1
Development Team
Paper Coordinator : Prof. Namita Agrawal
Department of Zoology, University of Delhi Principal Investigator : Prof. Neeta Sehgal
Head, Department of Zoology, University of Delhi
Content Writer : Dr. Sudhida Gautam, Hansraj College, University of Delhi
Dr. Renu Solanki, Deen Dayal Uppadhyaa College, University of Delhi Mr. Kiran Kumar Salam, Hindu College, University of Delhi
Content Reviewer : Dr. Surajit Sarkar, Department of Genetics South Campus, Delhi University
Co-Principal Investigator : Prof. D.K. Singh
Department of Zoology, University of Delhi Paper : 16 Molecular Genetics
Module : 01 Changing Concepts of Genes: Part 1
Description of Module
Subject Name ZOOLOGY
Paper Name Molecular Genetics; Zool 016 Module Name/Title Changing concepts of genes Module Id M01 Changing concepts of genes
Keywords Genes, unit factor, one gene one enzyme, polypeptide, cistron, muton
Contents
1. Learning Objectives 2. Introduction
3. Classical genetics/Unit factors-Mendel
4. Gene and metabolism: one gene one enzyme: Garrod’s classical experiment (life history) 5. One gene one polypeptide: Beadle and Tatum
6. Gene types on the basis of their behaviour
7. Modern concept of gene: Proposed by S. Benzer
8. Benzer’s Experiment: Cistron concept (Complementation assay) 9. Summary
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1. Learning Objectives
Concepts of classical genetics, unit factors mentioned by Mendel.
Theories of genetic; i.e., Theory of Dominant/recessive genes, Theory of Independent Assortment and Theory of Segregation given by Mendel.
Concept of “One gene one polypeptide: Beadle and Tatum”
Various types of genes.
Complementation experiment by Seymour Benzer.
2. Introduction
Field of genetics has opened large avenue for the young scientist. Genetics deals with the study of genes. With the advancements in field of cytology, and the discovery of nucleus, by 1885 scientists were sure that the nucleus contained the hereditary material/information. The dilemma of germplasm and somatoplasm was made crystal clear by the classical experiment of Augustus Weissman. He chopped the tails of mice for 22 generations and showed that it had no impact in the tail length of the progeny.
Protein/Enzyme coding regions of the DNA are known as genes. "GENE” was coined in 1909 by Johannsen. Gene Theory was proposed by T.H. Morgan in 1911. In 1949, the role of genes in protein synthesis was established by L. Pauling and Ingram. The fine structure of gene was proposed in 1969 by Seymour Benzer. Mendel’s experiment acted as an initial study to provide information that genes are the actual hereditary material which he called as the “Unit Factors”. These factors are passed on from parents to the progeny via the germ cells and are responsible for transmission of characters in generations. Discovery of (gene) unit factor by Mendel was followed by various experiments providing evidences of them being the hereditary material. Sutton and Boveri gave “The Chromosomal Theory of Inheritance”
i.e., genes are located on the chromosomes. Studies of Morgan helped in understanding the concept of crossing over and creation of genetic maps. In addition to Sutton (1902), Morgan (1913) other scientists Bridges (1923) and Muller (1927) and many others outlined the Classical concept of gene (which included gene positioning, crossing over and their pattern of inheritance). The gene concept was modernized by Benzer in 1957, by coining the term
“Cistron”, “Recon” and “Muton”. He emphasized about the different nature of genes and their location with respect to inheritance pattern.
Founder of modern genetics, Gregor Johann Mendel was an Austrian monk (Fig. 1) He has been credited for discovering the basic principles of heredity. It took approximately 35 years for people to recognize and appreciate his work which formed the basis of modern genetics.
Thus, he is recognized as the Father of Genetics. He worked with garden pea; he observed that the garden pea follows a particular pattern while passing on the traits from one generation to another. Around 1854 he himself began the experimental work by hybridizing the pea plants and recording all the results in a well-planned manner. He chose pea plant for his plant hybridization studies as it had many varieties and the offspring took less time in completing a generation so it was easy and quickly reproducible. Until 1900, Mendel’s work was not appreciated by the scientific community and directly rejected when he presented his findings. However, three botanist Hugo De Vries, Erich von Tschermak and Karl Correns are credited for Rediscovering the pioneer work of Mendel. Three of them worked independently and came to the same conclusion as Mendel. They interpreted their results in accordance to Mendel’s findings and drew attention to Mendelian studies on pea.
