during gamete formation (meiosis)

o    Fertilization restores the chromosome number to diploid condition.

o    The chromosomal theory of inheritance claims that, it is the chromosomes that segregate and assort independently.

Experimental verification of chromosomal theory:

·         Experimental verification of chromosomal theory of inheritance by Thomas Hunt Morgan and his colleagues.

·         Morgan worked with tiny fruit flies, Drosophila melanogaster.

Why Drosophila?

·         Suitable for genetic studies.

·         Grown on simple synthetic medium in the laboratory.

·         They complete their life cycle in about two weeks.

·         A single mating could produce a large number of progeny flies.

·         Clear differentiation of male and female flies

·         Have many types of hereditary variations that can be seen with low power microscopes.

Linkage and Recombination:

·         Morgan hybridized yellow bodied, white eyed females to brown-bodied, red eyed male and intercrossed their F1 progeny.

·         He observed that the two genes did not segregate independently of each other and the F2 ratio deviated very significantly from 9:3:3:1 ratio (expected when the two genes are independent).

·         When two genes in a dihybrid cross were situated on the same chromosome, the proportion of parental gene combinations was much higher than the non-parental type.

·         Morgan attributed this due to the physical association or linkage of the two genes and coined the term linkage.

·         Linage: physical association of genes on a chromosome.

·         Recombination: the generation of non-parental gene combinations.

·         Morgan found that even when genes were grouped on the same chromosome, some genes were very tightly linked (showed very low recombination) while others were loosely linked (showed higher recombination).

·         The genes white and yellow were very tightly linked and showed 1.3 percent recombination.

·         The genes white and miniature wing showed 37.2 percent recombination, hence loosely linked.

·         Alfred Sturtevant used the frequency of recombination between gene pairs on the same chromosome as a measure of the distance between genes and ‘mapped’ their position on the chromosome.

POLYGENIC INHERITANCE:

·         Human have no distinct tall or short instead a whole range of possible heights.

·         Such traits are generally controlled by three or more genes and are thus called polygenic trait.

·         Besides the involvement of multiple genes polygenic inheritance also takes into account the influence of environment.

·         Human skin color is another classic example of polygenic inheritance.

·         In a polygenic trait the phenotype reflects the contribution of each allele i.e. the effect of each allele is additive.

·         Assume that three genes A, B, C control the skin colour in human.

·         Dominant forms A, B; AND C responsible for dark skin colour and the recessive forms a, b, c for light color of the skin.

·         Genotype with dominant alleles (AABBCC) will have darkest skin color.

·         Genotype with recessive alleles (aabbcc) will have lightest skin colour.

·         Other combinations always with intermediate colour.

PLEIOTROPY:

·         A single gene can exhibit multiple phenotypic expression, such gene is called pleiotropic gene.

·         The mechanism of pleiotropy in most cases is the effect of a gene on metabolic pathways which contributes towards different phenotypes.

·         Phenylketonuria a disease in human is an example of pleiotropy.

·         This disease is caused due to mutation in the gene that code for the enzyme phenyl alanine hydroxylase.

·         Phenotypic expression characterized by:-

o    Mental retardation

o    Reduction in hairs.

o    Reduction in skin pigmentation.

SEX DETERMINATION:

·         Henking (1891) traced specific nuclear structure during spermatogenesis of some insects.

·         50 % of the sperm received these specific structures, whereas 50% sperm did not receive it.

·         Henking gave a name to this structure as the X-body.

·         X-body of Henking was later on named as X-chromosome.

Sex-determination of grass hopper:

·         Sex-determination in grasshopper is XX-XO type.

·         All egg bears one ‘X’ chromosome along with autosomes.

·         Some sperms (50%) bear’s one ‘X’ chromosome and 50% do not.

·         Egg fertilized with sperm (with ‘X’ chromosome) became female (22+XX).

·         Egg fertilized with sperm (without ‘X’ chromosome) became male (22 + X0)

Sex determination in insects and mammals (XX-XY type):

·         Bothe male and female has same number of chromosomes.

·         Female have autosomes and a pair of X chromosomes. (AA+ XX)

·         Male have autosomes and one large ‘X’ chromosome and one very small ‘Y-chromosomes. (AA+XY)

·         This is called male heterogammety and female homogamety.

Sex determination in birds:

• Female birds have two different sex chromosomes designated as Z and W.
• Male birds have two similar sex chromosomes and called ZZ.
• Such type of sex determination is called female heterogammety and male homogamety.

Sex determination in Honey bee:

• Sex determination in honey bee based on the number of sets of chromosomes an individual receives.
• An offspring formed from the fertilization of a sperm and an egg developed into either queen (female) or worker (female).
• An unfertilized egg develops as a male (drone), by means of parthenogenesis.
• The male have half the number of chromosome than that of female.
• The female are diploid having 32 chromosomes and males are haploid i.e. having 16 numbers of chromosomes.
• This is called haplodiploid sex determination system.
• Male produce sperms by mitosis, they do not have father and thus cannot have sons, but have grandsons.

