MOLECULAR GENETICS Introduction Molecular biology is a branch of modern biology concerned with explaining biological phenomena in molecular terms and often utilizing techniques of physical chemistry to investigate genetics and other biological problems. It is an indivisible subject that crosses traditional boundaries between genetics, biochemistry, cell biology, physics, organic chemistry and biophysical chemistry. ‘The study and birth of genetics originated with Gregor Mendel’s experiment with garden pea in 1866. The study suffered a gestation period until 1900 when it was rediscovered in his experiment the first are monohybrid characters. He uses a pair of contrasting character, where he discovered that there is consistence pattern of inheritance and each of the contrasting factors or trait is controlled by a unit factor (now called the gene) that exist in more than one form (or alleles), one is dominant and the other recessive. This led to Mendel’s first law of inheritance that allele segregate randomly. characters, he discovered that the pairs or contrasting In his second experiment, dihybri character (yellow round peas, RRYY dominant and wrinkle green peas rryy) are inherited independently. This led to the Mendet's second law of inheritance that different pairs of alleles segregate independently. Many other scientists after Mendel conceived the idea of experiments similar to Mendel's to test their own theories of heredity and found that the processes follows predictable rules. These rules can be rationalized as the passage of physical factors, each controlling a separate heritable trait from parents to offspring during reproduction. These factors were proposed to be ‘genes’ by W Johansen in 1909. These genes are carried by chromosomes (chromosome theory) in higher organisms in 1903 by WS Sutton.After the establishment of the experimental basis of heredity and chromosome theory rapid advances in the understanding of genetics continued. One of the important advances is the work of Thomas Hunt Morgan and his colleagues with fruit fly Drosophila melanogaster. The fly has many stable variant forms or characters and each having many several varieties. Morgan and colleagues discovered the way in which different combination of genes work together in controlling inheritance of individual characteristics and developed techniques of genetics analysis including gene mapping. These early geneticists were mainly interested in how genes are transmitted from parents to their offspring during reproduction and how different genes act together to control variable traits. During the 1930s when it was recognized that if genes are physical entities then, like other cell components they must be made of molecules and it should therefore be possible to study them directly by biophysical and biochemistry methods. Experiment like that of Max Delbruck in 1040s uses the infective cycle of bacteriophage to study what genes are and how they work. This has led to a new branch of genetics called molecular biology which ceases to regard gene as simply unit of inheritance and instead as a unit of biological information with the entire complement of genes in an organism containing the total amount of information needed to construct a living, functioning example of that organism. The concern of the molecular biologists and genet over the years has been to understand the way in which biological information is stored in genes and how that information is made available to the living cell. Most of the pioneering work in molecular genetics or biology was carried with bacteria E. coli cells than eukaryotic cells that are more complex. But by the 1980s work on lower eukaryotic, S. cerevisiae stated. Genes and Genetic Materials Today we say that the gene is the smallest unit of an organisms that is capable of transi itting genetic information and isa segment of chromosomes (from chromosome theory). Cytochemical studies later revealed the chromosomes to contain two biological compounds, protein and nucleic acid. One or other (perhaps and combination of both nucleoprotein) must therefore be the genetic material. Initially protein was considered as the genetic material, asa macromolecule that is able to exist in almost infinite variety of forms, with each cell containing large number ofdifferent traits, with different chemical characteristics. These properties were assumed not to have been exhibited by DNA as its structure was misunderstood at that time believing that DNA was relatively small (MW 1227), invariant, though to be exactly the same as every other DNA molecule and as structural material to hold the protein gene together. Protein as a ‘macromolecule is made up of long polymers of amino acids, with 20 different amino acids linked together in variety of