3. Classical genetics/Unit factors-Mendel
Fig. 1: Gregor Johann Mendel (Source: http://knowgenetics.org/wp-content/uploads/2013/01/Gregor_Mendel- 210x300.jpg)
5 Being a well learned botanist Mendel could identify characteristics traits which were opposite in two pea plants: e.g., tall & short, smooth seed or wrinkled seed, colour of seed yellow or green, shape of pod wrinkled or smooth, pod colour yellow or green, flower position terminal or axial and flower colour white or purple (Table 1; Fig. 2). He cross-fertilised the pea plants with opposite traits to obtain the progeny and recorded the observation. After analyzing his results he concluded three important principles referred to as the “Laws of Mendel” or the
“Mendelian principles” which were;
1. Law of Dominance: This principle states that one factor in a pair of traits is dominant over the other in inheritance unless both factors in the pair are recessive. The progeny expresses the dominant trait (Fig. 3)
2. Law of Segregation: This principle states that the offspring acquires one factor from each parent and during the production of gametes the two copies of each hereditary factor segregate. The segregation of each factor is responsible for the combination of the traits from both the parents.
3. Law of Independent assortment: This principle states that the laws of chance govern which particular characteristics of the parental pairs will occur in each individual offspring. The pairing and expression of the traits in the offspring are a chance factor.
Table 1: Various characteristics of pea plant observed by Mendel
Characteristics Traits Observed Dominant trait expressed in the Progeny Flower Colour Violet and White Violet
Flower Position Axial and Terminal Axial Plant Height Tall and Dwarf Tall Seed Texture Round and Wrinkled Round Seed Color Green and Yellow Yellow Pea Pod Texture Inflated and Constricted Inflated Pea Pod Color Green and Yellow Green
Fig. 2: Diagrammatic representations of the study by Mendel (Source: Departmental Artist and Author)
He concluded from his analyses that the inheritance of these traits followed a basic statistical pattern. He also added that these statistical patterns shown by the pea plant were also present in other organisms. His work was published in 1866 but remained unnoticed for a large period of time until (as it was considered insignificant at that time by the scientific community), 1900 when similar findings studies were rediscovered/ presented by Hugo De Vries, Erich von Tschermak and Karl Correns botanist/scientist the significance of his work was appreciated and it came to be known as the Mendel’s Laws. In the Mendelian times mechanism of meiosis and role of chromosomes were not known. Mendel had concluded that there were units of inheritance which were responsible for transferring the parental traits to the offspring. With the advancements in the molecular biology his postulates were welcomed and field of genetics was discovered.
He chose true breeding (plant in which traits remain same for many generations) pea plant because of its short life span (matured within one season) and he could artificially hybridize them (naturally they are self-fertilized). He had successfully discovered the basis of transmission of hereditary traits by his meticulous experiments (mostly involving monohybrid crosses) and accurate quantitative records.
Mendel crossed the pea plants having opposite traits- i.e., tall with short, wrinkled with smooth, yellow seeds with green seeds etc. - and analyzed the results which lead to his two most important conclusions “The Law of Segregation” and “The Law of independent
7 assortment”. He gave the results using simple statistical laws and conveyed that these patterns were followed not only in pea plant but also all living beings having such traits.
Unit factors in Pairs: Traits which used for the experimental analysis he called them as unit factors, which existed in pairs in individual organisms. Each unit factor came from either parent and the two forms were complimentary to each to other. For height of the plants traits were tall and small for a monohybrid cross and the output consisted of three combinations i.e., tall, small and intermediate (Table: 2).
Sample Trait Unit factor
Parent 1 TT (Tall) T
Parent 2 tt (small) T
Progeny TT (Tall), tt (small), intermediate (Tt)
T, T, t, t, T, t (any two)
Table.2: Experimental results for the unit factor experiment
Dominance or Recessiveness: When complimentary unit factors (inherited from either parent) are present in a single progeny, one unit factor is dominant unit factor (which is able to express itself in the F1 progeny phenotype) and other is known as recessive unit factor (which is not expressed in the phenotype of F1 progeny). We should remember that term dominant and recessive are just designatory for individual traits in the parents’ genotype. For example, if we consider the height of a plant tall plants are considered dominant over the recessive short/dwarf plants.
Segregation: Formation of gametes leads to the segregation of the individual traits; in order to equalize the chances for each trait in the individual gametes. Depending upon the fusion of gametes an individual may contain a pair of similar traits (TT-all tall progeny, tt- all short progeny) and the progeny will be decided accordingly. Because of segregation and random fusion of the gametes there is equal or 50% probability of receiving either of the traits. To understand it better let us illustrate it through a monohybrid cross as shown in Table 2: Parent 1 is tall and produces only T trait whereas parent 2 is short and produces only tt trait (unit
factor). Consider the gametes for Parent 1; it will be all tall “T” unit factor and for parent 2 it will be all short “t” unit factor. Following fertilization the progeny will receive one trait from each parent i.e., a tall factor and a short factor but because tall factor is a dominant to short, all the progeny are tall. On analyzing the gametes of the F1 progeny each progeny has T/tall factor which contributes to the tall height of the plant. However, selfing the F1 progeny we get four combinations for the F2 progeny which are:
1. tall/tall (TT) 2. tall/dwarf (Tt) 3. dwarf/tall (tT) 4. dwarf/dwarf (tt)
A combination 1, 2, 3 yields tall plants whereas 4 yields short plant. Thus, we see that F2 progeny consists of both tall and short plants in a ratio 3:1. This ratio was well maintained in all other crosses for other traits observed by the Mendel. Mendel emphasized that it was these factors which were responsible for passing on the characteristics among the populations from one progeny to other. With time these traits/ unit factors were identified as individual alleles and we know that a person can be homozygous or heterozygous for a particular trait.
