This document discusses gene function and the relationship between genes and proteins. It begins by explaining the one-gene-one-enzyme hypothesis proposed by Beadle and Tatum, which demonstrated that mutations in specific genes resulted in defects in single enzymes. The document then discusses protein structure and how amino acid sequences encode higher-level structures. It explores how gene mutations can result in single amino acid changes in proteins. Finally, it discusses how genes control cellular metabolism and provides examples of genetic diseases caused by enzyme deficiencies.

1. 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

2. Contents
1. How does gene work?
 Gene
 One-gene-one-enzyme hypothesis
2. Gene-proteinrelationship
 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

3. 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.

4.  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.

5. 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)

6. 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.

7. 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)

8. 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.

9. 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)

11.  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.

12. 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.

13. 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.

14. 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

15. 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.

16.  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 BeenDemonstrated 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

17. 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

18. 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.

19. 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

20. 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.

21.  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.

22. 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.

23. Reference:
 An introduction to genetic analysis, 5th edition, Griffiths AJF, Miller JH, Suzuki
DT, et al. New York: W. H. Freeman; 2000.