ways giving rise to different proteins distinct from one another by virtue of their different amino acid sequence. The assumption has no experimental evidence to back it. Experimental evidences to identify the genetic materials intensified in the late 1930s. Gradually it became evident that DNA rather than being simple molecule was a long polymer and could exist in variable forms. Two experimental evidences, that of Fredrick Griffith in 1928, the transformation principle and that of Menthan Chase and Alfred Hershey in 1951-2 using bacteriophage finally led to the conclusion that the genetic material is DNA not protein. EXPERIMENTAL PROOF THAT DNA IS THE GENETIC MATERIAL A. Transforming Principle | In 1928, British microbiologist Frederick Griffith made a series of unexpected observations while experimenting with pathogenic (disease-causing) bacteria. The experiment uses the bacteria strain S. pneumonia the causative agent of pneumonia. He designed four experiments as follows: a. Amouse was injected with a sample of virulent, smooth bacteria of the Type | stereotype (Type 1S}, as expected the mouse contacted with pneumonia and died. b. Asecond mouse was injected with avirulent, rough bacteria of Type Il stereotype (Type IR), again as expected the mouse remained healthy and live. c. Type IS bacteria were killed by heating at 60°C for 3 hours and then injected into the mouse. The mouse remained healthy and live. 4d. A mixture of heat-killed Type IS and Type IIR was injected into the mouse. The mouse contacted pneumonia and died. In addition, large number of living IS bacteria were isolated, even though the only living bacte mR in the original inoculum were avirulent Typeeee ieee acapaigan am sinc Sesame RE, Satteemenee, bale ie OB « < @ Mowe Grif’ discovery of ranformaton. (1) The pahogenic of he bacerium Soxpuserpeemtonae kill many of the mice iisinjecced into, The bares cells ae covered with apulpacchande cot which the aseriathemlves ytaedze. 2) Ineresingy, a injec of live, comlese baer produced nol efecs. However, te coat iselFs nt the ages of tease. (2) When Griffth jected mie with dead Dacera hac poesed polyacharde coms, the mice were unharmed. (Bue when Grit njered amare of dead bacteria with polysaccharide cous and ive Baca wahcor such cos, many ofthe mice ded ndvirene Bucena vith conte were recovered, Gafith ‘onclided:harthelive calls iad been “wansfermes* by she dea ons; his, geneac ifermancn specling the payesea ide com had pasted fram the dead eels vorhelring ane.B. Transforming principle (I!) ‘Averygo and McCarty at the Rockfeller institute in New York provided further proof that DNA was the transformation principle. They prepared a filtrate from heat-killed S cells containing the transforming principle and digest the individual components (protein, DNA and RNA) in the filtrate with specific degradative enzymes. Protease was used to degrade protein, ribonucleases to destroy RNA and deoxyribonuclease to destroy DNA. After digestion of one or more component, the filtrate was tested for retention of transforming ability. a. Treatment with protease had no effect on the transforming activity b. Ribonucleases treatment had no effect on transforming activity © Digestion of filtrate with DNase totally destroyed the transforming activity so that the filtrate was no longer able to convert one cell type into the other. From this observation, it become clear to Averygo and his colleague that the transforming principle must be the DNA. C. Bacteriophage genes are made of DNA This experiment was to characterize the genetic material. To identify the hereditary material injected into bacterial cells at the start of an infection, Hershey and Chase used the bacteriophage 2, which contains DNA rather than RNA. They labeled the two parts of the viruses, the DNA and the protein coat, with different radioactive isotopes that would serve as tracers. In some experiments, the viruses were grown on a medium containing an isotope of phosphorus, 2P, and the isotope was incorporated into the phosphate groups of newly synthesized DNA molecules. In other experiments, the viruses were grown on a medium containing 5, an isotope of sulfur, which is incorporated into the amino acids of newly synthesized protein coats. The 22P and 255 isotopes are easily distinguished from each other because they emit particles with different energies when they decay.After the labeled viruses were permitted to infect bacteria, the bacterial cells were agitated violently to remove the protein coats of the infecting viruses from the surfaces of the bacteria. This procedure removed nearly all of the 2°5 label (and thus nearly all of the viral protein) from the bacteri However, the P label (and thus the viral DNA) had transferred to the interior of the bacteria and was