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Microbiology and molecular genetics 1.How does gene work? 2.Gene-protein relationship 3.Genetic fine structure Submitted to: Dr. Ambreen Ahmed Submitted by: Jannat Iftikhar B11-16 Semester: 5 th

How do gene work1

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Microbiology

and

molecular

genetics

1.How does gene work? 2.Gene-protein

relationship

3.Genetic fine structure

Submitted to:

Dr. Ambreen Ahmed Submitted by:

Jannat Iftikhar B11-16

Semester: 5th

Contents

1. How does gene work?

Gene

One-gene-one-enzyme hypothesis

2. Gene-protein relationship

Protein structure

Protein motifs

Determining protein sequence

Relationship between gene mutation and altered protein

Colinearity of gene and proteins

Gene and cellular metabolism

Genetic observations obtained by enzyme structure

3. Genetic fine structure

Bead theory

The rII system

Destruction of bead theory

1.How do gene works?

People have known for many years that living things inherit traits from their parents. That

common-sense observation led to agriculture, the purposeful breeding and cultivation of animals

and plants for desirable characteristics. Firming up the details took quite some time, though.

Researchers did not understand exactly how traits were passed to the next generation until the

middle of the 20th century. But now it has become clear that genes are responsible to carry traits

through generations. Genes are often called blueprints of life, because the fate of every cell is

destined by genes. Before going into details that how gene works it is important to know what is

gene.

Gene:

A gene is a molecular hereditary unit of all living organisms. Living beings are dependent on

gene because they specify all proteins and functional RNA chains. Gene carries all the

information to build and maintain the cell and pass genetic traits to the generations. The ultimate

phenotype of all living organisms depends on genes. Fig.1 shows the position of gene in a cell.

Fig.1 position of chromosome in cell

Following points gives us a model relationship between genotype and phenotype.

The characteristics features of organisms are determined by the phenotype of its parts,

which in turn are determined by the phenotype of the component cells.

The phenotype of the cell is determined by its internal chemistry, which is controlled by

enzymes that catalyze its metabolic reactions.

The function of an enzyme depends on its specific three dimensional structures, which in

turn depends on the specific linear sequence of amino acids in the enzyme, since all

enzymes are proteins.

The enzymes present in the cell, and structural proteins as well, are determined by

genotype of the cell.

Genes specify the linear sequence of amino acids in polypeptides and hence in proteins,

thus genes determine phenotype.

One-gene-One-Enzyme Hypothesis

The actual function of gene became clear from the research in the 1940s on Neurospora by

George Beadle and Edward Tatum. Later on they received Nobel Prize for their work. One gene

one enzyme hypothesis, the name was given based on the idea that a gene act by the production

of the enzymes and each gene is responsible for production of the single enzyme that in turn

effect a single step in metabolic pathway.

Beadle and Tatum experiment

Beadle and Tatum analyzed mutants of Neurospora, a fungus with a haploid genome. They

first irradiated Neurospora to produce mutations and then tested cultures from ascospores for

interesting mutant phenotype. They detected numerous auxotrophs. In each case, the mutation

was inherited as a single gene mutation: each gave a 1:1 ratio when crossed with a wild type.

One set of mutant strains required arginine to grow on minimal medium. These strains

provided the focus for much of Beadle and Tatum’s further work. First they found that mutations

mapped out in three different regions on separate chromosomes. Even though the same

supplement (arginine) satisfied the growth requirement for each mutant. Let’s call the three loci

the arg-1, arg-2, and arg-3 genes. Beadle and Tatum discovered that the auxotrophs for each of

the three loci differ in their response to the chemical compounds ornithine and citrulline, which

are related to arginine. The arg-1 mutant will grow if supplemented with either ornithine or

citrulline or arginine in addition to minimal medium. The arg-2 mutant will grow only if

supplemented with citrulline or arginine but not the ornithine. The arg-3 mutant will grow only if

supplied with arginine.

It was already known that cellular enzymes often interconvert related compounds such as

these. Based on the properties of arg mutants, Beadle and Tatum and their colleagues proposed a

biochemical model for such conversion in Neurospora. Fig.2 gives us a view how a precursor is

converting into arginine.

