Pre-Medical Sex Determination

Pre-Medical
SEX D ET ERMINAT ION

Establishment of sex through differential development in an individual at an early stage of life, is called sex
determination. There are different methods for sex determination in organisms like environmental, non-allosomic
genetic determination, allosomic sex determination and haplodiploidy.
Sex Determination on the basis of fertilization.
Three types –
1. Progamic – Sex is determined before fertilization.
eg. - drone in honey bee
2. Syngamic - Sex is determined during fertilization.
eg. - most of plants & animals
3. Epigamic - Sex is determined after fertilization.
eg. - Female in honey bee.
Mechanism of sex determination :
[1] Allosomic determination of sex –
Chromosomes are of two types -
(a) Autosomes or somatic chromosomes -
These regulate somatic characters.
(b) Allosomes or Heterosomes or Sex chromosomes -
These chromosomes are associated with sex determination. Term "Allosome" & "Heterosome" were given
by M o ntg o mer y.
Sex chromosomes first discovered by "Mc Clung" in grass hopper
X- Chromosome discovered by "Henking" and called 'x-body'.
Wilson & Stevens proposed chromosomal theory for sex determination.
(1) XX - XY type or Lygaeus type : - This type of sex determination first observed by Wilson & Stevens
in Lygaeus insect. Two types–
(a) XX female and XY male :- In this type of sex determination female is Homogametic i.e produces only
one type of gamete
A+X
2A + XX (Female)  gametes
A+X
– Male is heterogametic (male produces two types of gamete)
A + X NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2
2A + XY(Male)  gametes
A + Y
In male X-chromosome containing gametes is called "Gynosperm" and Y- chromosome containing

gamete is called " Andros perm" .
eg. Man and dioecious plants like Coccinea, Melandrium
(b) XY female and XX male or ZW female and ZZ male :- In this type of sex determination female is
Heterogametic i.e produces two types of gamete and male individual is homogametic i.e produces one
type of gamete.
It is found in some insects like butter flies, moths and vertebrates like birds, fishes and reptiles.
In plant kingdom this type of sex determination is found in Fragaria elatior.
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Biology
(2) XX female and XO male :- or "Protenor type" :- In this type of sex deternination deficiency of one
chromosome in male. In this type, female is homogametic and male is heterogametic.
A + X

Female (2A + XX)  homogametic
A+X 
A + O 
Male (2A + XO)  heterogametic
A + X 
Example :–
– Grass hopper
– Squash bug Anasa
– Cockroach
– Ascaris and in plants like - Dioscorea sinuta & Vallisneria spiralis
Haploid - diploid mechanism –

In insects of order Hymenoptera which includes ants,honey bees, wasps etc.
Sex determination takes place by sets of chromosomes.
Diploid (two sets)  Female
Haploid (One set)  Male

In honey bee, male individual (Drone) develops from unfertilized eggs (Haploid). Male is always parthenote.
Queen and worker bees develop from diploid eggs i.e. fertilized egg.
Feeds on Royal jelly

Queen
(Fertile female)
Fertilized egg  diploid larva
(2n)
Worker
Bee bread (Sterile female)
[2] Genic balance theory :- C.B. Bridges proposed genic balance theory for sex determination in Drosophila.
– According to Bridges in Drosophila Y-chromosome is heterochromatic so it is not active in sex determination
In Drosophila sex determination takes place by sex index ratio.
No. of x chromosomes X
Sex index ratio= 
No. of set of Autosomes A
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In Drosophila gene of femaleness (Sxl- gene) (Sxl=Sex lethal gene) is located on x-chromosome and gene of
maleness is located on autosome
Gene of male fertility is located on y-chromosome and in Drosophila, y-chromosome plays additional role in
spermatogenesis and development of male reproductive organ, so y-chromosome is essential for the production
of fertile male.
X
Sex index ratio (a) = 1  female (2A + XX), (3A + XXX)
A
(2A + XY) = Fertile male
X
(b) = 0.5  male
A
(2A + XO) = Sterile male
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X
(c) = 1.5  Super female or meta female (sterile) (2A + XXX)
A
X
(d) = less than 0.5  Super male or meta male (Sterile) (3A + XY)
A
X
(e) = In between 0.5 and 1  Intersex (Sterile) (3A+XX)
A
[3] Environmental Determination of Sex. It is non-genetic determination of sex which is based purely on envi-
ronmental conditions. The organisms are potentially hermaphrodite and capable of expressing any of the two
sexes. Some examples are as follows –
1. In marine worm Bonellia, larva develops into female if it settles down alone in an isolated place. Any
larva coming in contact with the already grown female, it changes into male, and lives as a parasite in the
uterus of female.
2. Crepidula (marine mollusca) where larva develops into male in the company of female and develops
into female if left alone.
3. In crocodiles low temperature induces femaleness and high temperature maleness.
4. ln turtles temperature below 28°C induces maleness, above 33°C femaleness while between 28
- 33°C equal number of male and female animals are formed.
5. In marine fish Medusa sex changes according to environmental condition, becoming male in cold water
and female in warm water.
[4] Sex determination by Hormone –

Dizygotic twins are common in cattle like cow, sheep, goat etc. Some times the placentae of the two dizygotic
twins fuse forming blood vascular connections between two developing foetus. If twins are dizygotic, one foetus
may be male and the other female.
– Male hormone produced before female hormone by male twins which suppresses the differentiation of
female internal sex organ. Such a sterile female with Under developed ovaries, oviducts, Uterous etc. is called
free martin.
In free martin conditions, female is sterile & male is normal.
Gynandromorph –
Body of some Drosophila has some cells with male genotype (X0) and some cells with female genotype (XX).
Body of such type of Drosophila has half lateral part of male and half lateral part of female and it is called NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2
bilateral gynandromorph. It is formed due to loss of one x-chromosome at metaphase plate during first
zygotic division. Formation of gynandromorph is the best evidence that y-chromosome does not play any role in
sex differentiation.
this X chromosome will be lost
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Biology
Cytological bas is of sex determination –
Barr body technique or Lyon's hypothesis -
Interphasic nucleus of human female contains two X- chromosomes. Out of two, one X- chromosome becomes
heterochromatin and other X- chromosome is euchromatin. By staining X- heterochromatin, it appears as a
dense body which is called Barr body. (Facultative hetrochromatin)
No. of Barr body  (No. of X chromosomes – 1)
So in a Normal female (2A + XX)  One Barr body
Normal male (2A + XY)  Barr body absent
Turner syndrome (Sterile female) (2A + XO)  No. Barr body
Klinefelter syndrom (Sterile male)(2A + XXY)  One Barr body
Drum stick which occurs in blood of female of mammals, is also a type of barr body. Drum stick is absent in
neutrophils of Male.
Sex determination in human –