MUTATION:

• Mutation is a phenomenon which results in alteration of DNA sequences and consequently results in changes in the genotype and phenotype of an organism.
• In addition to recombination, mutation is another phenomenon that leads to variation in DNA.
• Loss (deletion) or gain (insertion/duplication) of a segment of DNA results in alteration in chromosomes.
• Since genes are located on the chromosome, alteration in chromosomes results in abnormalities or aberration.
• Chromosomal aberrations are commonly observed in cancerous cells.
• Mutations also arise due to change in a single base pair of DNA. This is known as point mutation. E.g. sickle cell anemia.
• Deletion and insertions of base pairs of DNA causes frame shift mutations.

GENETIC DISORDERS:

Pedigree Analysis:

• Analysis of traits in several of generations of a family is called the pedigree analysis.
• In the pedigree analysis the inheritance of a particular trait is represented in the family tree over generations.

Autosomal Dominant:

• Affected individuals have at least one affected parent
• The phenotype generally appears every generation
• Two unaffected parents only have unaffected offspring
• Traits are controlled by dominant genes
• Both males and females are equally affected
• Traits do not skip generations
• e.g. polydactyly, tongue rolling ability etc.

Autosomal recessive:

• Unaffected parents can have affected offspring
• Traits controlled by recessive  genes and
• Appear only when homozygous 
• Both male and female equally affected
• Traits may skip generations
• 3:1 ratio between normal and affected. 
• Appearance of affected children from normal parents (heterozygous)
• All children of affected parents are also affected.
• e.g.- Albinism, sickle cell anaemia etc.

Mendelian Disorder:

• Genetic disorders grouped into two categories –
• Mendelian disorder
• Chromosomal disorder
• Mendelian disorders are mainly determined by alteration or mutation in the single gene.
• Obey the principle of Mendelian inheritance during transmission from one generation to other.
• Can be expressed in pedigree analysis.

E.g. Haemophilia, colorblindness, Cystic fibrosis, Sickle cell anemia, Phenylketonuria, Thalasemia etc.

Hemophilia:

In this disease a single protein that is a part of the cascade of proteins involved in the clotting of blood is affected. Due to this in an affected individual a simple cut will result in non-stop bleeding.

• Sex linked recessive disease.
• The diseases transmitted from unaffected carrier female to some of the male progeny.
• Female becoming hemophilic is extremely rare because mother of such a female at least carrier and the father should be hemophilic.
• Affected transmits the disease only to the son not to the daughter.
• Daughter can receive the disease from both mother and father.

Sickle cell anaemia:

• The defect is caused due to substitution of Glutamic acid (Glu) by Valine (Val) at the sixth position of the beta globin chain of the haemoglobin molecule.
• Substitution of amino acid takes place due to the single base substitution at the sixth codon of the beta globin gene from GAG to GUG.
• The mutant haemoglobin molecule undergoes polymerization under low oxygen tension causing the change in the shape of the RBC from biconcave disc to elongated sickle like structure.
• This is an autosomes linked recessive trait.
• Transmitted from parents to the offspring when both the parents are carrier for the gene (heterozygous).
• This disease is controlled by single pair of allele, HbA, and HbS.
• There are three possible genotypes (HbA HbA, HbA HbS, and HbSHbS.
• Only homozygous individuals for HbS (HbS HbS) show the diseased phenotype.
• Heterozygous (HbA HbS) individuals appear apparently unaffected but they are carrier of the disease as there is 50 percent probability of transmission of the mutant gene to the progeny.

Phenylketonuria:

• Autosomal recessive trait.
• Inborn error of metabolism.
• The affected individual lack one enzyme called phenyl alanine hydroxylase that converts the amino acid phenyl alanine to tyrosine.
• In the absence of the enzyme phenyl alanine accumulated and converted into phenylpyruvic acid and other derivatives.
• Accumulation of these results in mental retardation.
• These derivatives excreted through kidney.

Chromosomal disorders:

• Caused due to absence or excess or abnormal arrangement of one or more chromosome.
• Failure of segregation of chromatids during cell division cycle results in the gain or loss of chromosome(s), called Aneuploidy.
• Failure of cytokinesis after telophase stage of cell division results in an increase in a whole set of chromosome in an organism and this phenomenon is called polyploidy.

Trisomy: additional copy of a chromosome may be included in an individual (2n+1).
Monosomy: an individual may lack one of any one pair of chromosomes (2n-1)

Down syndrome:

• Caused due to presence of an additional copy of the chromosome number 21 (trisomy of 21).
• This disorder was first described by Langdon Down (1866).
• Short stature with small round head.
• Furrowed tongue
• Partially opened mouth
• Palm is broad with characteristic palm crease.
• Physical, psychomotor and mental development is retarded.

 Klinefelter’s syndrome:

• Caused due to the presence of an additional copy of X-chromosome resulting into a karyotype of 47, (44+XXY).
• Overall masculine development.
• Also develop feminine character (development of breast i.e. Gynaecomastia)
• Individuals are sterile.