He believed that single trait was controlled by a single gene. However, modern genetics has revealed the concept of multiple genes and the role of environmental factors in the expression of genes. Mendel confined his work to the pea plants and explained the inheritance of discrete hereditary material (factors) which are now known as genes. He laid the foundation of genetics and later on it was found that his observations were true for a variety of traits in many plants and animals. However, the explanations were not simple and easy for all the inherited patterns and a lot of complexities were observed by the later workers. These workers even tried to resolve any of the complexities by following his principles with suitable modifications. With time they developed a lot of other patterns of inheritance and genetic expressions which are discussed below:
9 A. Incomplete dominance: It is a form of intermediate inheritance in which an allele
specific for a particular trait is not able to express completely over its paired allele. As a result the progeny expresses a phenotype having a combination of both the parental phenotype i.e., an intermediate phenotype. This F1 phenotype is the result of partial expression of both the parental alleles (mixing of trait) and unlike the complete dominance inheritance in which only the dominant allele is expressed. This is observed is polygenic inheritance of traits such as eye colour and skin colour in humans. In Snapdragon plant (Mirabilus jalapa or 4’O clock plant) the parental phenotype of the flower is either red or white in colour but when red and white are crossed with each other we get pink colour for the F1 progeny (Fig. 3). In this monohybrid cross the allele which produces red colour (R) is not completely expressed over the allele producing white colour (r). The resulting offsprings are pink in colour. When these pink coloured off springs are self-pollinated we get all three phenotypes red: pink: white in a ratio of 1:2:1.
However, when the F1 generation is crossed with the parental generation we get equal numbers of the parent and F1 generation in a ratio of 1:1. As shown in Fig. 3 when pink flower (F1 generation) is crossed with white flower (parental generation) the F2 progeny contains both pink and white flowers in equal proportion. The pink flowers are heterozygous with the (Rr) genotype. The red and white flowers are homozygous with the (RR) and (rr) genotype, respectively.
Fig. 3: Incomplete Dominance in Snapdragon
B. Codominance: In codominance an additional phenotype is produced, as both the allels are expressed in the progeny. It is exhibited in the human blood groups: AB blood type inheritance
Codominance in Human Blood Types: Four different phenotypes are observed in humans with three different alleles. The three alleles are A, B and O denoted by IA, IB and i, respectively. Both IA and IB are dominant alleles to the allele i, i.e. IA and IB are codominant (Table:3). These alphabets correspond to a particular carbohydrate found on the surface of the person’s red blood cells and give four different phenotypes namely; A, B, AB and O.
Therefore a person having blood group A has RBC’s coated with carbohydrate A, if the RBC’s are coated with carbohydrate B, the blood group is B type and when both A and B carbohydrates are present the blood group of the individual is AB. Cells which are not coated with any of the carbohydrates are known to exhibit blood group O.
Genotype Blood Type
IA IA A
IAi A
IA IB AB
IBi B
IB IB B
ii O
Table 3: Blood group and Genotypes
Because both A and B are dominant alleles both express mutually and determine an individual’s phenotype. If the F1 progeny receives one A allele from one parent and one B allele from the other, then the blood type and genotype would be AB. On the contrary allele O is recessive, which means it will not affect the phenotypic expression in the progeny. The only way to have blood type O is to have genotype OO, because there is no dominant allele to mask the recessive allele. Differences between incomplete dominance and co-dominance are given below in Table 4:-
11
Incomplete Dominance Codominance
Partial combined expression of both the alleles
Effect of both the alleles are equally conspicuous
Blending of traits are observed No blending of traits are observed The hybrid is an intermediate of the two
alleles
Both the alleles express themselves and no concept of hybrid
Phenotype produced is exclusive and has no allele of its own
Phenotype produced is a combination of the parental phenotype and the alleles
Expression in the progeny is more or less a quantitative effect.
No quantitative effect is observed.