There occur a special gene on differential region of Y-chromosome of human, called Sry - gene (Sex determine
region on y chromosome ). This gene forms a proteinaceous factor called TDF (testes determining factor). TDF
responsible for the development of male reproductive organs. So presence and absence of Y- chromosome
determines sex.
Sex determination in plant –

H.E. Warmke discovered sex determination in Melandrium plant.
In Melandrium Y- chromosome is long as compare to X- chromosome.
In plant sex chromosomes are found only in unisexual plant.
Pro. R.P. Roy gave the importance of Y-chromosome in plant.
He discovered sex determination in Coccinea indica (Family- cucurbitaceae)
PHENOT YPIC EXPRESSION IN HAPLOID ORGANISMS

(Neurospora Genet ic s)
Diploid organisms such as pea and Drosophila, have two alleles for each gene (the exceptions are for the X
linked genes in XY or XO males). With the result, the recessive allele is not expressed in the phenotype in
presence of the dominant one. However, this is not so in the case of haploid organisms. Contrary to diploid
organisms, the genetics of haploid organisms exhibit the following features:
1. Haploid organisms contain only one allele of a gene, so there is no complication of dominance. All the
genes, whether dominant or recessive, expresses itself in the offsprings.
2. In absence of dominance, any new mutation is immediately expressed in the phenotype, in haploid
organisms.
3. Study of inheritance of the mutated gene, its linkage, crossing over and biochemical consequence of a
mutation can easily be studied in haploid.
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Haploid parent A x a AB x ab
Diploid zygote Aa AaBb
(Zygotic meiosis)
Haploid offspring A a AB A b aB ab
Linkage And Rec ombination in Neuros pora (Drosophila of plant kindgom)

Detection of linkage and recombination of genes in haploid organisms as in fungi, bacteria etc. is comparatively
simple. Fungus Neurospora is one of the favourite material with geneticists, because :-
1. The life cycle of Neurospora is the product of a single meiosis.
2. The life cycle is of a short duration.
3. The meiotic products are linearly arranged in ascus as 8 ascospores as ordered tetrads (i.e, the eight
ascospores are arranged in the same order in which chromatids were on the meiotic metaphase plate).
Tetrad Analysis in Ordered Tetrads –

In Neurospora , the nuclei from hyphae of opposite mating type (+) and (–) fuse to form a diploid zygote. The
zygote is the only diploid stage in the life cycle of Neurospora . The zygote nucleus divides meiotically producing
four haploid nuclei, each of which then undergoes mitosis. The eight cells produced this way, form 8 haploid
ascospores enclosed in the ascus. The three divisions proceed along the longitudinal axis, so the ascospores are
arranged in a line in a specific order that indicates the order of arrangement of chromatids on the meiotic
metaphase plate. This is called linear or ordered tetrad. Each of the four products of meiosis can be cultured
separately to study their phenotypes and genotypes. This is called tetrad analysis.
Meiosis - I Meiosis - II Mitosis

  
2n
diploid
4-haploid POM
POM = Products of
8-ascospores
meiosis
(Ordered Tetrad) NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2
1. Firs t Divis ion Seg regation Between Centromere and gene-a.

A cross between two strain of Neurospora , one normal (a+) and other mutant (a) strain produces 8-ascospores,
out of which four are normal (a+) and other four mutants (a). The linear arrangement of ascospores in ascus is
4a+ : 4a. It indicates the absence of crossing over between locus-a and centromere. This is described as first
division segregation.
2. Second Division Segregation Between Centromere and Gene-a .

In a similar cross if crossing over takes place leading to paired arrangement of ascospores with a particular
gene, it is described as second division crossing over. The arrangement of ascospores in the sequence ( 2 :
2 : 2 : 2) is as follows :
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(i) a+ : a + : a : a : a : a : a + : a +
(i i) a : a : a + : a+ : a+ : a+ : a : a
(ii i) a+ : a + : a : a : a + : a + : a : a
a+
a+
a+
a+
a
a
a
First division segregation (4a +; 4a) a
a+
a+
a
a
a+
a+
a
Second division segregation (2a + : 2a : 2a + : 2a)
a
Single Gene Mapping in Neurospora
In Neurospora centromere behaves as a gene for mapping gene pair. In such a case distance of gene from the
centromere is calculated by calculating the percentage of cross overs between centromere and gene.
Que. If 10% asci show crossing over in ascocarp what will be distance between gene and centromere.
If total 100 asci are present in a Neurospora
90 non crossing over type
10 0
10 crossing over type
1 asci is derivative of 4 chromatids
100 asci are derivative of 400 chromatids = total chromatids
10 asci are derivative of 40 chromatids
(Out of 40 only 20 will be the recombinant type)
recombinant chromatid
% C.O. =  100
Total chromatid
20
=  100  5% Ans. 5 centi Morgan
400
GENE EXPRESSION
The second important characteristic (first is transmission) of the gene is to store and express the genetic informa-
tion that will contribute towards the phenotype.
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DNA carries information for the synthesis of all proteins required for the function of a cell. A close relationship
between genes and enzymes (or gene control the metabolism) was first discovered by a British physician Archibald
Garrod in 1909. He observed that certain hereditary diseases in man such as alkaptonuria (black urine
disease), albinism (absence of melanin pigment) phenylketonuria etc. are ‘inborn errors of metabolism and
are due to the defect in the enzyme that catalyses the conversion of one metabolic substance to another. Thus,
the inherited genetic defect is reflected in the deficiency of an enzyme.
Beadle and Tatum’s Hypothe sis (One gene one enzyme hypothe sis)
The concept that genes have the information to produce enzymes, or gene metabolism relationship was
experimentaly proved by Beadle and Tatum (1948), on the basis of experiments conducted on pink bread
mould (Neurospora crassa) . This mould can normally grow in a simple minimal medium containing salts and
sugar, making all other chemicals such as amino acids, purines, pyrimidines etc. through enzyme catalysed
reactions. This wild type of the mould is called 'prototroph'. Beadle and Tatum exposed the pink bread mould
to X-rays, which can bring about a change in the nucleotide sequence of DNA and thus causes mutation. They
found that the mutants were unable to grow on a minimal medium. Each type of mutant required some extra
nutrient in the minimal medium for it's normal growth. Such nutritional mutants are called 'auxotrophs'. They
obtained different nutritional mutants requiring amino acids ornithine or citrulline or arginine for growth. The
mutants could be classified into three types:
(i) some could grow on ornithine -, or citrulline -, or arginine - containing medium,
(ii) some could grow on citrulline - or arginine containing medium, and
(iii) some could grow only on arginine supplemented medium
Effect of medium supplements on the growth of mutants Neurospora c rassa