Turner’s syndrome:

• Caused due to the absence of one of the X- chromosomes i.e. 45 (44 + X0).
• Such females are sterile as ovaries are rudimentary.
• Lack of other secondary sexual characters.

MOLECULAR BASIS OF INHERITANCE

THE DNA:

·         DNA is a long polymer of deoxyribonucleotides.

·         The length of the DNA depends on, number of nucleotide pair present in it.

·         Characteristics of the organism depend on the length of the DNA.

·         Bacteriophage ø174 has 5386 nucleotides.

·         Bacteriophage lambda has 48502 base pairs.

·         Escherichia coli have 4.6 X 10⁶ base pairs.

·         Human genome (haploid) is 3.3 X 10⁹ bp.

Structure of polynucleotide chain:

·         A nucleotide has three component:-

o    A nitrogen base

o    A pentose sugar ( ribose in RNA and deoxyribose in DNA)

o    A phosphoric acid.

·         There are two types of nitrogen bases:

o    Purines ( Adenine and Guanine)

o    Pyrimidines ( Cytosine, Uracil and Thymine)

·         Adenine, Guanine and Cytosine is common in RNA and DNA.

·         Uracil is present in RNA and Thymine is present in DNA in place of Uracil.

·         Pentose sugar is ribose in RNA and Deoxyribose in DNA.

·         A nitrogen base attached to the pentose sugar at C¹ of pentose sugar by

 N-glycosidic linkage to form a nucleoside.

·         According to the nature of pentose sugar, two types of nucleosides are formed ribonucleoside and deoxyribonucleotides.

·         Ribonucleosides are:

o    Adenosine

o    Guanosine

o    Cytidine

o    Uridine

·         Deoxyribonucleosides are:

o    Deoxyadenosine

o    Deoxyguanosine

o    Deoxycytidine

o    Deoxythymidine.

·         Phosphoric acid attached to the 5’ OH of a nucleoside by Phosphodiester linkage a corresponding nucleotide is formed. (Ribonucleotide or deoxyribonucleotides depending on the sugar unit).

·         Two nucleotides are joined by 3’-5’ Phosphodiester linkage to form dinucleotide.

·         More than two nucleotides joined to form polynucleotide chain.

·         Polynucleotide chain has a free phosphate moiety at 5’ end of sugar, is referred to as 5’ end

·         In the other end of the polymer with 3’-OH group called 3’ end.

·         The backbone of the polynucleotide chain is sugar and phosphate.

·         Nitrogen bases linked to the sugar moiety project from the backbone.

·         In RNA every nucleotide has an additional –OH group at 2’ of ribose.

·         In RNA Uracil is found in place of thymine.

·         5-methyl uracil is the other name of thymine.

History of DNA:

·         DNA is an acidic substance in the nucleus was first identified by Friedrich Meischer in 1869. He named it as ‘Nuclein”

·         1953 double helix structure of DNA was given by James Watson and Francis Crick, based on X-ray defraction data produced Maurice Wilkins and Rosalind Franklin.

·         Hallmark of their proposition was base pairing between two strands of polynucleotide chains. This was based on observation of Erwin Chargaff.

·         Chargaff’s observation was that for a double stranded DNA, the ratio between Adenine and Thymine, and Guanine and Cytosine are constant and equal one.

Salient features of Double helix structure of DNA:

·         Made of two polynucleotide chains.

·         Sugar and phosphate forms the backbone and bases projected to inside.

·         Two chains have anti-parallel polarity.

·         Two strands are held together by hydrogen bond present in between bases.

·         Adenine of one strand pairs with Thymine of another strand by two hydrogen bonds and vice versa.

·         Guanine of one strand pairs with Cytosine of another strand by three hydrogen bonds and vice versa.

·         A purine comes opposite to a pyrimidine. This generates approximately uniform distance between the two strands of the helix.

·         The two chains are coiled in a right – handed fashion.

·         The pitch of the helix is 3.4 nm or 34 A⁰

·         There are roughly 10 bp in turn.

·         The distance between the bp in a helix is 0.34nm or 3.4 A⁰.

·         The plane of one base pair stacks over the other in double helix.

·         H-bond confers stability of the helical structure of the DNA.

·         Central dogma of flow of genetic information: DNA→ RNA→ Protein.

Packaging of DNA Helix:

·         Distance between two conjugative base pairs is 0.34nm, the length of the DNA in a typical mammalian cell will be 6.6 X10⁹ bp X 0.34 X10^-9 /bp, it comes about 2.2 meters.

·         The length of DNA is more than the dimension of a typical nucleus (10-6m), how is such a long polymer packaged in a cell?

Packaging in prokaryotes:

·         They do not have definite nucleus.

·         The DNA is not scattered throughout the cell.

·         DNA is held together with some proteins in a region is called ‘nucleoid’.

·         The DNA in nucleoid is organized in large loops held be proteins.