found in viruses subsequently released from the infected bacteria. Hence, the hereditary information injected into the bacteria that specified the new generation of viruses was DNA and not protein. Protein coat DNA Jabeled labeled with 9s with a are labeled with EB radioactive isotopes. bacteria cells, Bacteriophages Infect | | Gactenal cell are | Sgtated to remove | peieincoals, - a ay a = 35g radioactivity Sp radioactivity tound found in the medium in the bacterial ceils “The Hershey and Chase experiment. Hershey and Chase found thar ?5S radioactivity did not enter infected bacterial cells and 22 radioactivity did. "They concluded thar viral DIVA, not protein, was responsible for directing the production of new viruses.Chemical Nature of Hereditary Materi Nucleic acid — consists of DNA and RNA DNA is a long polymeric molecule, comprised of numerous individual units linked together in a series. These individual units (monomers) are called nucleotides. They either exists in cells as components of nucleic acids or as individual molecules where they play different role: metabolism. A nucleotide is made up of three distinct components ~ a sugar, nitrogenous base and phosphoric acid. Sugar: The sugar component of nucleotide is a pentose (containing five carbon atom). Pentose Sugar exists in two forms, the straight chain or Fischer structure and the ring or Haworth structure. It is the ring form that occurs in the nucleotide. In DNA the sugar is 2’-deoxyribose while in RNA the sugar is ribose HOCH, 4 OH Lee e OHH On OH Deoxyribose (in DNA} Ribose (in RNA} Nitrogenous base: DNA has four different nitrogenous base of purine and pyrimidines origin exists in DNA. In RNA it ha: addition to cytosine, adenine, guanine, a pyrimidine base uracil replacing thymine. Pyrimidines Ha 2 g é. ye ge tse tte e eye ge FNy SH gol CH 4 4 " sine Thymina (in DNA) Uracil (in RR cytes ymin fp ReiayNucleoside: A molecule comprising of sugar joined to a base (at N9 of purines or at N1 of pyrimidines) DNA RNA * Adenosine — 2'- deoxyadenosine > Uridine * Guanosine —2’- deoxyguanosine > Adenosine + Cytosine —_2’- deoxycytosine > Guanosine + Thymine — 2’- deoxythymidine > cytosine ° oa, on 2-day Tyne deze -deory ror (ore see DNA prerton) RNA presen) Phosphoric acid: a phosphoric acid group attached to nucleoside at 5’ carbon of the sugar forms anucleotide. DNA + 2'-deoxyadenosine 5’-triphosphte Adenylic acid + 2'- deoxyguanosine 5'- triphosphate Guanilic acid + 2'- deoxycytosine 5°- triphosphate Cytidylic acid + 2'- dexoythymidine 5’-triphosphate Thymidilic acid RNA + Uridine 5'- triphosphate Uridylic acid + Adenosine 5’ triphosphate Adenylic acid + Guanosine 5'-triphosphate Guanylic a + Cytosine 5'- triphosphate Cytidylic acidBase | Nucleoside Nucleotide RNA DNA | Code | (monophosphate) | (monophosphate) | ‘Adenine | Adenosine | (Adenylic acid) AMP, damp | A Guanine | Guanosine _| (Guanylic a | dp | Gs | Cytosine | Cytidine | (Cytidylicaciay | | cmp. c | Thymine | Thymidine | (Thymidylic acid) | TMP T Uracil © [(Uridyticaciay | UMP u oun oak 2'-Deoxyadenosine 5'-monophosphate 2'-Deoxyguanosine 5'-monophosphate {Deoxyadenylate, dAMP) (Deoxyguanylate, dGMP) oe e eo I 6 I CH og o ky e " oh ok 2'-Deoxycytidine 5'-monophosphate —_2'-Deoxythymidine 5'-monophosphate (Deoxycytidylate, (CMP) (Thymidylate, dTMP) Polynucleotides: Individual nucleotides are linked. together to form a polymer by attaching one nucle le to another through phosphate groups. This polymer is called polynucleotide. The nucleotides monomers is linked by joining the alpha (a) phosphate group attached to the 5’ carbon ofnucleotide to the 3’carbon to the next nucleotide in the chain. Normally polynucleotide is build up from nucleoside triphosphate subunit, so during polymerization the B and y phosphates are cleaved off. The -OH group attached to the 3’carbon of the second nucleotide is also lost. The linkage between the nucleotides is called a phosphodiester bond: ‘phospho’ indicating the presence of a phosphorus atom and ‘diester’ referring to the two ester (C-O-P) bonds in each linkage. Stend 0 ' So-bmo or a-s' phosphodiester linkage yas phosphodiester linkage v9! ete phosphodiester | °° Hinks sited cytosine (ct AngThe polynucleotide will have two ends that are not the same, A 5’ end that has not participated ina phosphodiester bond formation and the B and y phosphate groups are still in place. This end is also called 5'-P terminus. At the other end is the unreacted 3'-OH. This end is called 3’ or 3'OH terminus. This means the polynucleotide can be looked on 5’->3' or 3'-» 5’ directions. There is apparently no limitation to the number of nucleotide that can be joined to form an individual DNA polynucleotide. Polynucleotide can be of any length and at any point in the chain. The nucleotide can