Growth on Medium supplemented with
Mutant Type Ornithine Citrulline Arginine
I + + +
II – + +
III – – +
+ = growth, – = no growth.
Biochemical Pathway
Precursor  Ornithine  Citrulline  Arginine
encoded enzyme Enzyme A Enzyme B Enzyme C
Location of gene Gene A Gene B Gene C

on chromosome
– This shows that all mutants could grow on arginine supplemented medium suggesting that arginine is the final
product of this pathway.
– Since some mutants could not grow on ornithine, this compound must be synthesized earlier than citrulline and/
or arginine.
– Beadle and Tatum taking clue from this growth behaviour of Neurospora and other experiments, proposed the
arginine biosynthetic pathway. The precursor compound first gives rise to ornithine, ornithine then gives
rise to citrulline and the latter is finally converted into arginine; each of these steps is mediated by an enzyme.
– The mutant of class I, could grow on ornithine, citrulline or arginine, suggesting that they lacked the capacity
to synthesize ornithine, but beyond that they could complete the pathway.
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– Similarly, mutants of class II, could not convert ornithine to citrulline, but if supplied citrulline could synthesize
arginine.
– The last mutants of class III could not convert citrulline to arginine and had to be supplied latter for growth.
– Beadle and Tatum reasoned that these defects could arise due to defective enzymes in each case. Since such
changes were mutational, they held that one gene controls one enzyme in a pathway leading to their famous
‘one gene one enzyme hypothesis’.
The hypothesis states that each gene controls the synthesis of a specific enzyme or protein. Beadle and Tatum
were awarded with Noble prize for this work in 1958. Their work founded the new science of ‘biochemical
genet ics’. Beadle and Tatum used total isolat ion method to detect the nutritional mutant of Neurospora.
Modification of Beadle and Tatum’s Hypothesis - One gene one enzyme hypothesis held that a gene has
information to produce one enzyme. The enzymes are proteins, but all proteins are not enzymes. Recently some
RNAs have also been found to manifest enzyme activity. Proteins are complex molecules and may be composed
of one or more polypetide chains. For example, haemoglobin consists of four polypetide chains-2  and 2 
chains. Thus, more than one gene may control the synthesis of a protein. It is now held that one gene is
responsible for the formation of one polypetide chain. Therefore, one gene one enzyme hypothesis has been
replaced by ‘one gene one polypetide hypothesis’. Further modification came when one gene was identified
as a functional unit or cistron and the same was called ‘one cistron-one polypeptide hypothesis’.
TYPES OF GENE
All the genes do not play the same role nor all genes are active all the time. With regard to their role and
activity, the genes are of following types:
(1) Jumping genes –
It is a segment of DNA which moves from one chromosome to another chromosome wihtin the genome of an
indivisual. McClintock (1983) got nobel prize for the discovery of jumping gene in maize. Two transposable
controling elements (Ds) and activator (Ac), which can jump to any chromosome from their original location on
chromosome 9. Also in bacteria plasmid transposone carry gene for antibiotic resistance (ampicilline).
Other Examples :
Transposable elements (TE) in Drosophila– As much as 10% of the genome consist of transposons, most impor-
tant of these are copia like element, Fold back (FB) (for eye colour), and P and I element ( for sterlity).
Ty - element in yeast.
(2) Overlapping gene – A few genes in some animal viruses code for two different polypeptides. These are called
overlapping genes. For example–in  x 174 virus, SV-40 virus.
(3) Constitutive genes (House-keeping genes) – These genes are expressed constantly, because their products
are constant needed for cellular activity. e.g. genes for glycolysis, gene of ATPase enzyme.
(4) Non-constitutive genes (Smart gene or Luxary gene) – These genes remain silent and are expressed only
when the gene product is needed. They are switched ‘on’ or ‘off’ according to the requirement of cellular
activities. Non-constitutive genes are of two types; inducible and repressible. The inducible genes are switched-
on in presence of a chemical substance called inducer, required for the functioning of gene activity. The
repressible genes continue to express themselves till a chemical, often an end product of the metabolism inhibits
or represses their activity. Such type of inhibition is called feed back inhibition or feed back repression.
(5) Homeotic genes – Homeotic gene regulates the organ differentiation in embryo.
Homeobox related to transcription of homeotic gene.
If mutation takes place in homoetic gene organ formation is disturbed.
(6) Pseudoallele – Genes lying side by side, producing related phenotypic effect distinguishable through a rare
crossing over. Eg. star (dominant) and asteroid (recessive) traits in Drosophila.
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(7) Isoallele – If several alleles exhibit same phenotype then they are said to Isoallele.
In Drosophila allele W+c W +s W+g  produce red eye colour
REGUL AT ION OF GENE EXPRERSION

– The mechanism which stimulates the expression of certain genes and inhibits that of others is called regulation
of gene expression.
– It is possible only if the organism has a mechanism of regulating gene activity by allowing some to function and
others to restrain their activity through switching on and switching off system. This means, the genes are turned
‘on’ or ‘off’ as per requirement.
– A set of genes is ‘switched on’ when enzymes are required to metabolise a new substrate. The enzymes produced
by these genes metabolise the substrate.
– The molecules of metabolite that come to switch on of the genes are termed as inducers and the phenomenon
is called induction.
– Similarly, certain genes which are in their ‘switch on’ state, continue to synthesise a metabolite till the later is
produced in amount more than required or else, it is supplied to the cell from outside. In other words, certain
genes continue to express themselves till the end product of inhibits or repress their expression. Inhibition by an
end product is known as ‘feed back repression’.
OPER ON CONCEP T
– In 1961, two French microbiologist Francis Jocob and Jacques Monad at the Pasteur Institute in Paris,
proposed a mechanism called operon model for the regulation of gene action in E. coli.
– An operon is a part of genetic material or DNA, which acts as a single regulated unit having one or more
structural genes-an operator gene, a promoter gene, a regulator gene.
– Operons are of two types (i) inducible (ii) repressible.
1. Inducible System (Lac operon of E. coli)
– An inducible operon system normally remains in switched off condition and begins to work only when the
substance to be metabolised by it is present in the cell. Inducible operon system generally occurs in catabolic
pathways. e.g. Lac operon of E. coli.
Active repressor + inducer = inactive repressor
RNA–polymerase Structural gene
i  p o 
R.G. P.G. O.G. z y a
   
m–RNA No–Transcription NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2
 No–Translation
Repressor protein
[OPERON – OFF]
RNA–polymeraseStructural gene
  