Packaging in Eukaryotes:

• In eukaryotes the packaging is more complex.
• There is a set of positively charged, basic protein called Histones.
• Histones are positively charged due to rich in basic amino acids like Lysines and arginines.
• Histones are organized to form a unit of eight molecules called histone octamere.
• Negatively charged DNA wrapped around positively charged histone octamere to form a structure called nucleosome.
• A typical nucleosome contains 200 bp of DNA helix.
• Nucleosome constitutes the repeating unit of a structure in nucleus called chromatin, thread like stained bodies seen in the nucleus.
• The nucleosomes are seen as ‘beads-on-string’ structure when viewed under electron microscope.
• The chromatin is packaged to form chromatin fibers that are further coiled and condensed at metaphase stage to form chromosome.
• Packaging at higher level required additional set of proteins called Non-histone Chromosomal (NHC) proteins.
• In a typical nucleus some loosely coiled regions of chromatin (light stained) is called euchromatin.
• The chromatin that more densely packed and stains dark are called Heterochromatin.
• Euchromatin is transcriptionally active chromatin and heterochromatin is inactive.

THE SEARCH OF GENETIC MATERIAL:

Transforming principle:

• Given by Frederick Griffith in 1928.
• His experiment based on Streptococcus pneumoniae (caused pneumonia).
• There is change in physical form of bacteria.
• There are two colonies of bacteria:
• Smooth shiny colonies called S strain.
• Rough colonies called R strain.
• S-strain bacteria have a mucous (polysaccharide) coat.
• R-strain does not have mucous coat.
• S-strain is virulent and caused pneumonia in mice and died when infected.
• R-strain is non-virulent and dose caused pneumonia in mice when infected.
• Heat killed S-Strain is non-virulent and dose not causes pneumonia.
• The heat killed S-Strain mixed with live R-Strain injected into mice; the mice developed pneumonia and died.
• He recovered live S-Strain bacteria form the dead mice.

Conclusion of experiment:

• R – Strain bacteria had somehow been transformed by the heat killed S-Strain bacteria.
• Some ‘transforming principle’, transferred from heat killed S-Strain bacteria, had enabled the R-Strain to synthesize smooth polysaccharide coat and become virulent (S Strain).
• The transformation of R-Strain to S-Strain is due to transfer of Genetic material.
• However the biochemical nature of genetic material was not defined from his experiment.

Biochemical characterization of transforming principle:

• Biochemical nature of transforming principle was discovered by Oswald Avery, Colin Macleod and Maclyn McCarty. (1933-44)
• Prior to their work genetic material was thought to be protein.
• They worked to determine the biochemical nature of the ‘transforming principle’ of Griffith’s experiment.
• They purified biomolecules (proteins, DNA and RNA) from the heat killed S cells to see which one could transform live R cells to S cells.
• Heat killed S-Strain + protease + Live R-Strain → transformation.
• Heat killed S-Strain + RNase + Live R-Strain → transformation.
• Heat killed S-Strain + DNase + Live R-Strain → transformation.

Conclusion of the experiments:

• Protein of heat killed S-Strain is not the genetic material
• RNA of heat killed S-Strain is not the genetic material.
• DNA of heat killed S-Strain is the genetic material, because DNA digested with DNase mixed with R-strain unable to transform R-Strain to S-Strain.

The Genetic Material is DNA:

• ‘DNA is the genetic material’ is proved by Alfred Hershey and Martha Chase (1952).
• They worked on the virus that infects bacteria called bacteriophage.
• During normal infection the bacteriophage first attaches the bacteria cell wall and then inserts its genetic material into the bacterial cell.
• The viral genetic material became integral part of the bacterial genome and subsequently manufactures more virus particle using host machinery.
• Hershey and Chase worked to discover whether it was protein or DNA from the viruses that entered the bacteria.

Experiment :( blenders experiment)

• They grew some viruses on a medium having radioactive phosphorus and some others on medium having radioactive sulfur.
• Viruses grown in radioactive Phosphorus have radioactive DNA but not radioactive protein because Phosphorus present in DNA not in protein.
• Viruses grown in radioactive sulfur have radioactive protein not radioactive DNA because sulfur present in protein but not in DNA.
• Infection: radioactive phages were allowed to attach to E.coli bacteria; the phages transfer the genetic material to the bacteria.
• Blending: the viral coats were separated from the bacteria surface by agitating them in a blender.
• Centrifugation: The virus particles were separated from the bacteria by spinning them in a centrifuge machine.

Observation:

• Bacteria infected with viruses that had radioactive DNA were radioactive and no radioactivity in the supernatant.
• Bacteria infected with viruses that had radioactive protein were not radioactive, but radioactivity found in the supernatant.

Conclusion of Experiment:

• DNA is the infecting agent that made the bacteria radioactive hence DNA is the genetic material not the protein.

PROPOERTIES OF GENETIC MATERIAL (DNA VERSUS RNA):

Criteria for genetic material:

• It should be able to generate its replica (replication)
• It should be chemically and structurally stable.
• It should provide the scope for slow changes (mutation) that required for evolution.
• It should be able to express itself in the form of ‘Mendelian Character’.
• Protein dose not fulfill the criteria hence it is not the genetic material.
• RNA and DNA fulfill the criteria.

