MOLECULAR BASIS OF INHERITANCE (NOTES)

ATS COACHING CLASSES
REVISION NOTES
MOLECULAR BASIS OF INHERITANCE

At the time of Mendel the nature of those factors regulating the pattern of inheritance was not clear, over the next 100 years the nature of the putative genetic material was investigated culminating in the realisation that DNA is the genetic material at least for the majority of organisms.

DNA and RNA are the two types of nucleic acids found in living systems, DNA acts as the genetic material in most of the organisms while RNA though acts as genetic material in some virus mostly functions as a messenger. 

Deoxyribonucleic acid:

The two kinds of nucleic acids present in living beings are DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid).

 In most species, DNA serves as genetic material. In other creatures, such as viruses, RNA also serves as genetic material and as a messenger. It serves as an adaptor, structural molecule, and in certain situations a catalytic molecule.

 DNA is a lengthy polymer of deoxyribonucleotides. Base pairs are another name for a pair of nucleotides.

 The length of DNA is often defined as the number of nucleotides present. Human DNA has a haploid content of 3.3x109 bp, while Escherichia coli has 4.6 x 106 bp. 

The polynucleotide Chain: A nucleotide is made up of three parts:

 • a nitrogenous base 

• a pentose sugar (ribose in the case of RNA and deoxyribose in the case of DNA), and 

• a phosphate group. Purines (Adenine and Guanine) and Pyrimidines (Cytosine, Uracil and Thymine) are the two kinds of nitrogenous bases.

Cytosine is found in both DNA and RNA, whereas Thymine is found in DNA. Uracil is found in RNA in place of Thymine. A nucleoside is formed when a nitrogenous base is connected to a pentose sugar via an N-glycosidic bond. Nucleotide is generated when a phosphate group is connected to the 5'-OH of a nucleoside via phosphodiester linkage. To make a dinucleotide, two nucleotides are connected together via a 3'- 5' phosphodiester bond. More nucleotides combine to produce polynucleotide. 

Salient Features of DNA:

DNA contains the sugar D -2- deoxyribose. Cytosine, uracil and thymine are pyrimidine bases in DNA, while guanine and adenine are purine bases.

The structure of DNA is a double strand -helix. Because DNA molecules are so big, their molecular mass can vary greatly. DNA has a unique replicating characteristic. The transmission of hereditary effects is controlled by RNA.

Double Helical Structure of DNA by Watson and Crick James Watson and Francis Crick suggested the double helix concept for the structure of DNA based on X-ray diffraction evidence collected by Maurice Wilkin and Rosalind Franklin. 

According to this model: 

• DNA is made of two polynucleotide chains in which backbone is made up of sugar-phosphate and bases projected inside it.

• Two chains have antiparallel polarity. One 5’ to 3’ and 3’to 5’. 

• The bases in two strands are linked together by H-bonds. Guanine and Cytosine form triple hydrogen     bonds, whereas Adenine and Thymine form double hydrogen bonds.

 • Two chains are coiled in the right hand. The pitch of the DNA helix is 3.4 nm, with each turn containing around 10 bp. 

• To provide stability, the plane of one base pair stacks over the plane of the other in a double helix. Other than this hydrogen bonding and the presence of thymine in place of uracil confers additional stability to the DNA.

The Central dogma of molecular biology, developed by Francis Crick, holds that genetic information flows from DNA — –> RNA —–> Protein.

In RNA, nucleotide residues have an extra –OH group at the 2'-position in ribose, and uracil replaces Thymine. Packing of DNA Helix

In prokaryotes, the nucleus is not clearly defined, and negatively charged DNA is mixed with positively charged proteins known as nucleoids.

Packaging of DNA Helix

 In eukaryotes, histones are positively charged proteins that are arranged into 8 molecules termed histone octamers. To construct a nucleosome, negatively charged DNA wraps around a histone octamer. Histones contain a high concentration of the basic amino acid residues lysines and arginines. The side chains of both amino acid residues are positively charged. 

A single nucleosome includes around 200 base pairs. 

Chromatin is the nucleosome's repeating unit. 

Some regions of chromatin in the nucleus are loosely packed (and stain light) and are referred to as euchromatin. Heterochromatin is chromatin that is more densely packed and stains dark. Euchromatin is active transcriptionally, whereas heterochromatin is inactive. 