Table 4: Differences between Incomplete Dominance and Co-dominance
Co-dominance is helpful in certain cases e.g. Sickle cell anemia. The disease is due to presence of an abnormal protein (in the beta chain of Haemoglobin, glutamic acid is replaced by valine at the sixth position). Normal Haemoglobin is (RR) whereas as that of an infected person is sickle shaped cells (SS). Carrier or the heterozygous have a mixture of both normal and sickle shaped cells (RS) and are produced when healthy and infected persons interbreed (Fig.4). Sickle cell anemia is widely spread in the African malarial zone (Sub-Saharan Africa). These areas also prone to malaria, a disease caused by blood borne parasite (Plasmodium falciparum) and transmitted through mosquitoes. This sickling allele was not produced by natural selection; instead it’s a result of random mutations. The heterozygous (RS) sickle cell trait has a moderately good resistance to malaria as these misshapen and deflated red blood cells rarely develop the life threatening anemia and related problems observed in the homozygous (SS) sicklers. The otherwise harmful sickling allele provides an advantage for heterozygous individuals. The homozygous (RR) dominants have normal RBC’s which are excellent hosts for malaria. As, the environment is highly favourable to the falciparum malarial environment, the nature selects for heterozygous sicklers against homozygous sicklers and humans with normal red blood cells. The sickle
cell mutation occurred thousands of years ago and helped to overcome malarial outbreaks.
The resulting survivors then passed on the mutation to their offspring and this trait became established throughout the sub-Saharan African regions.
Fig. 4: Sickle cell anemia (Source: Author and Departmental artist)
C. Multiple alleles: When a gene has three or more alternate forms at the same gene locus it is known as a multiple allele gene. A well-known example is of Human Blood group where three alleles give rise to four phenotypes in the humans as discussed above in the example of codominance (Table: 4). Multiple alleles have genetic benefits as they give a variety of genetic combinations and phenotypes. It is important to known our own blood group as it helps us to identify suitable donors. If blood is transfused from an incompatible donor there are chances of severe immune response. As each blood group has its own specific type of antibody (Fig. 5). Thus if we have blood group A and receive the blood from a donor having blood group B, our body will produce antibodies and destroy the new RBC’s having carbohydrate B. Antibodies are specific proteins produced within the body to identify and destroy the foreign invaders (proteins, viruses). When a mismatched blood type appears these antibodies bind to the unknown carbohydrates and kill the blood cells.
13 Fig. 5: Human Blood Groups (Source: Department Artist and Author)
Human Genetics: The inheritance patterns of the diploid genes are same in humans as observed in pea and Drosophila. But in humans breeding experiments are not very feasible;
usually the information about human inheritance is largely derived by studying the pedigree of naturally occurring traits. The identification of the trait might be of importance to human health and well-being or help in understanding health-related genetic defeats. Knowing the nature of the defect (gene is dominant or recessive) or of those bearing it (carrier is homozygous or heterozygous) enables us to take appropriate steps to minimize its severity and counseling can be given to reduce, if not eliminate, its propagation into future generations. Various traits include blood grouping, eye colour, hair texture, albinism, colour blindness, ability to taste PTC (Phenylthiocarbamide), Haemophilia, Sickle cell anemia, inborn errors of metabolism and mental ability and so on.
4. Gene and metabolism: one gene one enzyme: Garrod’s classical experiment (life history)
Fig. 6: Sir Archibald Garrod (Source: http://www.dnaftb.org/images/13/13bio.gif)
Sir Archibald Garrod studied medicine at Oxford University (Fig.6). His Father Alfred Baring Garrod was also a physician who studied rheumatoid arthritis. Garrod studied human disorder “Alkaptonuria” by collecting family history and urine samples from his patients. He deduced from his observations that alkaptonuria is a recessive genetic disorder and in early 1900s introduced the phrase inborn error of metabolism. Tyrosine was not been broken down into carbon dioxide and water. He observed the accumulation of an intermediate product i.e.
homogentisic acid in the urine of patients, which turned black when exposed to air (oxidation of homogentisic acid). He found that homogentisic acid was an intermediate product in the breakdown of phenylalanine. The patients lacked an enzyme namely; homogentisate oxidase responsible for converting the homogentisic acid into malylacetoacetate (Fig.7) He even published his studies in a book called “the incidence of alkaptonuria: a Study in Chemical Individuality” (1902) stating the links between genetics and metabolic pathways.
Fig. 7: Classical experiment by Archibald Garrod and his interpretation Source: Department Artist and Author
Garrod said that genes are responsible for certain diseases like sickle cell anemia and Huntington’s disease, alkaptonuria, cystinuria, pentosuria and albinism are due to defects in
15 proposed that lack of functional enzymes was responsible for these inherited traits/ defects.
He explained that certain defects are a result of gene mutations in biochemical pathways for eliminating the liquid wastes. The change in the colour of the urine is an indication of the error. In 1909 Archibald Garrod gave the concept of “Inborn Errors of Metabolism” which was proved by the studies of George Beadle and Edward Tatum in 1941.
Gene action refers to the expression of genetic character influenced by the gene. Genes code for proteins which are associated/ result in the enzyme production. Enzymes are responsible for maintaining the homeostasis of the body cells through a process of autocatalysis. As a rule one gene affects one enzyme. The various actions of genes are expressed in their development of pigments, colors, hormones, size and form, production of proteins, antigen and antibody production, decisive effect on human disease like albinism, tyrosinosis etc. This idea was given by Beadle and Tatum (1941) (Fig. 8 (a) & (b)).