RNA is unstable:

• 2’-OH group present at every nucleotide (ribose sugar) in RNA is a reactive group and makes RNA liable and easily degradable.
• RNA is also now known as catalyst, hence reactive.
• RNA is unstable and mutates faster. Consequently the viruses having RNA genome and having shorter life span mutate and evolve faster.

DNA is more stable:

• Stability as one of the properties of genetic material was very evident in Griffith’s ‘transforming principle’ itself that heat, which killed the bacteria at least did not destroy some of the properties of genetic material.
• Two strands being complementary if separated by heating come together, when appropriate conditions are provided.
• Presence of Thymine in place of uracil confers additional stability to DNA
• DNA is chemically less reactive and structurally more stable when compared to RNA.
• Therefore among the two nucleic acids the DNA is a better genetic material.

Better genetic material (DNA or RNA)

• Presence of thymine at the place of uracil confers more stability to DNA.
• Both DNA and RNA are able to mutate.
• In fact RNA being unstable mutate at a faster rate.
• RNA can directly code for the synthesis of proteins, hence easily express.
• DNA however depends on RNA for protein synthesis.
• The protein synthesis machinery has evolved around RNA.
• Both RNA and DNA can functions as genetic material, but DNA being more stable is preferred for storage of genetic information.
• For the transmission of genetic information RNA is better.

RNA WORLD:

• RNA is the first genetic material.
• Essential life processes evolved around RNA.
• RNA used to act as a genetic material as well as catalyst.
• But RNA being catalyst was reactive and hence unstable.
• Hence DNA has evolved from RNA with chemical modifications that make it more stable.
• DNA being double stranded and having complementary strand further resists changes by evolving a process of repair.

REPLICATION: THE PROCESS:

• Watson and Crick proposed a scheme for replication of DNA.
• The Original statement that “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material (Watson and Crick, 1953)
• The scheme suggested that the two strands would separate and act as template for the synthesis of new complementary strands.
• New DNA molecule must have one parental strand and one new strand.
• This scheme of replication is called Semiconservative type of replication.

  Experimental Proof of semiconservative nature of replication:

• It is now proved experimentally that replication is semiconservative type.
• It was first shown in Escherichia coli and subsequently in higher organism.
• Mathew Messelson and Franklin Stahl performed the following experiment in 1958.

STEPS OF THE EXPERIMENTS:

• They grew E.coli in 15NH4Cl medium for many generations. (15N is heavy nitrogen not radioactive element)
• The result was that 15N was incorporated into newly synthesized DNA and other nitrogen containing compound as well.                                                        
• This heavy DNA molecule could be distinguished from normal DNA by centrifugation in a cesium chloride (CsCl) density gradient.
• Then they transferred the E.coli into a medium with normal 14NH4Cl and let them grow.(E.coli divides in 20 minutes)
• They took samples at definite time intervals as the cells multiplied, and extracted the DNA that remained as double-stranded helices.
• Various samples were separated independently on CsCl gradients to measure the densities of DNA.
• The DNA that was extracted from the culture one generation after the transfer from 15N to 14N medium had a hybrid or intermediate density.
• DNA extracted from the culture after another generation (after 40 min.) was composed of equal amount of this hybrid DNA and of ‘light ‘DNA.

Experiment by Taylor and colleagues:

• Used radioactive thymidine to detect distribution of newly synthesized DNA in the chromosomes.
• They performed the experiment on Vicia faba (faba beans) in 1958.
• They proved the semiconservative nature of DNA replication in eukaryotes.

Replication Machinery and Enzymes:

• In all living cells such as E.coli replication requires a set of enzymes.
• E.coli completes the replication of its DNA in within 38 min.
• The average rate of polymerization has to be approx. 2000 bp per sec.
• The polymerization process must be accurate; any mistake during replication would result into mutation.
• Deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP) serve dual purposes:
• Provide energy for polymerization.
• Acts as substrates for polymerization.
• The replication process occurs within a small opening of the DNA helix called replication fork.
• The region where, replication fork formed is called origin of replication.
• The replication fork is formed by an enzyme called helicase.
• Two separated strand is called template strands.
• Main enzyme is DNA-dependent DNA polymerase, since it uses a DNA template to catalyze the polymerization of deoxyribonucleotides.
• DNA polymerase catalyses polymerization only in one direction i.e. 5’→3’.
• On one strand (template with 3’→5’ polarity) the replication is continuous hence called leading strand.
• In another strand (template with 5’→3’ polarity) the polymerization takes place in the form of short fragment called Okazaki fragment.
• The short fragments are joined by DNA ligase, hence called lagging strand.
• In eukaryotes replication takes place in S-phase of cell cycle.
• A failure of cytokinesis after replication results into polyploidy.

TRANSCRIPTION:

‘The process of copying genetic information from one strand of the DNA into RNA is termed as transcription’.
Transcription vs. Replication:

• Principle of complementarity governs the process of transcription except Adenosine of DNA forms base pair with the Uracil instead of thymine. During replication Adenine pairs with thymine instead of uracil.
• During replication once started the whole DNA is duplicated, whereas transcription takes place only a segment of DNA.
• In replication both strand acts as template, where as in transcription only one strand is acts as template to synthesize RNA.
• In replication DNA copied from a DNA, where as in transcription RNA copied from the DNA.