Search for Genetic Material: 

The Transforming Principle: Frederick Griffith conducted an experiment on the microorganisms Streptococcus pneumoniae in 1928. (bacterium responsible for pneumonia). This bacterium has two strains: 

• those that generate smooth shining colonies (S) and

• those that form rough colonies (R) (R). Mice infected with the S strain (virulent) develop pneumonia, but mice infected with the R strain do not. In brief his experiment was as follows: Inject S strain into mice, it dies.

(i) R strain injected into mice, it survives.

(ii) Inject S strain (heat-killed) into mice, it survives.

(iii) Inject S strain (heat-killed) plus R strain (living) into mice, it dies.

(iv) Griffith came to the conclusion that R strain bacteria had been transformed by heat-killing S strain bacteria. Some transforming factors were transmitted from the S strain to the R strain, allowing the R strain to produce a smooth polysaccharide coat and become virulent. This must be the result of genetic material transfer.  

Biochemical Characterisation of Transforming Principle Oswald Avery, Colin MacLeod, and Maclyn McCarty worked together to determine the biochemical basis of Griffith's transformative principle. They extracted biochemicals (proteins, DNA, RNA, and so on) from heat-killed S cells to determine which ones may turn living R cells into S cells. They determined that DNA from S bacteria alone enabled R bacteria to convert. As a result, they came to the conclusion that DNA is the genetic material.

  Experimental Proof that DNA is the genetic material

In one formulation, the protein component was rendered radioactive, whereas the nucleic acid (DNA) component was not. These two phage preparations were allowed to infect an E.coli culture. Before cell lysis, the E.coli cells were gently agitated in a blender to release the clinging phage particles, and the culture was centrifuged. The heavier infected bacterial cells settled to the bottom, while the lighter virus particles remained in the supernatant. When a bacteriophage with radioactive DNA was utilized to infect E.coli, the pellet contained radioactivity. When a bacteriophage with a radioactive protein coat infected E.coli, the supernatant held the majority of the radioactivity. His work demonstrates that protein does not penetrate the bacterial cell and that the only genetic substance is DNA.

Properties of A Genetic Material 

• It should be able to replicate itself (replication).

 • It needs to be chemically and structurally stable.

 • It should allow for the gradual changes (mutation) essential for evolution. 

• It should be able to express itself using 'Mendelian Characters.' When compared to RNA, DNA is chemically less reactive but structurally more stable. As a result, DNA is superior genetic material. 

RNA is employed as a genetic material as well as a catalyst, and because it is more reactive, it is less stable. As a result, DNA has evolved from RNA.

 Replication: 

Watson and Crick proposed that two strands of DNA split and serve as a template for the creation of new complementary strands. After replication, each DNA molecule would contain one parental and one freshly synthesised strand; this is known as semiconservative replication.

 • Messelson and Stahl demonstrate semiconservative replication experimentally by growing E.coli on nutritional media containing nitrogen salts ( 15NH4Cl) tagged with radioactive 15N. 

• Alfred Hershey and Martha Chases (1952) studied bacteriophages, which are viruses that infect bacteria. 

• 15N was integrated into both strands of DNA, resulting in DNA that was heavier than DNA obtained from E.coli cultured on 14N-containing media. The E.coli cells were then moved to a 14N-containing media.

 • They extracted the DNA and measured its density after one generation when one bacterial cell multiplied into two. Its density was halfway between that of heavier 15NDNA and lighter 14N-DNA.

• Because a new DNA molecule with one 15N-old strand and a corresponding 14N-new strand was generated during replication (semi-conservative replication), its density is intermediate between the two. 

Replication of DNA

Enzyme DNA polymerase is required for DNA replication, which catalyses polymerisation on one strand 5' to 3' after unwinding with the help of Helicase enzyme.

 As a result, replication in one strand is continuous while replication in the other strand is discontinuous in order to synthesise Okazaki fragments that are linked together by the enzyme DNA ligase.

 Replication fork:

1. Replication forks are Y-shaped structures that arise as the DNA splits open.
2. The DNA-dependent DNA polymerases catalyzes polymerization only in 5' - 3' direction.
3. Some additional complications get created at the replicating fork.
4. Helicase activity, primer synthesis, single-strand binding protein binding, and synthesis of new strands are the events going on at the replication fork.
5. The replication fork won't be extended if the helicase gene is altered.
6. Helicase unwinds the DNA strands.
7. This process requires the hydrolysis of ATP.

Role of enzymes in replication fork :

1. DNA Helicase : It unwinds the double helix into two single strands, there by allowing single strands to replicate.
2. Primase: This enzyme adds a small segment of RNA sequences called primer.
3. DNA Polymerase: It catalyzes the synthesis of new DNA strands from nucleoside triphosphates.