Note how this relationship easily explains the three classes of mutants. The arg-1 mutants

have a defective enzyme A, so they are unable to convert precursor into ornithine as the first step

in producing arginine. However, they have normal enzymes B and C, and so the arg-1 mutants

are able to produce arginine if supplemented with ornithine or citrulline. The arg-2 mutants lack

enzyme B and arg-3 mutants lack enzyme C. thus a mutation at a particular gene is assumed to

interfere with the production of the single enzyme. The defective enzyme then creates a block in

some biosynthetic pathway. The block can be overcome by supplying to the cells any compound

that normally comes after the block in the pathway.

Now we can diagram a more complete biochemical model. (fig.3)

Fig.3.Diagrammatic representation of production of arginine

This entire model was inferred from the properties of the mutant classes detected through

genetic analysis. This model, which is known as one-gene-one-enzyme hypothesis, provides the

first evidence of function of the gene; some genes were responsible for the functions of the

enzymes and each gene apparently control one specific enzyme.

One-gene-one-enzyme hypothesis became one of the great unifying concept in biology

because it provides a bridge that brought together the concept and research techniques of

genetics and biochemistry. (Fig.4)

Fig.4. Beadle and Tatum experiment

2.Gene – Protein relationship

One-gene–one-enzyme hypothesis was an important step in our understanding of gene

function, but how do genes control the functioning of enzymes? All enzymes are proteins, and thus we must recall the basic of protein structure to understand the next step in the study of gene function.

Protein structure:

A protein is a macromolecule composed of amino acids attached end to end in a linear sequence.

The general formula for the amino acid is H2N-CHR-COOH, in which R group can be anything

from hydrogen to a complex ring. Amino acids are linked together by peptides bond. A peptide

bond is formed through a condensation reaction that involves the removal of a water molecule.

Several amino acids are join together by peptide bond to form a molecule called polypeptide; the

proteins found in living organisms are all large polypeptides. Proteins have a complex structure

that is traditionally thought of having four levels.

The linear sequence of amino acid in a polypeptide chain is called primary structure of protein.

Fig.5 shows the linear sequence of tryptophan synthase and beef insulin.

Fig.5. linear sequence of two proteins. (a) The E.coli tryptophan synthase (b) bovine insulin

protein.

The secondary structure of protein refers to the interrelationship of amino acids that are close

together in the linear sequence. This spatial arrangement often results from the fact that

polypeptides can bend into regularly repeating (periodic) structures, created by hydrogen bonds

between the CO and NH groups of different residues. Two of the basic periodic structures are α

helix and β pleated sheet. (Fig.6)

fig.6 (a) α helix, a common basis of secondary protein structure.

Fig.6 (b) Two views of the antiparallel β pleated sheet, another common form of secondary

protein structure.

A protein also has a three dimensional pattern termed as the tertiary structure, which is

generated by electrostatic, hydrogen, and Van der Waals bond that form between various amino

acid groups, causing the protein chain to fold back on itself. (fig.7) Often two or more folded

structures will bind together to form a quaternary structure; this structure is multimeric

because it is composed of several separate polypeptide chains or monomers.

Fig.7. Folded tertiary structure of myoglobin.

Many proteins are compact structures; such proteins are called globular protein. Enzymes and

antibodies are among the important globular proteins. Other unfolded proteins called fibrous

proteins are important components of cell structure as hair and muscle. Here is given a diagram

showing primary, secondary and tertiary structure of protein. (Fig.8)

Protein motifs:

Several elements of secondary structure often combine to produce a pattern, or motif, that is

found in many other proteins. Sometimes we can recognize motif by their amino acid sequence

pattern and other times by observing the three dimensional structure. The helix-loop-helix motif

is found in calcium binding protein and a variant of it is found in regulatory proteins that bind

DNA. The zinc binding motif also found in DNA binding proteins, is termed the zinc finger,

because of the way that residue protrudes out like a finger. (fig.9)

Determining the protein sequence:

If we purify a particular protein, we find that we can specify a particular ratio of the various

amino acid that make up the specific protein. But the protein is not formed by random hookup of

fixed amounts of various amino acids; each protein has a unique characteristic sequence. For a

small polypeptide, the amino acid sequence can be determined by clipping of one amino acid at a

time and identifying it. However the large polypeptides cannot be readily sequenced in this way.

Method for sequencing the large polypeptide:

Fredrick Sanger worked out a brilliant method for deducing the sequence of large polypeptides.