R.G. P.G. O.G. z y a
    Polycistronic
Repressor protein
m–RNA
+   
–ga lact osidase Permease Transacetylase
Inducer (lactose) [OPERON – ON]
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An inducible operon system consists of four types of genes
(i) Structural genes - These genes synthesise mRNAs, which in turn synthesise polypeptide or enzyme
over the ribosomes. An operon may have one or more structural genes. Each structural gene of an
operon is called cistron. The lac operon (lactose operon) of Escherichia coli contains three structural
genes (Z, Y and A). These genes occur adjacent to each other and thus are linked. They transcribe a
polycistronic mRNA molecule (a single stretch of mRNA covering all the three genes), that helps in the
synthesis of three enzymes- galactosidase (breaks lactose into glucose and galactose), lactose permease
(helps in entry of lactose in cell from outside) and transacetylase (transfers an acetyl group from acetyl
Co A to  galactosidase).
(i i) Operator gene - It lies adjacent to the structural genes and directly controls the synthesis of mRNA over
the structural genes. It is switched off by the presence of a repressor. An inducer can take away the
repressor and switch on the gene that directs the structural genes to transcribe.
(ii i) Promoter gene - This gene is the site for initial binding of RNA polymerase. When the operator gene is
turned on, the enzyme RNA polymerase moves over it and reaches the structural genes to perform
transcription.
(i v) Regulator gene - It produces a repressor that binds to operator gene and stops the working of the
operator gene.
(v) Repressor - It is a protein, produced by the regulator gene. It binds to the operator gene so that the
transcription of structural gene stops. Repressor has two allosteric site (1) operator gene (2) effective
molecule (inducer/corepressor)
(v i) Inducer - It is a chemical (substrate, hormone or some other metabolite) which after coming in contact
with the repressor, forms an inducer repressor complex. This complex cannot bind with the operator
gene, which is thus switched on. The free operator gene allows the structural gene to transcribe mRNA
to synthesise the enzymes.
The inducer for lac operon of Escherichia coli is lactose (in fact allolactose an isomer of lactose). When the
sugar lactose is added to the culture of E. coli, a few molecules of lactose gets into the bacterial cells by the
action of the enzyme permease, a small amount of this enzyme is present in the cell even when the operon is not
working. These few lactose molecules are then converted into an active form which acts as an inducer and binds
to the repressor protein. The inducer repressor complex fails to join with the operator, which is turned on. The
three genes are expressed as three enzymes to metabolise lactose. Allolactose is real inducer of lac operon.
Gratuitous inducer  Some molecules resemble with natural inducer but are not metabolize by the enzyme.
Example : Isopropyl thio galactoside (IPTG) : This resembles lactose and thus has the property of induction.
Such inducer which induces enzyme synthesis, without getting metabolized are called gratuitous inducer.
2. Repre ssible System (Tr yptophan operon of E. coli.)
– A repressible operon system is normally in it's switch on state and continue to synthesise a metabolise till the
latter is produced in amount more than required, or else it becomes available to the cell from outside. Repressible
operon system is commonly found in anabolic pathway. e.g. Tryptophan operon of E. coli.
[Inactive repressor + co-repressor = acitve repressor]
Tryptophan operon of Escherichia coli is an example of repressible system. It consists of the following:
(i) Structural genes. These genes are meant for transcription of mRNA, which in turn synthesise enzyme.
Tryptophan operon has five structural genes E, D, C, B and A. They lie in continuation and synthesise
enzymes for five steps of tryptophan synthesis.
(ii) Operater gene (trp O). It lies adjacent to the structural genes and controls the functioning of the
structural genes. Normally, it is kept switched on, because the apo-repressor produced by the regulator
gene does not bind to it. The operator gene is switched off when a co-repressor is available alongwith
apo-repressor.
(iii) Promoter gene (trp P). It marks the site at which the RNA polymerase enzyme binds. When the
operator gene is switched on, it moves from promotor gene to structural genes for transcription.
(iv) Regulator gene (trp R). It produces a regulatory protein called apo-repressor for (Inactive repressor)
possible blocking the activity of operator gene.
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(v) Apo-repressor. It is a regulatory protein synthesised by regulator gene. When a co-repressor substrate
is available in the cell, the apo-repressor combines with the co-repressor to form a apo-repressor co-
repressor complex. This complex binds with the operator gene and switches it off. Presence of apo-
repressor alone, the operator gene is kept switched on because, by itself the apo-repressor is unable to
block the working of operator gene.
(vi) Co-repressor. It is an end product of reactions catalysed by enzymes produced by the structural genes.
In the presence of tryptophan some molecules of tryptophan act as co-repressor, co-repressor-bind with
inactive repressor. co-repre ssor repre ssor complex bind with-operator region and prevent the binding of
RNA polymerase to the promoter, the trp-operon is off.
Structural
 gene

R.G. P.G. O.G. E D C B A
      Transcription
Polycistronic m -RNA
Aporepressor

     Translation
(inactive repressor) Polypeptides
[OPERON – ON] E1 E2 E3 Enzymes

Chorismic acid Tryptophan
Structural gene

R.G. P.G. O.G. E D C B A
     
Aporepressor No–Transcription
+ No–Translation
Co–repressor
(Tryptophan)
[OPERON – OFF]
Differenc es between Induc tion and R epression
Induc tion Repression

1. It is switching-on of an operon which 1. It is switching-off an operon which
is normally in switched-off state. is normally in switched-on state: NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2
2. It starts transcription and translation. 2. It stops transcription and translation.
3. It is caused by a new metabolite which 3. It is caused by increased formation
needs enzymes to get metabolised. or availability of a metabolite.
4. It generally operates in a catabolic 4. It generally operates in an
pathway. anabolic pathway.
The repressor molecule has key role in regulation of lac-operon. Repressor molecule active or inactive.
Active repressor may be rendered inactive by addition of an inducer while the inactive repressor can be made
active by addition of a co-repressor.
34 E
Biology
Because the product of regulator gene the repressor act by shutling off the transcripition of structural gene the
operon model, as originally proposed by Jocob & Monad is referred as –negative control system.
GENE EXPRESSION IN EUK ARYOT ES