Fig. 8: (a) American Genetist George Wells Beadle; (b) American Biochemist Edward L. Tatum Source: (a)http://academic.pgcc.edu/~kroberts/biotech/chapt2/beadle.jpg
(b)http://academic.pgcc.edu/~kroberts/biotech/chapt2/tatum.jpg
In 1958, Nobel Prize was awarded to Beadle and Tatum, shared with Joshua Lederberg (1925-2008) in the field of physiology. Beadle and Tatum are credited with the discovery of one gene-one enzyme hypothesis, but a third scientist named Boris Ephrussi, who worked at the Institute de Biologie Physico-chimique (Institute of Physico-chemical Biology) in Paris, France, and studied the genes of the fruit flies Drosophila melanogaster. Beadle was working from 1934-1935 at the Caltech in Pasadena, had an opportunity to work with Ephrussi. They worked together from 1934-1937 with mutant flies to understand the eye colour through genetic components (They transferred the larval tissue destined for eye formation in adults
from one larval embryo to another embryo and recorded the results. They used 26 mutants with different eye colour and transplanted the fetal eye tissue in the abdominal region of the wild type or normal, fruitfly. They were able to observe the growth of mutant eye in the transplanted abdomens, except two cases. Thus, the larvae had the normal eye and an additional eye in the abdomen. They deciphered that it was the larval genes of the transplanted tissue responsible for the mutant eye colour rather than the environment of the larval tissue. The two exceptions were of fly larvae having vermilion (v) and cinnabar (cn) eye colour which were quite distant from the normal eye colour brown. When the tissues of these mutants were transplanted in the wild type larvae the additional eye in the abdomen was of brown colour, rather than the colour of the mutant. They concluded from these findings that some substance was diffusing from the host tissue into the mutant larval tissue transplant which leads to the development of the wild type eye colour. From their studies they hypothesized that the wild type vermilion and cinnabar factors were genes that coded for enzymes responsible for the production of substances capable of causing wild type eye development.
Fig. 9: Neurospora crassa: Eukaryotic multicellular fungus commonly known as red bread mold, Source : http://modelorganisms.nih.gov/neurospora/images/neurospora.jpg
The idea of one gene-one enzyme was introduced in early 1940’s and was proved by American Geneticist George Wells Beadle and American biochemist Edward L. Tatum.
17 the mold on different media preparations (Fig.10). The mold Neurospora was first exposed to x-rays to obtain various mutations. Mutations are the sudden heritable change in the genome of the organism having the capability of disrupting the mold’s ability to synthesis amino acids/nutrients and grow. They subsequent germinated the mutant spores in tubes having complete medium (i.e. amino acid, vitamins and organic substances) along with proper physical environment. Later on growing these mutant strains on minimal growth mediums (Medium having sugars, salts and vitamins which were utilized by the enzymes of the mold to synthesis the necessary amino acids for sustaining the mold) containing only the basic nutrients in which the wild type or the non-mutated, strain of Neurospora could survive. They observed that the mutants were not able to grow on the minimal media instead they required additional supplements of specific amino acids in order to grow. The effect of mutation was identified by adding the supplements one by one to test which supplement could fulfill the requirement of the missing enzyme in the mold and helped it to grow. These mutants when crossed with the normal molds were able to grow in the minimal medium revealing that these metabolic defects were outcome of recessive traits and proved the alteration in the genes.
Thus, they concluded that mutants have disruption in specific gene which was responsible for the production of specific amino acid (which was being added as the supplement for the mutants to grow). Producing identifiable nutritional mutations with x-ray technique was a herculean task and they achieved success in their 299th attempt, which grew only when the minimal medium was supplemented with Vitamin B6. Thus, with patience and continued perseverance they were able to identify many more mutants revealing that genetic mutations affect metabolic pathways and gave detailed multi-step synthesis pathway for many amino acids and vitamins. The information obtained from the experiments on Neurospora confirmed what Beadle had witnessed in Drosophila when he worked with Ephrussi. They published their results in “Genetic control of biochemical reactions in Neurospora,” in 1941, proposing the concept of one gene-one enzyme hypothesis. This hypothesis was verified but since 1940s it has undergone a slight change and today it is known as one gene one polypeptide hypothesis as stated by Yanofsky (1965). As with the advancement in modern science we know that an enzyme is not coded by a single gene/polypeptide.
Fig. 10: One gene one enzyme experiment, Beadle ad Tatum (1941)
Source: Departmental Artist and Author, http://academic.pgcc.edu/~kroberts/biotech/chapt2/1gene.jpg
5. One gene one polypeptide: Beadle and Tatum
The one gene one polypeptide hypothesis states that a single gene is responsible for coding and production of a single polypeptide. A gene consists of specific sequence of nucleotides.