Transcription: 

It is the process of copying genetic information from one strand of DNA into RNA. In transcription only one segment of DNA is copied in RNA. The Adenosine forms base pair with Uracil instead of Thymine.

 • A promoter, a structural gene, and a terminator are all involved in DNA transcription.

 • The strands with polarity 3' to 5’ operate as templates and are referred to as template strands, whereas the other strand is referred to as coding strands.

• The promoter is positioned at the 5' end and binds the RNA polymerase enzyme to initiate transcription. 

• The sigma factor also aids in the initiation of transcription.

 • The terminator is normally positioned at the 3'end of the coding strand and defines the end of transcription where the rho factor will attach to halt transcription. 

• Exons are sequences found in mature and processed RNA. Exons are broken up by introns. In mature and processed RNA, introns do not exist. 

• In eukaryotes, three RNA polymerase enzymes, I, II, and III, catalyse the production of all kinds of RNA.

RNA polymerase I : rRNAs

RNA polymerase II : messenger RNA

 RNA Polymerase III: tRNA 

• The mRNA serves as a template, the t-RNA transports amino acids and reads the genetic information, and the rRNA performs structural and catalytic functions during translation. 

• The primary transcript is non-functional and contains both exons and introns. It goes through the splicing process, in which introns are deleted and exons are connected in a certain order. 

• Capping and tailing are processes that hnRNA (heterogeneous nuclear RNA) goes through. Capping the 5' end of hnRNA with an uncommon nucleotide (methylguanosine triphosphate). Tailing polyadenylate is the addition of a tail at the 3'end of a template in an autonomous way.

Genetic Code: 

The link between amino acid sequences in polypeptides and nucleotide/base sequences in mRNA is known as the genetic code. It governs the sequencing of amino acids during protein synthesis. 

• George Gamow proposed that the genetic code be a combination of three nucleotides that code for 20 amino acids. 

• H.G. Khorana developed chemical method for synthesising RNA molecules with defined combination of bases. 

• Marshall Nirenberg’s cell free system for protein synthesis finally helped the code to be deciphered.

Features of the Genetic Code.

 • The code is triplet. There are 61 codons that code for amino acids and three stop codons that do not code for any amino acids (UAG, UGA and UAA). 

• Codon is clear and specific; it codes for a single amino acid. 

• The code is degenerate. Some amino acids are coded by multiple codons. 

• The codon is read in mRNA in a continuous, punctuated form.

• The codon is almost ubiquitous. AUG has two purposes. It encodes methionine and serves as an initiator codon.

Mutations and Genetic Code The shift of amino acid residue glutamate to valine leads in a single base pair alteration (point mutation) in the 6th position of the Beta globin chain of Haemoglobin. This develops in a disorder known as sickle cell anaemia. 

Insertion and removal of three or more bases, Insert or delete one or more codons, resulting in one or more amino acids and the reading frame remaining unchanged. Frameshift insertion or deletion mutations are examples of such mutations.

The Adapter molecule-tRNA 

The t-RNA molecules are known as adaptor molecules. It has an anticodon loop with bases corresponding to the coding found on mRNA, as well as an amino acid acceptor to which amino acid attaches. Each amino acid has its own t-RNA. The clover-leaf secondary structure of t-RNA is illustrated. The t-RNA molecule is a compact molecule that looks like an inverted L.

Translation: 

The process of polymerisation amino acids to generate a polypeptide is known as translation. The sequence of nucleotides in the mRNA determines the order and sequence of amino acids. Peptide bonds joins the amino acids. The following steps are involved: 

• Charging of tRNA .

• Peptide bond formation between two charged tRNA.

 • AUG is the start codon. Untranslated regions are extra sequences in an mRNA that are not translated (UTR). 

• The ribosome attaches to mRNA at the start codon to initiate translation. Ribosomes migrate from codon to codon along mRNA in order to extend the protein chain.

 • At the end of the process, release factors bind to the stop codon, halting translation and releasing polypeptides from the ribosome.

1. Translation is the process by which ribosomes in the cytoplasm or endoplasmic reticulum make proteins after the process of converting DNA to RNA in the cell's nucleus, as defined by molecular biology and genetics.
2. To create a specific amino acid chain, or polypeptide, during translation, messenger RNA (mRNA) is decoded in a ribosome outside the nucleus.
3. Later, after folding into a functioning protein, the polypeptide carries out its specific tasks within the cell.
4. By encouraging the binding of complementary tRNA anticodon sequences to mRNA codons, the ribosome makes decoding easier.
5. As the mRNA goes through and is "read" by the ribosome, the tRNAs transport particular amino acids that are strung together into a polypeptide.