1. In eukaryotes, functionally related genes may not be clustered together constituting an operon.
2. The most popular model is known as ‘Britten-Davidson model’ or ‘Gene-battery model’ proposed by
Britten and Davidson in 1969.
3. A set of structural genes controlled by one sensor site is termed as battery.
4. Gene-battery model assumes the presence of four classes of sequences:
(i) Producer gene: A producer gene is comparable to structural gene of prokaryotic operon.
(ii) Receptor site: A receptor site is comparable to operater gene of bacterial operon and one such
receptor site is assumed to be present adjacent to each producer gene.
(iii) Integrator gene: Integrator gene is comparable to regulator gene and is responsible for synthesis of
an activator RNA. It activates the receptor site.
(iv) Sensor site: A sensor site regulates the activity of integrator gene. Activator gene can be transcribed
only when the sensor site is activated.
The sensor sites are recognized by agents which change the patterns of gene expression like hormones and
proteins. When a transcription factor (protein, hormone) bind to the sensor site it cause the transcription of
integrator.
Activator RNA mRNA
regulator gene) (Operater gene) (Structural gene)
Sensor site Integrator gene Receptor site Producer gene
Britten-Davids on model or Gene-battery model
HUMAN GENETICS
The study (analysis) of genetic characters and aspects like genetic improvements among humans are included in
human genetics. This is also known as eugenics (=well born).
Eugenics is a term derived from Greek language 'Eugenes' meaning "Well born". First of all, Sir Francis
Galton, 1883 proposed the idea of improvement in human species through change in hereditary characters in
a scientific manner and named it Eugenics. Because of this Sir Francis Galton is known as "father of Eugenics".
For improvement of hereditary characters in future generations there should be an increase in number of
best individuals and reduction in number of defective individuals. Presently, eugenics is studied under
following types -
1. P os itive Eug enic s
In positive eugenics the main approach is to increase the number of progenies having best hereditary characters.
This is achieved through genetic counselling, selective mating (planned marriages), arranging marriages at right
age, freedom from social bonds etc.
Preservation of ovum of superior quality, artifical insemination and preserving the best quality sperms with aid
of modern techniques are also included in positive eugenics.
Euphenics and gene engineering techniques are also being used to establish the best quality of hereditary traits
in human society.
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2. Neg ative Eug enic s
In negative eugenics the carriers of undesirable inferior traits (or characters or genes) are not allowed to produce
their progenies by various checks such as marriage control, segregation, birth control, sterilization and control of
consanguineous marriages.
To find out various facts, scientists have to perform a number of experiments, but it is not possible to do so in humans.
The folowing problems are faced in studying human genetics -
1. Cells of human body are relatively smaller and number of chromosomes present in them is more.
2. It is not possible to perform various experiments on humans in the laboratory.
3. Due to greater life span of humans, a lot of time is required to study their genetic characteristics.
4. Rate of reproduction is slow in humans.
5. Individuals of human species are generally heterozygous for various characters and it is very difficult to
find homozygous individuals.
6. Due to controlled hybridization and long lived life among humans, it is not possible to study many generations
in easy way.
Despite of above mentioned problems humans are considered suitable for genetical experiments due to-
1. Longer life span of humans, the abnormalities that require relatively long period to express themselves
can be easily studied.
2. By pedigree study and analysis of families, many genetic characters of man can be traced out.
3. To study many diseases (like haemophilia, colour blindness etc.) and intelligence quotient (I.Q.) humans
are more preferable than any other organisms.
Examples of some autosomal c haracters in human
Cha racter Domina nt R e c e s s iv e
Eye colour Brown / Black Blue
Eye size Large Small
Ear lobes Free Fused
Hair Curly hair Straight
Cheek Dimple cheek Normal
Hand Right Handed Left handed
Rolling of tongue Rollar Non Rollar
Rh Factor Rh + Rh—
PTC (Phenyl thiocarbamide) taster Taster Non Taster
Skin pigmentation Normal Albino

Devices Used in Human Genetical Studies
The study and analysis of human genetics is performed by many methods like pedigree analysis, statistical
analysis and human karyotyping. Of these the important ones, that is, pedigree analysis and human karyotype
is being described here.
1. P edig ree Analys is
Study of ancestoral history of man of transmission of genetic characters from one generation to next, is pedigree
analysis. Dwarfism, albinism, colour blindness, haemophilia etc. are genetically transmitted characters. To study
and analyse them a pedigree of genetic facts/data and following symbols are used.
36 E
Biology
Symbol used in Pedigree :
1. – Normal Male
2. – Normal Female
3. – Mating (marriage)
4. The siblings are indicated in chronological order of birth
5. Sex unspecified
6. Twin
If monozygotic
If dizygotic
7. , Affected male and female individual
8. , Heterozygous for autosomal recessive
9. Carrier female of sex linked recessive character or disease
10. Death of individual
11. Abortion or still birth (sex unspecified)

12. Consanguineous marriage
13. Parent with male child affected with disease
14. 5 – Five unaffected offsprings
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Pedigree analysis provides valuable informations regarding genetical make up of human beings. If any genetic
disease is occuring in a family, then pedigree analysis provides guidance to forthcoming parents about their
future progenies for example- polydactyly in humans.
Example : Given pedigree shows inheritance of albinism which is an autosumal recessive disorder. If 4th
individual is homozygous normal then find out the carrier individual.
1 2
– Normal male
– Normal Femal
4 – Albino male
3
– Albino female
5 6
Ans. : 1, 2, 5, 6 (Aa)
2. Human K aryotype
Humans have 23 pairs (46) chromosomes. In this method, the chromosomes (autosomes and sex chromosomes)
are arranged according to their size and structure. Based on the position of centromere and relative lengths of
both arms of chromosome, three types of chromosomes are found in human - metacentric, submetacentric and
acrocentric. Karyotype helps to know the relative structures (morphology) of chromosomes. Besides, it helps in
chromosomal identification and it's nomenclature. It is also used in studying chromosomal abnormalities.
POPUL AT ION GENET ICS
Study of gene frequency in a population is called population genetics.
1. Gene pool – A gene pool is the sum total of genes in reproductive gametes of a population.
2. Gene-flow – Migration of gene from one population to another population by cross fertilization.
3. Genetic load – The existance within the population of disadvantegeous allele in heterozygous genotype is
known as genetic load
4. Gene frequency – Gene frequency is defined as proportion of different alleles of a gene in a population.
Ex . In a population of 100 individuals of MN blood group 50 MM, 20 MN, 30 NN find out the frequency of
M & N.
MM – 50
MN – 20 NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2
NN – 30
Total M gene– 50 × 2 + 20 = 120
Total N gene – 30 × 2 + 20 = 80
Total gene – 200
M 120
Freq. of M gene P=   0.6
M+ N 200
N 80
Freq. of N gene   0.4
M+ N 200
0.6 + 0.4 = 1
p + q =1
38 E
Biology
Hardy Weinberg Law –
1908 G.H.Hardy (English methematician) & German Physician, W.Weinbergh independently discovered that an
equilibrium is established between frequencies of allele in random mating population and these gene frequency
remain constant from generation to generation.
This law applicable when factors like mutation, selection & migration, are absent.
Hardy Weinberg theorem or Hardy weinbergh law

p2 + 2pq + q2 = 1 (A  p, a  q, p+q = 1)
AA Aa aa
In this equation frequency of A  P or the Frequency of Homozygous dominant will be AA–P2
In this equation frequency of a  q the frequency of homozygous recessive – q2
the frequency of Aa = 2pq
The frequency of different genotype produced due to random mating will depend upon the gene frequency and
equillibrium is stablished after one single generation of random mating.
Factors affecting gene frequency :