Each triplet codes for an amino acid, which form the monomeric units of polypeptides joined to each other by peptide bonds. Earlier it was known that a single gene is responsible for polypeptide (which codes the enzyme). They studied the growth of fungus Neurospora crassa. In laboratory the fungus grows on minimal medium containing agar, sucrose, nitrate, vitamin (biotin) and inorganic minerals. This means that all supplements are present within these minimal medium for the fungal development. However, when treated with x-ray the fungus is unable to grow in minimal medium due to lack of certain growth component. The mutant spores are given individuals component of the minimal medium as a growth supplement to determine the deficient component which the mutants are unable to synthesis.
The mutants give a healthy progeny when crossed with a normal/wild type and the ascospores (8) of the progeny are individually tested for their nutritional requirements. A single gene mutation is determined crossing the single arginine deficient mutant strain (a-) with wild type (a+), all the 8 ascospores of the progeny survive on a medium containing arginine, but only four ascospores survive in absence of arginine from the medium. Indicating the mutation of a
19 each of which is controlled by a single gene. The mutation of gene leads to the suppression of the specific step. This is demonstrated by the development of mutants on the minimal medium when the deficient component is added to it. Thus, on identifying the mutant we know the defect in the biosynthetic pathway. The mutation occurs in a specific chromosomal region responsible for the functionality loss of that particular enzyme (encoded within that chromosomal region). This conveys the basic enzyme relationships. The modern researchers have identified that gene is a part of DNA and responsible for synthesis of particular protein.
Currently the concept of “one gene one enzyme “given by Beadle and Tatum has been revised to “one gene one polypeptide chain (protein molecule)” in view of the complexity in the structure, functions of the enzymes and the genetic variability of proteins (such as sickle cell haemoglobin).
However, later on realizing the phenomenon of splicing (with which it became evident that a single gene codes for specific nonenzymatic proteins as well as individual polypeptide chains). In 1945 George Beadle modified the one gene one enzyme hypothesis to one gene one polypeptide hypothesis. Now we know that certain genes code for different types of RNA involved in protein synthesis. Especially in eukaryotes a single gene can produce several RNA products depended on differential splicing. The direct relationship between a gene and enzyme was put forth by the Neurospora genetics. However later on finding that an enzyme can consists of various polypeptides it was modified to one gene one polypeptide hypothesis.
6. Gene types on the basis of their behaviour
A variety of gene types have been proposed on the basis of experimental evidences after identification of genetic material and studying the behavior of genes and interaction during hybridization (Table:2). The gene type depends on its expression type (single or cumulative), number of factors which a single influences, ability to influence the character of other genes in addition to the genes (Table:5) which regulate the process of protein synthesis (Transcription and Translation).
Gene Definition
Basic genes: Gene which bring out the expression of a particular character.
Lethal genes: Genes responsible for the death of their possessor.
Multiple gene: When two or more pairs of independent genes act together to produce a single phenotypic trait.
Cumulative gene: Genes expressing additive effects on the action of other genes.
Pleiotropic genes: Genes capable of producing changes in more than one character.
Modifying gene: Gene which cannot produce a character by itself but interacts with other to produce a modified effect.
Inhibitory gene: Gene who affect the expression of another gene by suppressing or inhibiting it.
Table 5: Gene types on the basis of their behaviour
Structural genes: These genes regulate production of specific m-RNA and determine the kind of protein to be synthesized.
Operator genes: These genes act as switches to turn on or turn off the activities of structural genes, regulating the elongation and termination of polypeptide chain.
Regulator genes: These genes produce certain proteinaceous substance called repressors which prevent the operator genes from their action.
Table 6: Genes in protein synthesis and genetic code:
The Neoclassical Concept of the Gene: Identification of gene and genetic material done by different geneticist who brought a blow to the classical concept of gene are given in Table: 7.
Griffith had discovered transformation in 1928. He had revealed that killed bacterial cells when injected into healthy mice were able to transform genetically different living bacteria into their own kind (Fig. 11).
21 Fig. 11: Griffith’s Experiment (Source: Departmental Artist and Author)
Substance responsible for transformation in bacteria was DNA proved by Avery et.al.(1944).
Thus, it was concluded that DNA consisted of genes (Fig. 12).
Fig. 12: Transformation in bacteria was proved by Avery et. al. (Source: Department Artist and Author)
Hershey and Chase (1952) were able to prove that the only component of bacterial virus was its DNA which enters the bacterial host, and therefore it was presumed that DNA is the sole bearer of viral genetic information.
Fig. 13: Experimental evidence that DNA is the solebearer of viral genetic material Source: Department artist and Author
Scientist Name Observation
Oliver (1940) and Lewis (1941)
The phenomenon of intragenic recombination in Drosophila melanogaster
Green and Green (1949)
Map mutations of the lozenge locus of D. melanogaster into linear order
Roper (1950) and Pontecorvo (1952)
Observed intragenic recombination within genes in the ascomycete Aspergillus nidulans, which were known to control the synthesis of one single enzyme.