There are several proteolytic enzymes-that break the peptide bonds only between specific amino

acids in proteins. Proteolytic enzymes can break a large protein in a number of fragments, which

can then be separated according to their migration speed in a solvent on chromatographic paper.

Because different fragments will move at different speed in various solvents.

Two dimensional chromatography can be used to enhance the separation of fragments. In this

technique a mixture of fragments is separated in one solvent; and then paper is turned 900, and

another solvent is used. When the paper is stained, the polypeptides appear as spots in a

characteristic chromatographic pattern called the finger print of protein. Each of the spot can be

cut out and the polypeptide fragments can be washed from the paper. Because each spot contains

only a small polypeptide, their amino acid sequence can be determined.

Using different proteolytic enzymes to cleave the protein at different points, we can repeat the

experiment to obtain other sets of fragments. The fragments from the different treatments

overlap, because the breaks are made in different places with each treatment. The problem of

solving the overall sequence then becomes one of fitting together the small-fragment

sequences—almost like solving a tricky jigsaw or crossword puzzle. Using this elegant

technique, Sanger confirmed that the sequence of amino acids (as well as the amounts of the

various amino acids) is specific to a particular protein. In other words, the amino acid sequence

is what makes insulin insulin.

Fig.10. Two dimensional chromatographic fingerprinting of a polypeptide fragment

mixture A protein is digested by a proteolytic enzyme into fragments that are only a few amino

acids long. A piece of chromatographic filter paper is then spotted with this mixture and dipped

into solvent A. As solvent A ascends the paper, some of the fragments become separated. The

paper is then turned 90° and further resolution of the fragments is obtained as solvent B ascends.

Relationship between gene mutation and altered protein:

Change of just one amino acid is sometimes enough to alter the protein function. This was first

shown by Vernon Ingram, who studied the globular protein hemoglobin. (fig.11) hemoglobin is

made up of four polypeptide chains; two identical α chains each containing 141 amino acids, and

two identical β chains, each containing 146 amino acids.

Fig.11. The difference at the molecular level between normalcy and sickle-cell disease.

Ingram compared hemoglobin A (HbA), the hemoglobin from normal adults, with hemoglobin S

(HbS) the protein from the people homozygous for the mutant gene that causes sickle-cell

anemia, the disease in which red blood cells take on a sickle shape. Using Sanger’s technique,

Ingram found that the finger print of HbS differs from that of HbA in only one spot. Sequencing

that spot from the two kinds of hemoglobin, Ingram found that only one fragment differs in two

kinds. Apparently of all the amino acids known to makeup a hemoglobin molecule, a substitution

of valine for glutamic acid at just on point at position 6 on the β chain, is indeed responsible to

produce the defective hemoglobin. Unless patients with HbS receive medical attention, this

single error in one amino acid in one protein will hasten their death. Fig.12 shows how this gene

mutation ultimately leads to the pattern of sickle-cell disease.

Fig.12. The compounded consequences of one amino acid substitution in hemoglobin to produce

sickle-cell anemia.

A gene mutation that had been well established through genetic studies was connected with an

altered amino acid sequence in a protein. Subsequent studies identified numerous changes in

hemoglobin, and each one is the consequence of a single amino acid difference. We can

conclude that one mutation in a gene corresponds to a change of one amino acid in the sequence

of a protein

Colinearity of gene and protein:

By determining the structure of DNA it became clear that the structure of protein is encoded

in the linear sequence of nucleotides in DNA. Ingram’s demonstration shows that one

mutation alters one amino acid in a protein; we found that there is a linear relationship

between the sequence of mutant sites in genes and the linear sequence of amino acid in a

protein. Charles Yanfosky fond the relationship between mutant genes and altered proteins

by studying the enzyme tryptophan synthase in E.coli. This enzyme is responsible for

conversion of indole glycerol phosphate into tryptophan. This enzyme is controlled by two

genes trpA and trpB. Each gene controls a separate polypeptide; after the A and B

polypeptide are formed, they combine to form a multimeric protein. Yanofsky observed

mutation in the trpA gene which resulted in change in tryptophan A subunit. He induces

mutation by P1 transduction to produce a detailed gene map. He also determined the

sequence of amino acid of each respective changed tryptophan synthase. His results were

similar to Ingram‘s: each mutant has a defective polypeptide associated with a specific amino

acid substitution at a specific point. However he was able to demonstrate an exciting

correlation between that Ingram was unable to show due to some limitations of his system.