(1) Mutation (2) Selection (3) Migration (4) Randon drift
Directional-mutation, selection, migration
Nondirectional = random drift
Nondirctional which can not be predicted from one generation to the other.
1. Mutation : Mutation lead to introduction of new gene leading to genetic difference. Change in gene frequency
due to mutation, will depend upon mutation rate.
2. Selection (Natural selection) : By the natural selection there is increase in frequency that are more fit to
nature.
Selections are associated with superior fitness will increase in frequency in the population.
3. Migration : Migration recessive or dominant allele can be introduced into a population from a nearby popula-
tion.
This causes difference in frequency between the two population.
4. Random drift or Genetic drift.: Allele frequency can change randomly, sometime increasing and sometime
decreasing such deviation in gene frequency will be due to sampling error.
Due to sampling error, random changes in gene frequencies are called random drift.
population  sampling error 

population  sampling error 
Q. In a random population frequency of recessive phenotype is 0.09. What is the frequency of heterozygous
genotype?
S ol . q 2 = 0.09
q = 0.3
p = 1 – q
p = 1 – 0.3  0.7
Frequency of heterozygote = 2pq = 2 × 0.7 × 0.3 = 0.42 = 42%
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GENETIC ENGINEERING
INTRODUCTION :
– Genetic engineering also referred as ‘recombinant DNA technology’ or ‘gene splicing’ is one kind of
biotechnology involving manipulation of DNA.
– It deals with the isolation of useful genes from a variety of sources and the formation of new combinations of
DNA (recombinant DNA) for repair, improvement, perfection and matching of a genotype.
– Thus, genetic engineering may be defined ‘as a technique for artificial and deliberately modifying DNA (gene) to
suit human needs’.
– In genetic engineering breakage of DNA molecule at two desired places is done with the help of restriction
endonuclease to isolate a specific DNA segment and then insert it in another DNA molecule at a desired
position.
– The new DNA molecule is recombinant DNA and the technique called genetic engineering. Genetic engineer-
ing aims at adding, removing or repairing of a part of genetic material. Genetic engineering can be used to
improve the quality of human life.
Paul bergh (Father of genetic engineering). He transferred gene of SV-40 virus (simian virus) in to E.coli
with the help of  – phage. (Nobel prize - 1980)
The concept of genetic engineering was the outcome of two very significant discoveries made in bacterial
research. These were–
(i) presence of extrachromosomal DNA fragments called plasmids in the bacterial cell,which replicate along with
chromosomal DNA of the bacterium.
(ii) presence of enzymes restriction endonucleases which cut DNA at specific sites.
These enzymes are, therefore, called ‘molecular scissors’.
TOOLS AND TECHNIQUES OF GENETIC ENGINEERING

To o l s
Genetic engineering involves cutting of desired segments of DNA and pasting of this D.N.A in a vector to
produce a recombinant DNA (rDNA). The ‘biological tools’ used in the synthesis of recombinant DNA include
enzymes, vehicle or vector DNA, passenger DNA and host cells.
1. Enzymes. A number of specific kinds of enzymes are employed in genetic engineering.
These include lysing enzymes, cleaving enzymes, synthesising enzymes and joining enzymes. NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2
(i) Lysing enzymes. These enzymes are used for opening the cells to get DNA for genetic experiment.
Bacterial cell wall is commonly dissolved with the help of lysozyme.
(ii) Cleaving enzymes. These enzymes are used for DNA molecules. Cleaving enzymes are of three types;
exonucleases, endonucleases and restriction endonucleases.
40 E
Biology
(a) Exonucleases cut off nucleotides from 5' or 3' ends of DNA molecule.
(b) Endonucleases break DNA duplex at any point except the end.
(c) Restriction endonucleases cleave DNA duplex at specific points in such a way that they come to
possess short single stranded free ends. For example, a restriction endonuclease ECOR-l (from Escherichia
coli) recognizes the base sequence GAATTC/CTTAAG in DNA duplex and cleaves it's strands between G
and A.
Restriction enzymes are obtained from bacteria. They are useful to bacteria because the enzyme bring about
fragmentation of viral DNA without affecting the bacterial genome. This is an adaptation against bacteriophages.
Restriction enzyme (Eco R-I) was discovered by Arber, Smith & Nathans (1978 Nobel prize). These enzymes
exist in many bacteria beside cleavage some restriction endonuclease, also have capability of modification.
Modification in the form of methylation, by methylation the bacterial DNA modifies and therefore protects it's
own chromosomal DNA from cleavage by these restriction enzymes.
Restriction enzymes are used in recombinant DNA technology because they can be used in vitro to recognize
and cleave within specefic DNA sequence typically consisting of 4 to 8 nucleotides. This specific 4 to 8 nucle-
otide sequence is called restriction site and is usually palindromic, this means that the DNA sequence is the
same when read in a 5'-3' direction on both DNA strand
 
AND MADAM DNA
As a result the DNA fregments produced by cleavage with these enzymes have short single stranded overhang
at each end these kinds of ends are called sticky or cohesive ends because base pairing between them can stick
the DNA molecule back together again.
5' G A A T T C 3' 5' G 3' 5' A A T T C 3'

  + 
3' C T T A A G 5' 3' C T T A A 5' 3' G 5'
Therefore by cutting two different DNA samples with the same restriction enzyme and mixing the fragments
together a recombinant DNA molecule can be generated.
Exceptionally, some enzymes cleave both strand of DNA at exactly the same nucleotide position, typically in the
center of the recognition sequence resulting in blunt end or flush end.
S ma I (S erratia marc es cens )


5' C C C G G G 3' 5' C C C 3' + 5' G G G 3'

3' G G G C C C 5' 3' G G G 5' + 3' C C C 3'

Nomenclature of enzyme – The first letter used for the enzyme is the first letter of the bacterium genus name
(in Italics) then comes the first two letter of it's species (In Italics), next is the strain of the organism, last is Roman
numerical signifing the order in which the enzymes were isolated from that strain of bacteria.
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EXAMPLES OF RES TRICTION ENZYME
Recognition sequences of some res triction endonucleas es
Name R e c o g ni ti on End after cleavage Sou rce
s e qu en c e


Eco RI –GAATTC– – G AATTC– Escherichia coli - containing drug
–CTTAAG– –CTTAA G– resistant plasmid RI.