Bonner (1950) and Giles (1952)
Observed intragenic recombination within genes in Neurospora crassa.
Pritchard (1955) He was the first to demonstrate with microbic fungi that mutations within a single gene could be mapped into linear order by means of recombination.
Table 7: The Breakdown of the Classical View of the Gene
23
7. Modern concept of gene: Proposed by S. Benzer
Twentieth century has witnessed a lot of development in biological sciences, it begun with the Mendelian units of hereditary and also the entire human genome. However, Benzer’s studies played an important role in the shifting of genetic studies from the classical to advanced molecular genetics. Benzer was born in Brooklyn to a Jewish Polish immigrant parents in 1921. Even though he graduated in physics from Purdue University (Fig.14), he read the book “What is life?” in later days of his graduation after which he grew a lot of interest in biology. Benzer took the summer bacteriophage course at Cold Spring Harbor Laboratory in 1948 with Gunther Stent. In 1953, after Watson and Crick published their model of DNA, Benzer hatched his plan to get inside the gene by using bacteriophage with mutant rII genes. In 1993, he received the Crafoord Prize for his extraordinary work in genes and behavior. He won the American Nobel Prize i.e. the Albany medical Centre Prize in Medicine and Biomedical Research (2006) for the behavior changes in Drosophila due to single gene mutations.
Fig. 14: Seymour Benzer
Source: http://www.nejm.org/na101/home/literatum/publisher/mms/journals/content/nejm/2006/
nejm_2006.355.issue-26/nejmbkrev57338/production/images/small/nejmbkrev57338_f1.gif
8. Benzer’s Experiment: Cistron concept (Complementation assay)
Benzer studied the bacteriphage T4 genome having mutations on the rII region. The mutants have distinct host range and plaque morphology. Growing E. coli cells when infected with wild type T4 phage produce small, turbid plaques with fuzzy ends in contrast to the plaques produced by rII mutants which are larger in size and possess a distinct boundary. Wild type T4 phage has an ability to form plaques on E. coli strain B and E coli strain K12. The rII mutants were able to grow only in E. coli B strain (E. coli K12 was not infected by the T4 phage). This property of different plaque structure was used by Benzer to isolate small proportions of wild type phages among large populations of rII mutants (Fig.15).
Fig. 15: Complementation experiments showing possible outcomes
No complementation occurs since the coinfected cell is unable to make the normal product of gene A.The coinfected cells will not produce the viral particles, thus no bacterial cell lysis and no plague formation occurs.
Complementationoccurs since the coinfected cell is able to make the normal prduct of gene A and gene B. The coinfected cell will produce the viral particles that lysis the cell, resulting in the apperaence of clear plagues.
Source: Department Artist and Author
He was able to identify 60 different rII mutants which he crossed in all possible combinations, using E. coli as the host. He collected the progeny after the cell lysis. He consider x and y as the two rII mutants (each having a separate type of mutation). Benzer crossed rIIx with rII y and the progeny obtained consisted of two parental type: rIIx and rIIy, two recombinant type rIIx,y (double mutant) and the wild type (rII). His observations
25 However, the relative frequency of the parental and progeny populations was dependent on the distance between the two alleles. He plated the phage progeny on the permissive host i.e.
E.coli B and then from the total number of plagues produced he calculated the total number of phage progeny per milliliter. Later, he plated another sample of phage T4 on the non- permissive host (E.coli K12), and found the frequency of r+ recombinants. Thus, he was able to find the percentage of very rare r+ recombinants produced by crossing-over between closely linked alleles. The recombination frequency was given by the following formula:
2 x number of r+ recombinants x 100 % Total number of progeny
The number of r+ recombinants is multiplied by 2 to account for the double mutants (as they have same phenotype as single mutants). A control was set up for each cross in which rII parent was used to infect the E. coli B and later the progenies were used to infect both E. coli B and K12 separately. This was done to calculate the reversion frequencies. Because changes are that rII mutant undergoes a mutation to revert back to the r+ type. Benzer concluded that the reversion frequencies were of much lower magnitude than that of the smallest recombination frequency. He constructed a linear genetic map from the recombination data obtained from the 60 rII mutants. r+ recombinants were not obtained from certain crosses which revealed that these pairs carried the mutations at exactly the site. Homoallelic mutations were responsible to change the same nucleotide pair within a gene. Heteroallelic mutations were able to change different nucleotide pairs within a gene i.e. they produced r+ recombinants when crossed indicating that they carried altered nucleotide pairs in the DNA (Fig. 15).
Till now it was believed that the gene is a functional unit of DNA and each gene has a specific role. Benzer devised an experiment to find whether the two different rII mutants had same function (i.e., the mutants belonged to a single gene). Benzer adapted a cis-trans test/complementation test given by Edward Lewis to study the nature of gene in Drosophila.