be A, G, CorT. ‘The DNA Double Helix Structure ‘The double helical structure of DNA deduced by James Watson and Francis Crick in 1953 made use of results of several lines of e1 lence concerning the structure of DNA in living cells. They complemented these experimental results by building and assessing accurate scale models of ious possible DNA structures and only the double helix was compatible with all the data. Early physical studies of DNA using X-diffraction patterns, icate that the molecule is an ‘extended chain having ordered structure. The arrangement and dimensions of various parts of the molecule was also revealed. Most significant observation was that the molecule is helical and that the bases of the nucleotides are stacked with their planes separated by a spacing of 3.4A. Chemical analysis of the DNA content of purified samples from different tissues and different organisms reveal that A=T and G=C. this means that the total purine is equal to the pyrimidines. Combining these experimental evidences, James Watson and Francis Crick decided that the only structure that fit fed all the facts was the double helix with the following features: 1. The double helix comprises of two polynucleotides 2. The nitrogenous bases are stacked on the inside of the helix and the sugar phosphate backbone on the outside3. The bases of the two polynucleotides interact by H- bonding. Two bonds between A and T and three bonds between G and C. This made the two polynucleotides of the helix to be complementary. 4, Ten base pair (bp) occur per turn of the helix. The pitch is 34 that is the same space between adjacent bp 3.4A and the helix is 20A in diameter. 5. The two strands are anti-parallel, one polynucleotide runs 3'-5' direction and the other in 51-3" direction and only anti-parallel polynucleotide will form a stable helix. 6. The double helix is not regular. It has two different grooves, a major and a minor groove. Feature in the interaction between the double helix and the proteins involve in DNA replication and expression of genetic information. 7. The double heli is right-handed Of the features of the double helix, complementary base pairing is of fundamental importance as it provides a means which DNA retains sequence during replication. The double helical structure exists in several forms, A, B, C, D, E and Z forms. The B-form is the structure that DNA takes up in the cell while z-form has a different conformation, itis left-handed helix.Genes Organization on DNA Molecules Cells of higher organisms e.g, humans, all the genes are ca (23 pe 1d by small number of chromosomes ) each of which contain single DNA molecule. The DNA molecule carries 100s to thousands of the genes. In lower organisms and bacterium e.g. has one chromosome, so total complements of genes of the organisms are present on a single DNA. Genes along DNA molecule can be spaced out more or less randomly in some cases grouped into distinct cluster. Sometimes the individual genes in a cluster are related for particular biological function. Operons Operon is fairly common features of the organization of gene in bacteria. For example, lactose operon — contain genes arranged in cluster that code for series of enzymes that work in concert in an integrated manner in lactose metabolism. Multigene Family ‘They are found in many organisms. It is a cluster of related genes and each individual gene has identical or similar nucleotide sequence and therefore contain identical or similar piece of biological information. There are two types of multigene families: 1. Simple multigene families: the genes are exactly or virtually the same e.g. higher organisms have multiple copies of the genes for 5S rRNA. In human there are about 200 '5S FRNA genes on chromosome 1, These multiple genes not randomly spread throughout the chromosome, help satisfy demand for large quantity of 5S rRNA that cannot be met by one or two genes. 2. Complex multigene families: made up of similar but non-identical genes. E.g. Gene family for the globins polypeptides of humans each has a pair of alpha and beta globin. Each type of globin polypeptide exists as a family of related molecules different from one another at just few a position within the polypeptide chain. The types of globin found in blood stream depend on the stage of development of the organism.Pseudogene: These are genes similar to the members of their family but the biological information they contain scrambled and is no longer readable. Discontinuous gene: Common in higher organisms and many types of virus, where biological information carried by some genes is split into district units separated by intervening segments of DNA. The section containing biological information is called exon and the intervening sequence is referred to as introns.