 

Hind III –AAGCTT– –A AGCTT– Haemophilus influenzae
–TTCGAA– –TTCGA A–




Bam I –GGATCC– – G GATCC– Bacillus amyloliquefaciens
–CCTAGG– –CCTAG G–




Hae III –GGCC– –GG CC– Haemophilus aegyptius
–CCGG– –CC GG–


(iii) Synthesizing enzymes. These enzymes are used to synthesize new strands of DNA, complementary to
existing DNA or RNA template. They are of two types; reverse transcriptases and DNA polymerases.
(a) Reverse transcriptases help in the synthesis of complementary DNA strands on RNA templates;
(b) DNA polymerases help in the synthesis of complementary DNA strands on DNA templates.
(iv) Joining enzymes. These enzymes help in joining the DNA fragments. For example DNA ligase from
Escherichia coli is used to join DNA fragments. Joining enzymes are, therefore, called molecular glues.
(iv) Alkaline phosphatases. These enzymes cut off phosphate group from the 5' end of linearised circular
DNA and prevent its recircularisation.
2. Vehicle DNA or Vector DNA. The DNA used as a carrier for transferring a fragment of foreign DNA into a
suitable host is called vehicle or vector DNA.
You know that plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the
control of chromosomal DNA.
The following are the features that are required to facilitate cloning into a vector.
(i) Orig in of replic ation (ori) : This is a sequence from where

replication starts and any piece of DNA when linked to this sequence
Ecor I Cla I Hind III
can be made to replicate within the host cells. This sequence is also
Pvu I
responsible for controlling the copy number of the linked DNA. So, BamH I
Pst I
if one wants to recover many copies of the target DNA it should be amp
R R
tet
Sal I
cloned in a vector whose origin support high copy number.
pBR322
(ii) Selectable marker : In addition to 'ori', the vector requires a

ori
selectable marker. Normally, the gene s encoding resistance to rop
antibiotics such as ampicilin, chloramphenicol, tetracycline or

Pvu II
kanamycin, etc., are considered useful selectable markers for E.
coli.
42 E
Biology
(iii) cloning sites : In order to link the alien DNA, the vector needs, recognition sites for the commonly
used restriction enzymes. The ligation of alien DNA is carried out at a restriction site present in one of the
two antibiotic resistance genes. For example, you can ligate a foreign DNA at the Bam H I site of
tetracycline resistance gene in the vecter pBR322. The recombinant plasmids will loss tetracycline resistance
due to insertion of foreign DNA (insertional inactivation) now, it can be selected out from non-recombinant
ones by plating the transformants on ampicillin containing medium. The transformants (plasmid transfer)
growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The
recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, non-
recombinants will grow on the medium containing both the antibiotics. In this case, one antibiotic resistance
gene helps in selecting the transformants.
Selection of recombinants due to inactivation of antibiotics is a cumbersome (troublesome) procedure

because it requires simultaneous plating on two plated having different antibiotics. Therefore, alternative
selectable markers have been developed which differentiate recombinants from non-recombinants on
the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant
DNA is inserted within the coding sequence of an enzyme, which is referred to as insertional inactivation.
The presence of a chromogenic substrate X–gal (5–bromo–4chloro– D galacto pyranoside) gives blue
coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into
insertional inactivation of the  -galactosidase (reporter enzyme) and the colonies do not produce any
colour, these are identified as recombinant colonies.
(iv) Vectors for cloning genes in plants and animals : Agrobacterium tumifaciens, a pathogen of several
dicot plants deliver a piece of DNA known as 'T-DNA' to transform normal plant cells into a tumour .
Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells. A better
understanding of the art of delivering genes by pathogens in their eucaryotic hosts has generated knowledge
to transform these tools of pathogens into useful vectors for delivering genes of interest to humans. The
tumour inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector
which is no more pathogenic to the plants but is still able to use the mechanisms to deliver genes
(disarmed) and are now used to deliver desirable genes into animal cells. So, once a gene or a DNA
fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host
(where it multiplies).
(i) P las mids . They are extra chromosomal DNA segments found in bacteria which can replicate
independently. Plasmids can be taken out of bacteria and made to combine with desired DNA segments
by means of restriction enzymes and DNA ligase. A plasmid carrying DNA of another organism integrated
with it, is known as recombinant plasmid or hybrid plasmid or Chimeric plasmid.
(ii) Viruses. The DNA of certain viruses is also suitable for use as a vehicle DNA. Bacteriophage (bacterial
virus) has been used to transfer gene for  galactosidase from Escherichia coli to human cells. Lambda
phage ( phage) has been used for transferring lac genes of E. coli into haploid callus of tomato.
Vector t ype Ins ert size kb Antibiotic resist-