Why both strands of DNA not copied during transcription:

• If both strand of DNA acts as template, they would translated into two RNA of different sequences and in turn if they code for proteins, the sequence of amino acids in the protein would be different. Hence one segment of DNA would be coding for two different proteins.
• The two RNA molecules if produced from simultaneously would be complementary to each other, hence will form double stranded RNA. This would prevent RNA translation into protein.

Transcription unit:

• A transcription unit in DNA consists of three regions:
• A promoter
• The structural gene
• A terminator.
• DNA dependent RNA polymerase catalyses the polymerization in only one direction that is 5’→3’.

Structural gene:

• The DNA strand having polarity 3’→5’ is called template strand for transcription.
• The other strand of DNA having polarity 5’→3’ is called coding strand.
• The sequences of nitrogen base in the RNA transcribed from the template strand are same as the coding strand of DNA except having Thymine in place of Uracil.
• All the reference point defining a transcription unit is made with the coding strand only, not the template strand.

 Promoter:

• Promoter and Terminator present on either side of structural gene.
• The promoter located towards 5’ end (upstream) of the structural gene.
• It is a short sequence of DNA that provides binding site for RNA polymerase. (mostly TATA , Commonly called TATA box)
• Presence of the promoter defines the template and coding strands.
• If the position of promoter is changed with terminator the definition of coding and template strand will be reversed.

Terminator:

• The terminator located towards 3’ end (downstream) of coding strand.
• It terminates the process of transcription.
• It is also a short segment of DNA which recognizes the termination factor. (ρ-factor)

Transcription unit and the gene:

• Gene is defined as the functional unit of inheritance.
• Genes are located on the DNA.
• The DNA sequence coding for tRNA and rRNA molecule also define a gene.
• Cistron: a segment of DNA (structural gene) coding for a polypeptide.
• Monocistronic: most of eukaryotic structural gene codes for single polypeptide.
• Polycistronic: Most prokaryotic structural gene code for more than one polypeptides.
• In eukaryotes the monocistronic  structural gens have interrupted coding sequences, the genes are said to be split gene:
• The coding sequences or expressed sequences are called Exons.
• Exons are interrupted by Introns.
• Exons are said to be those sequences that appear in mature or processed mRNA.
• Introns never appear in mature of processed mRNA. They are spliced out.

Types of RNA:

• In prokaryotes there are three major types of RNAs: mRNA (messenger), tRNA (transfer), and rRNA (ribosomal).
• All three RNAs are required to synthesize protein in a cell.
• The mRNA provides the template and having genetic information in the form of genetic code.
• The tRNA brings the amino acids and read the genetic code of mRNA.
• The rRNA is the structural part of the ribosome and also as catalytic role during process of translation.

Process of transcription: prokaryotes.

• There is a single DNA dependent RNA polymerase that catalyses transcription or synthesis of all three types of RNAs in prokaryotes.
• The process of transcription completed in three steps:

Initiation:

• RNA polymerase binds to the specific site of DNA called promoter.
• Promoter of the DNA is recognized by initiation factor or sigma (σ).
• RNA polymerase along with initiation factor binds to the promoter.

Elongation:

• RNA polymerase unzipped the DNA double helix and forms an open loop.
• It uses ribonucleoside triphosphates as substrate and polymerizes in a DNA template following the rule of complementarity.
• Only a short stretch of polymerized RNA remains binds with the enzyme.
• The process of polymerization continued till the enzyme reaches the terminator gene.

Termination:

• RNA polymerase recognizes the terminator gene by a termination-factor called rho (ρ) factor.
• The RNA polymerase separated from the DNA and also the transcribed RNA.

Additional complexities in eukaryotes:

• There are three different types of RNA polymerases in the nucleus:
• RNA polymerase I transcribes rRNA (28S, 18S, and 5.8S)
• RNA polymerase II transcribes heterogeneous nuclear RNA (hnRNA).
• RNA polymerase III transcribes tRNA, 5srRNA and snRNA.
• Post transcriptional processing: (occurs inside the nucleus)

(a) Splicing:

• The primary transcript (hn RNA) contain both exons and introns and required to be processed before translationally active (mRNA).
• The introns are removed and exons are joined in a defined order.
• This process is catalyzed by SnRNP, introns removed as spliceosome.

(b) Capping: an unusual nucleotide called methyl guanosine triphosphate is added to the 5’ end of hnRNA.

(c) Tailing: Adenylate residues (200-300) are added at 3’ end of hnRNA in a template independent manner.

The processed hnRNA is now called mRNA and transported out of the nucleus for translation.