Complementation test showed that rII region consists of two genes namely, A and B (which correspond to two different regions on the chromosome). Any mutation within these two
region result in rII mutants having specific plaque morphology and host range property. By this we can say that rIIA and rIIB give different products when grown in E. coli K12.
Complementation test helps us to identify units of a gene which are having specific mutations leading to the expression of same mutant phenotype. Benzer deliberately infected the non- permissive host E. coli K12with a pair of rII mutant phages to see whether they were able to survive together and produce progeny phages (as they were not able to produce phage progeny when these mutants were infecting the E. coli K12 independently). If progeny are obtained when the paired mutants are used to infect the non-permissive host, it conveys that the mutants are complimentary to each other. Complimentary mutants tell us that the mutations are present in different genes which code for different products. Thus, when both rII mutants are present the combined products allow the progeny to propagate. In the absence of progeny the mutants are not complimentary, indicating the presence of mutations on the same functional gene (unit) i.e., the life cycle of phage cannot proceed because both the mutants are producing the defective gene and no progeny is observed.
27 S. Benzer (1957) coined specific terms to genes depending on their nature and genetic material in relation to their locations on chromosomes and the basis of genetic phenomenon to which they involve. Benzer reported the fine structure of gene based on its properties like the ability to mutate, recombine and the specific function of the gene. He classified genes into three structures: cistron, muton and recon.
i) Cistron/Genes as unit of transmission: The part of DNA specifying a single polypeptide chain is termed as cistron. A cistron can have 100 nucleotide pairs in length to 30,000 nucleotide pairs. It transmits characters from one generation to other as unit of transmission.
ii) Recon/ Genes as unit of recombination: The smallest segment of DNA capable of being separated and exchange with other chromosome is called recon. A recon consists of not more than two pairs of nucleotides.
iii) Muton/ Gene as unit of mutation: Muton is the smallest unit of genetic material which when changed or mutated produce a phenotypic trait. Thus muton is delimited to a single nucleotide.
9. Summary
Genetics deals with the study of genes which are passed on from the parents to the progeny. Genes are the coding regions of DNA which are transcribed to give different functional protein/enzymes according to the needs of the cell.
Mendel used garden pea as his experimental plant and gave the three “Mendelian principles” which were: Law of Dominance: This principle states that one factor in a pair of traits is dominant over the other in inheritance unless both factors in the pair are recessive. The progeny expresses the dominant trait. Law of Segregation: This principle states that the offspring acquires one factor from each parent and during the production of gametes the two copies of each hereditary factor segregate. The segregation of each factor is responsible for the combination of the traits from both the parents. Law of Independent assortment: This principle states that the laws of chance govern which
particular characteristics of the parental pairs will occur in each individual offspring. The pairing and expression of the traits in the offspring are a chance factor.
Incomplete dominance is a form of intermediate inheritance in which an allele specific for a particular trait is not able to express completely over its paired allele. As a result the progeny expresses a phenotype having a combination of both the parental phenotype i.e., an intermediate phenotype which is observed in Snapdragon.
Codominance is observed when both are alleles are dominant and express in the progeny.
ABO blood groups of humans exhibit this type of inheritance where both A and b are dominant allele and I is the recessive allele. If more than two alleles are responsible for a particular gene, the concept is known as multiple gene. In humans three alleles A,B and I code for four type of blood groups i.e. A,B, AB and O.
Sickle cell anemia has not eliminated by the natural selection because the sickelling allele provides an advantage to the heterozygous individuals. The heterozygous (RS) sickle- cell trait has a moderately good resistance to malaria as these misshapen and deflated red blood cells rarely develop the life threatening anemia and related problems observed in the homozygous (SS) sicklers.
Certain diseases like Huntington’s disease, alkaptonuria, cystinuria, pentosuria and albinism are due to defects in the metabolic pathways. These defects are a result of non- functional enzymes in the metabolic pathways. Inborn error of metabolism is because of gene mutations in biochemical pathways due to which a non-functional enzyme leads to the accumulation of its substrate.
Neurospora Genetics involved growing the mutants on minimal medium and realized that the mutants needed additional supplements of specific amino acids in order to grow.
These mutants when crossed with the normal molds were able to grow in the minimal medium revealing that these metabolic defects were outcome of recessive traits and proved the alteration in the genes. Thus, they concluded that mutants have disruption in
29 gene one polypeptide hypothesis states that a single gene is responsible for coding and production of a single polypeptide.
Now we know that certain genes code for different types of RNA involved in protein synthesis. Especially in eukaryotes a single gene can produce several RNA products depended on differential splicing. The direct relationship between a gene and enzyme was put forth by the Neurospora genetics.
Genes are classified into three structures: cistron, muton and recon; based on its properties like the ability to mutate, recombine and the specific function of the gene.