He found an exact match between the sequences of mutational sites in the gene map of trpA

gene and the location of the respective amino acid in the A polypeptide chain. More the

amino acids there were between the corresponding substitutions in polypeptide, the farther

apart were the mutational sites in the map units. Hence, Yanofsky was showed that there is

colinearity between the sequence of the gene and that of the respective polypeptide. Fig.13a

and fig.13b shows the complete set of data.

Fig.13a. Simplified representation of the colinearity of gene mutations.

Fig.13b. Actual colinearity shown in the A protein of tryptophan synthetase from E.

coli. There is a linear correlation between the mutational sites and the altered amino

acid residues.

Genes and cellular metabolism:

Genetic diseases

By thinking the enzyme activity in terms of cellular metabolism, we realize that inactivation of

any enzyme leads to staggering results. We have seen the metabolic charts on laboratory walls

that shows myriad interlocking, branched and circular pathway along which the chemical

intermediates form a specific enzyme. Every arrow on the metabolic chart is controlled by an

enzyme and each enzyme is produced under the instructions of the gene that specifies its

function. By changing on one critical gene the entire assembly can break down.

Human provides many such examples. The list in the table.1 gives some representative examples

and tells us that how much genetic involvement is there in human disease.

Table 1.Representative Examples of Enzymopathies: Inherited Disorders in Which Altered

Activity (Usually Deficiency) of a Specific Enzyme Has Been Demonstrated in Humans

Condition Enzyme with deficient

activity*

Condition Enzyme with deficient

activity*

Acatalasia

Acid phosphatase

deficiency

Albinism

Catalase

Acid phosphatase

Tyrosinase

Granulomatous disease Reduced nicotinamide adenine dinucleotide

phosphate (NADPH) oxidase

Aldosterone

deficiency

Alkaptonuria

18-

Hydroxydehydrogenase

Homogentisic acid

Hydroxyprolinemia

Homogentisic acid

Hydroxyproline oxidase

Lysine-ketoglutarate

reductase

Angiokeratoma, diffuse (Fabry

disease)

Ceramide trihexosidase Hypophosphatasia Immunodeficiency

disease

Alkaline phosphatase Adenosine deaminase

Condition Enzyme with deficient

activity*

Condition Enzyme with deficient

activity*

Apnea, drug-induced Argininemia

Pseudocholinesterase Uridine monophosphate

kinase

Argininosuccinic

aciduria

Ataxia, intermittent

Argininosuccinase

Pyruvate decarboxylase

Krabbe disease Galactosylceramide β-

galactosidase

Citrullinemia Argininosuccinic acid synthetase

Leigh necrotizing encephalomyelopathy

Pyruvate carboxylase

Crigler-Najjar

syndrome

Glucuronyl transferase Maple-sugar urine

disease

Keto acid decarboxylase

Cystathioninuria Cystathionase Niemann-Pick disease Sphingomyelinase

Ehlers-Danlos syndrome, type V

Lysyl oxidase Ornithinemia Ornithine ketoacid aminotransferase

Farber lipogranulomatosis

Galactosemia

Ceramidase

Galactose 1-phosphate

uridyl transferase

Pentosuria

Phenylketonuria

Xylitol dehydrogenase (L-xylulose reductase)

Phenylalanine hydroxylase

Gaucher disease Glucocerebrosidase Tay-Sachs disease Hexosaminidase A

Condition Enzyme with deficient

activity*

Condition Enzyme with deficient

activity*

Gout Hypoxanthine-guanine

transferase

Phosphoribosyl pyrophosphate (PRPP) synthetase (increased activity)

Wolman disease Xeroderma pigmentosum

Acid lipase

Ultraviolet-specific

Fig.14 shows a corner of metabolic map to show that how a set of disease can be a cause of the

blockage of adjacent step in the biosynthetic pathway.

Fig.14. One small part of the human metabolic map, showing the consequences of various

specific enzyme failures.

GENETIC OBSERVATION EXPLAINED BY THE ENZYME

STRUCTURE:

By understanding the gene-protein relationship and how does enzyme function, we can re-

examine some of the genetic findings in terms of biochemistry involved.

Temperature sensitive alleles are a good example. Some mutants appear to be wild type at

normal temperature but can be mutant at high and low temperature. Now it is clear that such

mutation are the result of the substitution of amino acid that produce a protein that is functional

at normal temperature, called permissive temperatures, but become distort and unable to perform

normal function at low or high temperature, called restrictive temperatures. (Fig.15)

Conditional mutations such as temperature sensitive mutations are very useful for geneticists.