GENETIC CODE:

Contribution to discovery:

• The process of replication and transcription based on complementarity.
• The process of translation is the transfer of genetic information form a polymer of nucleotides to a polymer of amino acids. There is no complementarity exist between nucleotides and amino acids.
• If there is change in the nucleic acid (genetic material) there is change in amino acids in proteins.
• There must be a genetic code that could direct the sequence of amino acids in proteins during translation.
• George Gamow proposed the code should be combination of bases, he suggested that in order to code for all the 20 amino acids, the code should be made up of three nucleotides.
• Har Govind Khorana enables instrumental synthesizing RNA molecules with desired combinations of bases (homopolymer and copolymers).
• Marshall Nirenberg’s cell – free system for protein synthesis finally helped the discovery of genetic code.
• Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful in polymerizing RNA with desired sequences in a template independent manner (enzymatic synthesis of RNA)

 Salient features of genetic code:

• The codon is triplet. Three nitrogen base sequences constitute one codon.
• There are 64 codon, 61 codes for amino acids and 3 codons are stop codon.
• One codon codes for only one amino acid, hence it is unambiguous.
• Degeneracy: some amino acids are coded by more than one codon.
• Comma less: the codon is read in mRNA in a continuous fashion. There is no punctuation.
• Universal: From bacteria to human UUU codes for phenyl alanine.
• Initiation codon: AUG is the first codon of all mRNA. And also it codes for methionine (met), hence has dual function.
• Non-overlapping: The genetic code reads linearly
• Direction: the code only read in 5’ → 3’ direction.
• Anticodon: Each codon has a complementary anticodon on tRNA.
• Non-sense codon: UAA, GUA, and UAG do not code for amino acid and has no anticodon on the tRNA.

Mutation and Genetic code:

• Relationship between DNA and genes are best understood by mutation.

Point mutation:

• It occurs due to replacement nitrogen base within the gene.
• It only affects the change of particular amino acid.
• Best understood the cause of sickle cell anemia.

Frame shift mutation:

• It occurs due to insertion or deletion of one or more nitrogen bases in the gene.
• There is change in whole sequence of amino acid from the point of insertion or deletion.
• Best understood in β-thalasemia.

tRNA-the Adaptor molecule:

• The tRNA is called sRNA (soluble RNA)
• It acts as an adapter molecule.
• tRNA has an anticodon loop that base complementary to the codon.
• It has an amino acid accepter end to which it binds with amino acid.
• Each tRNA bind with specific amino acid i.e. 61 types of tRNA found.
• One specific tRNA with anticodon UAC called initiator tRNA.
• There is no tRNA for stop codons. (UAA, UGA, UAG)
• The secondary structure is like clover-leaf.
• The actual structure of tRNA is compact, looks like inverted ‘L’.

TRANSLATION:

• It refers to polymerization of amino acids to form a polypeptide.
• The number and sequence of amino acids are defined by the sequence of bases in the mRNA.
• The amino acids are joined by peptide bond.
• Amino acids are activated in the presence of ATP and linked to their specific tRNA is called charging of tRNA or aminoacylation of tRNA.
• Ribosome is the cellular factory for protein synthesis.
• Ribosome consists of structural rRNA and 80 different proteins.
• In inactive state ribosome(70S) present in two subunits:-
• A large sub unit 50S.
• A small sub unit 30S.

Initiation:

• The process of translation or protein synthesis begins with attachment of mRNA with small subunit of ribosome.
• The ribosome binds to the mRNA at the start codon (AUG).
• AUG is recognized by the initiator tRNA.

Elongation:

• Larger subunit attached with the initiation complex.
• Larger subunit has two site ‘A’ site and ‘P’ site.
• Initiator tRNA accommodated in ‘P’ site of large subunit, the subsequent amino-acyl-tRNA enters into the ‘A’ site.
• The sub subsequent tRNA selected according to the codon of the mRNA.
• Codon of mRNA and anticodon of tRNA are complementary to each other.
• Formation of peptide bond between two amino acids of ‘P’ and ‘A’ site, catalyzed by ribozyme, (23S rRNA in bacteria)
• The moves from codon to codon along the mRNA called translocation.

Termination:

• Elongation continues until a stop codon arrives at ‘P’ site.
• There is no tRNA for stop codon.
• A release factor binds to the stop codon.
• Further shifting of ribosome leads to separation of polypeptide.
• An mRNA also has some additional sequences that are not translated called untranslated regions (UTR).

REGULATION OF GENE EXPRESSION:

• Regulation of gene expression in eukaryotes takes place in different level:
• Transcriptional level (formation of primary transcript)
• Processing level (regulation of splicing)
• Transport of mRNA from nucleus to the cytoplasm.
• Translational level.
• In prokaryotes control of rate of transcriptional initiation is the predominant site for control of gene expression.
• The activity of RNA polymerase at the promoter is regulated by accessory proteins, which affects its ability to recognize the start site.
• The regulatory proteins can acts both positively (activators) or negatively (repressor)
• The regulatory proteins interact with specific region of DNA called operator, which regulate the accessibility of RNA polymerase to promoter.