Regulation of Gene Expression 

All of the genes are not always required. The genes that are only needed sometimes are known as regulatory genes, and they are designed to function only when needed while remaining inactive at other times. Such controlled genes must thus be turned 'on' or 'off' when a certain function begins or ends. Here are some examples: 

The lac operon:

One regulatory gene (i) and three structural genes comprise the Lac operon (y,z and a). Gene i encodes the lac operon repressor. The z gene encodes beta-galactosidase, which hydrolyzes disaccharide, lactose into monomeric units, galactose, and glucose. Gene y codes for permease, which enhances cell permeability. Transacetylase is encoded by gene a. Lactose is the substrate for the enzyme beta-galactosidase, and it governs the operon's switching on and off, thus the name inducer. Negative regulation refers to the control of the lac operon by a repressor. 

The Human Genome Project: 

The Human Genome Project was launched in 1990 with the goal of discovering the whole DNA sequence of the human genome through the use of genetic engineering techniques and bioinformatics to extract and clone the DNA segment for determining DNA sequence.

Goals of Human Genome Project:

 • Identify all the genes (20,000 to 25,000) in human DNA. 

• Determine the sequence of the 3 billion chemical base pairs that make up human DNA. 

• Store this information in database. 

• Improve tools for data analysis.

 • Transfer related information to other sectors. 

• To address the legal, ethical and social issues that may arise due to project.

 

The US Department of Energy and the National Institute of Health oversaw the experiment.

The strategy included two key approaches:

(i) the first involves identifying all of the genes that express as RNA, known as Express sequence tags       (EST). 

(ii) The second step is to sequence all of the genome's coding and non-coding sequences, which is known as sequence annotation. Features of Human Genome Project: 

• There are 3164.7 million nucleotide bases in the human genome. 

• A typical gene has 3000 bases, although sizes vary widely, with dystrophin being the biggest known       human gene of 2.4 million bases. 

• Proteins are coded in less than 2% of the genome. 

• Repeated sequences account for a sizable component of the human genome. 

• Repetitive sequences are DNA sequence lengths that are repeated numerous times, often hundreds to       thousands of times. 

• Chromosome 1 has the most genes (2,968), whereas chromosome Y has the fewest (231).

 • Researchers have found around 1.4 million places in humans where single base DNA variations (SNPs - single nucleotide polymorphism) occur.

DNA fingerprinting

is a simple approach to compare the DNA sequences of two people. It entails finding changes in a specific section of a DNA sequence known as repetitive DNA because a tiny length of DNA is repeated multiple times in this region. 

Satellite DNA is divided into numerous groups based on its base makeup, segment length, and quantity of repeating units.

 Polymorphism in DNA sequence provides the foundation for both genetic mapping of the human genome and fingerprinting.

 Alec Jeffrey was the first to create fingerprinting technology. He employed a satellite DNA probe to detect such high polymorphism as Variable Number of Tendon Repeats (VNTR)

Process of DNA Fingerprinting:

Isolating the DNA.

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Digesting the DNA with the help of restriction endonuclease enzymes.

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Separating the digested fragments as per the fragment size by the process of electrophoresis.

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Blotting the separated fragments onto synthetic membranes like nylon.

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Hybridising the fragments using labelled VNTR probes.

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Analysing the hybrid fragments using autoradiography.

 

Applications of DNA Fingerprinting

• Utilizing the DNA fingerprinting strategy, the natural personality of an individual can be uncovered. For approving one's character, there is no other preferable alternative over DNA fingerprinting. 

• Gravely harmed dead bodies can be distinguished. 

• It is utilized to detect maternal cell contamination. 

• One of the significant downsides of pre-birth determination is maternal cell tainting. The amniotic liquid or CVS test contains the maternal DNA or maternal tissue, once in a while. Contamination expands the opportunity of false-positive outcomes, particularly on account of carrier recognition. Utilizing VNTRs and STRs markers with PCR-gel electrophoresis, maternal cell tainting can be recognized during pregnancy hereditary testing. 

• One of the most significant uses of the current strategy is in the crime scene examination and criminal check. The example is gathered from the crime site which could be salivation, blood, hair follicle, or semen. DNA is removed and investigated against the suspect, utilizing the two markers we clarified previously. By coordinating DNA band designs criminal's connected to wrongdoing can be built up.