Mutant cultures can easily be maintained under permissive conditions, and the mutant phenotype

can easily be studied under restrictive conditions. Such auxotrophs can be very useful in the

genetic dissection of biological system. Just for example due to a temperature sensitive allele,

various times we can shift to a restrictive temperature during development in order to determine

the time of activation of a gene.

Fig.15. illustration of protein conformational distortion

3. Genetic fine structure

Till 1950s, we regard the chromosome as a linear (one dimensional) array of gene strung like

beads on unfastened necklace, based on our cytological and genetic analysis. This model is

sometimes called bead theory. According to this theory, the existence of a gene as a unit of

inheritance is recognized through its mutant alleles. All of these alleles affect a single phenotypic

character, all map to one chromosome locus, all give mutant phenotypes when paired, and all

shows Mendelian ratios when inter crossed. Here are given some of the points of the bead theory

which are worth emphasizing.

BEAD THEORY

Gene is fundamental unit of structure, change and function.

1. Structure: gene is indivisible by crossing over. Crossing over always occurs between the genes but never within them.

2. Function: gene is the fundamental unit of function. Parts of gene cannot function. 3. Change: gene is also treated as a fundamental unit of change or mutation. It changes

from one allelic form to another. There are no smaller components within it that can be changed.

Seymour Benzer in 1950s showed that bead theory was not correct. Benzer was able to use

genetic system in which extremely small level of recombination could be detected. He

demonstrated that gene can be defined as a unit of function; a gene can be subdivided into a

linear array of sites that are mutable and can be recombined. The smallest units of mutation and

recombination are now known to be correlated with single nucleotide pairs.

The rII system

Seymour Benzer gave a most refined analysis of a single gene for a locus in T4 bacteriophage

that infects E.coli. This locus is known as rII locus and at this locus mutant is responsible for the

formation of rough plaques or colonies. This locus has the largest number of rapid lysing

mutants, and is called rII locus. It can be differentiated by other loci to produce plaques on

lysogenic K strain of E.coli. which carries X prophage. Though the rII mutants may infect may

infect the K strain but cannot cause lysis and therefore are unable to produce plaques.

On the other hand, rII mutants make sharp plaques on E. coli, strain B. the T4 (rII+) wild type

phage will make small and fuzzy plaques on both strains (B & K). Moreover, when K was

infected simultaneously by rII+ and rII. Large plaques were formed, as rII+ helps in lysis so that

rII may express. These features made the Benzer able to identify mutants and wild type phage

with high efficiency.

Destruction of bead theory

With the help of deletion mapping, Benzer was able to map an extraordinary number of

mutations in the rII locus against each other. He showed in his experiments that mutations in the

same gene can recombine with one another. This was against the bead theory of classical

genetics that said that recombination could occur between the genes but not within the gene.

Benzer’s analysis of fine structure of the gene shows that each gene consists of linear array

of sub elements and that these sites within a gene can be altered by mutation and can undergo

recombination. This finding is also against the bead theory, which said that gene as a whole is

mutable, not part of genes. Same work done by several other investigators identified that each

genetic site is a base pair in double stranded DNA. Therefore, Benzer’s contribution bridged the

gulf between classical genetics and the knowledge of the chemical structure of DNA revealed by

Watson and Crick. According to the bead theory, the Watson-Crick structure made no sense.

However, Benzer’s demonstration that gene do indeed have fine structures that can be revealed

solely by genetic analysis allowed a fusion of the two disciplines and helped to launch the

modern era of molecular genetics.

Fig.16. Fine-structure analysis of the rII locus. This mapping technique localizes the position of

a given mutation in progressively smaller segments of the DNA molecule contained in phage T4.

The rII region represents only a few percent of the entire molecule (top). The mapping is done by

crossing an unknown mutant with reference mutants having deletions (darker red) of known

extent in the rII region. Each site represents the smallest mutable unit in the DNA molecule, a

single base pair. The molecular segment (extreme bottom), estimated to be roughly in proper

scale, contains a total of about 40 base pairs.

Reference:

An introduction to genetic analysis, 5 th edition, Griffiths AJF, Miller JH, Suzuki

DT, et al. New York: W. H. Freeman; 2000.