Lac operon:

• Francois Jacob and Jacque Monod first to describe a transcriptionally regulated system of gene expression.
• A polycistronic structural gene is regulated by common promoter and regulatory genes. Such regulation system is common in bacteria and is called operon.
• Lac operon consists of:-
• One regulator gene ( i-gene)
• Three structural genes (z,y,a)
• Operator. (binding site of repressor protein)
• Promoter.(binding site of the RNA polymerase)

• The i-gene codes for repressor of the lac operon.
• The structural gene consist  of three gene (z, y and a)
• ‘z’-gene codes for beta-galactosidase, which hydrolyze lactose into Galactose and glucose.
• ‘y’ –gene codes for permease, which increases the permeability of bacterial cell to lactose.
• ‘a’-gene codes for transacetylase.

• All three genes are required for the metabolism of lactose in bacteria.
• Inducer: lactose is the substrate for β- galactosidase and it regulates the switching on and off of the lac operon. Hence it is called inducer.
• In the absence of glucose, if lactose is added in the growth medium of the bacteria, the lactose is transported into the cell by permease.
• Very low level of expression of lac operon has to be present in the cell all the time; otherwise lactose cannot enter the cell.

Mechanism of regulation of lac operon:

• The repressor protein is synthesized from i-gene (all time constitutively)
• In the absence of the inducer i.e. lactose the active repressor binds to the operator and prevents RNA polymerase from transcribing the structural gene
• In the presence of the inducer such as lactose or allolactose, the repressor is inactivated by interaction with inducer.
• This allows RNA polymerase access to the promoter and transcription proceeds.
• The regulation of lac operon by repressor is referred to as negative regulation.

HUMAN GENOMIC PROJECT:

• Genetic make-up of an organism or an individual lies in the DNA sequences.
• Two individual differs in their DNA sequences at least in some places.
• Finding out the complete DNA sequence of human genome.
• Sequencing human genome was launched in 190.

Goals of HGP:

• Identify all the approximately 20.000 – 25000 genes in human DNA.
• Determine the sequence of all 3 billion chemical base pairs.
• Store this information in data bases.
• Improve tools for data analysis.
• Transfer related technologies to other sectors, such as industries.
• Address the ethical, legal, and social issues (ELSI) that may arise from the project.

Methodology:

• To identify all the genes that expressed as RNA referred as Expressed Sequence Tags (ETSs).
• Simply sequencing the whole set of genome that contained all the coding and non-coding sequence, and later assigning different regions in the sequence with functions called Sequence Annotation.
• The commonly used hosts for sequencing were bacteria and yeast and vectors were called as BAC (bacterial artificial chromosome) and YAC (yeast artificial chromosome).

Salient features of Human Genome:

• The human genome contains 3164.7 million nucleotide bases.
• The average gene consists of 3000 bases.
• The largest known human gene being dystrophin at 2.4 million bases.
• The total number of gene is estimated at 30.000.
• 99.9 percent nucleotide base sequences are same in all peoples.
• The function of 50% genes discovered is unknown.
• Less than 2 percent of the genome codes for proteins.
• Repeated sequences make up very large portion of human genome.
• Chromosome I has most genes (2968) and the Y has the fewest (231).
• It is identified about 1.4 million locations where single-base DNA differences (SNPs – single nucleotide polymorphism) occurs in humans.

DNA FINGER PRINTING:

• DNA finger printing is a very quick way to compare the DNA sequences of any two individual.
• DNA fingerprinting involves identifying differences in some specific regions in DNA called repetitive DNA, because in these sequences, a small stretch of DNA is repeated many times.
• During centrifugation the bulk DNA forms major peak and the other small peaks are called satellite DNA.
• Depending on base composition (A:T rich or G:C rich), length of segment, and number of repetitive units, the satellite DNA classified into many types, such as mini –satellite and micro – satellite.
• These sequences dose not code for any proteins.
• These sequences show high degree of polymorphism and form basis of DNA fingerprinting.
• Polymorphism in DNA sequence is the basis of genetic mapping of human genome as well as of DNA fingerprinting.
• Polymorphism (variation at genetic level) arises due to mutations.
• If an inheritable mutation is observed in a population at high frequency it is referred as DNA polymorphism.

The process:

• DNA fingerprinting was initially developed by Alec Jeffreys.
• He used satellite DNA as the basis of DNA fingerprinting that shows very high degree of polymorphism. It was called as Variable Number Tandem Repeats.(VNTR)
• Different steps of DNA fingerprinting are:-
• Isolation of DNA.
• Digestion of DNA by restriction endonucleases.
• Separation of DNA fragments by gel electrophoresis.
• Transferring (blotting) of separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon.
• Double stranded DNA made single stranded.
• Hybridization using labeled VNTR probe.
• Detection of hybridized DNA fragments by autoradiography.

• The VNTR belongs to a class of satellite DNA referred to as mini-satellite.
• The size of VNTR varies from 0.1 to 20 kb.
• After hybridization with VNTR probe the autoradiogram gives many bands of different sizes. These bands give a characteristic pattern for an individual DNA. It differs from individual to individual.
• The DNA from a single cell is enough to perform DNA fingerprinting.

Applications:

• Test of paternity.
• Identify the criminals.
• Population diversity determination.
• Determination of genetic diversity.