ance gene Centromere
Plasmid 0.5 – 8
Bacterophage lambda 9 – 23 Ori
Cosmid 30 – 45
BAC 50 – 300
Origin of replication Telomere
YAC 1000 – 2500
(Ori) Plasmid
Yeat Articial
Chromosome (YAC)
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3. Passenger DNA. It is the DNA which is transferred from one organism into another by combining it with the
vehicle DNA. The passenger DNA can be complementary, synthetic or random.
(i) Complementary DNA (cDNA)- It is synthesized on mRNA template with the help of reverse tran-
scriptase and necessary nucleotides. The DNA strand is then separated from the hybrid RNA-DNA
complex by using alkali. Complementary DNA strand is then synthesized over the template of cDNA with
the help of DNA polymerase. cDNA formed through reverse transcription is shorter than the actual or in
vivo gene because of the absence of introns or non-coding regions.
(ii) Synthetic DNA (sDNA)- It is synthesized with the help of DNA polymerase on DNA template.
Kornberg (1961) synthesized first synthetic DNA from a mixture of deoxyribonucleotide triphosphates,
DNA polymerase enzyme, metal ions and a segment of viral DNA.
Khorana (1968) synthesized first artificial gene (DNA) without a template. They synthesized the gene
coding for yeast alanine t-RNA, which contained only 77 base pairs. However, it did not function in the
living system. In 1979, Khorana was able to synthesize a functional tyrosine t-RNA gene of E. coli with
207 nucleotide pairs. Since then a number of genes have been synthesized artificially.
(iii) Random DNA - It refers to small fragments formed by breaking a chromosome with the help of restriction
endonucleases.
Technique of Recombinant DNA
– The DNAs which are to be used as passenger DNA and the vehicle DNA are extracted out of their cells by lysing
the cells with the suitable enzyme.
– The extracted DNAs are isolated from other cell contents by ultra centrifugation and purified.
– Both the passenger and vehicle DNAs are then, cleaved by using the same restriction endonuclease so
that they have complementary sticky ends.
– The complementary sticky ends of the passenger and vehicle DNAs are joined with ligase enzyme. This gives
rise to a recombinant DNA.
– The recombinant DNA is now inserted into a host cell such as Escherichia coli. The bacteria to be used as hosts
should be without plasmids.
– The host cells are treated with calcium chloride or lysozyme. It creates transient (temporary) pores in their wall
and makes the latter permeable to recombinant DNA.
– The recombinant DNA is added to the culture in which such bacteria are growing. The recombinant DNA is
taken up by the bacteria. It replicates when the host bacteria divide and give rise to multiple copies of recombinant
DNA.
E.Coli Human
cell
Plasmid
DNA
Restriction
Restriction endonuclease endonuclease
Sticky end
Insulin
Gene
Complementary pairing
Ligase
Plasmid with insulin gene
Fig.: Mechanism of transferring insulin gene from human DNA to E.coli with a plasmid
44 E
Biology
GENE T RANSFER
Transfer of desired genes from one organism into another is an important aspect of genetic engineering. Gene
transfer involves essentially three stages:
(i) Isolating a useful DNA segment from the donor organism.
(ii) Inserting it to a suitable vector.
(iii) Splicing of this altered DNAs into a recipient organism or host cell.
The cell to begin making the appropriate product and then device an economical method for it's mass
production.
Gene transfer is achieved by two kinds of transfer methods:
(i) Indirect method throughvectors or carriers and
(ii) Direct or vector less transfer method.
(i) Gene Transfer Through Vectors. Plasmids and viruses are commonly used vectors for the transfer of
desired genes. The desired genes are first made to join suitable plasmid or virus which are then introduced
into the target cells.
Chromo-
somal DNA
T-DNA
Agrobacterium
T-DNA integrates
into host
DNA
Tumour
(crown gall)
Chromosome Ti plasmid
Transformed
plant cell
– A plant pathogenic bacterium- Agrobacterium tumefaciens produces crown galls or plant tumours in al-
most all dicotyledonous plants.
– This bacterium infects all broad leaved agricultural crops such as tomato, soyabean, sunflower and cotton but
not cereales.
– Tumour formation is induced by it's plasmid which is therefore called Ti plasmid (Ti for tumour inducing)
Agrobacterium tumefaciens naturally transfers some part of Ti-plasmid in to host plant DNA without any human
effort so it is called natural genetic engineer of plant.
In the transformation process two essential component in Ti-plasmid –
(a) T-DNA – (Transferred DNA)

(b) Vir-region – (Virulence region)
Inside the host plant cell T-DNA is seperated from Ti-plasmid, and integrated into host plant DNA that causes
crown gall tumour.
Vir-region contains genes which are essential for T-DNA transfer and integration to host plant DNA. Vir-region
contains 6-operons A, B, C, D, E, and G. (Acetosyringone is inducer of Vir-operon)
When we use Ti plasmid as a vector, first we remove the tumour causing gene from T-DNA region. Then desired
gene inserted inplace of it. Now, this plasmid is called disarmed plasmid.
Same as Ri plasmid of A.rhizogenes (causing hairy root disease) also used as vector for gene transfer to plant
cell.
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(ii) Vectorless Gene Transfer : Foreign genes can also be transferred directly by the following methods:
(a) Electroporation- It creates transients (temporary pores) in the plasma membrane to facilitate
entry of foreign DNA.
(b) Chemical mediated genetic transformation- It involves certain chemicals such as polyethylene
glycol (PEG), that help in the uptake of foreign DNA into host cells.
(c) Microinjection- It is the introduction of foreign genes into plant or animal cells using micropipettes
or glass needles.
(d) Particle gun/Biolistic method- It is a technique in which tungsten or gold particles coated with
foreign DNA are bombarded into target cells to facilitate entry of the foreign genes.
(e) Liposome mediatd gene transfer- In this method DNA encloses within lipid begs. These lipid
begs fused with protoplast.
GENE TRANSFER IN ANIMALS

In animals, the genes are transferred mostly through direct methods such as electroporation, microinjection or
using particle gun.
The desired foreign genes can be introduced into fertilised eggs or embryos through microinjection. These
transgenic eggs or embryos can be implanted into the uterus of another female, called surrogate (foster)
mother for their further development. Now, since most of the human genes have been identified through
‘human genome project’, it is hoped that a number of human genetic disorders such as Alzheimer, cancer,
haemophilia, thalaessemia and cystic fibrosis can now be cured through insertion of the correct genes into these
patients.
ACHIEVEMENT S OF GENET IC ENGINEERING

– The DNA recombinant technology or genetic engineering provides great benefits for advancement of science
and society.
(1) Gene Therapy :
– A new system of medicine gene therapy, may develop to treat some hereditary diseases such as SCID, haemophilia
etc.
– It is the technique for curing gentic disease through replacing a "Faulty Gene" by a normal healthy functional
gene.
– The first gene therapy used in severe combined immuno deficiency (SCID) pateint.
Infant with SCID disorder lack the enzyme adenosine deaminase (ADA)
– ADA enzyme involved in purine metabolism.
– These patients have no functioning T & B lymphocytes.
– The affected child of SCID develops recurrent infection early in life because they have no immune response
against invading pathogen.
– The ideal approach would be to give the patient a functioning ADA by gene therapy.
– Thus, genetic disorder can be over come by introducing specific gene.
(2) Format ion of Transgenic Organis am (GMO) :-
– Transgenic plant obtained through recombinant DNA technology. First transgenic plant was tobacco. It
contains resistant gene against weedicide (Glycophosate).
– First transgenic animal was mouse contain gene for growth hormone. This enlarged mouse was known as
S up e rm o u s e .
– First introduced transgenic crop in India (2002) is Bt-cotton.
46 E
Biology
– Bacteria may be used as "Living factories" for synthesizing vitamins, hormones and antibodies.
– Human insulin (Humulin) was first genetically engineered product produced by an American firm Eli Lilly –
5th July 1983.
– Charles weismann of university of Zurich, obtained interferon through recombinant E.coli (1980)
– Microbes have been engineered to produce Human growth Hormone (HGH) for curing dwarfism.
– Vaccines which are produced by genetic engineering e.g., for Hepatitis-B and Herpes virus).
– Nitrogen fixation genes may be transferred from bacteria to the major food crops to boost food production
without using expensive fertilizers.
– Bt-cotton is resistant for boll worm(Helicoperpa armigera - Larva of insect). It is formed by transfer of pest
resistant gene from Bacillus thuringiensis (bt-2 gene encoding Bt–toxin).
Bacillus thuringiensis produces a toxic protein called crystal protein (Cry-Protein) this protein is toxic for larva of
certain insect. This protein kills the insect by inhibiting ion transport in midgut (bt 2 gene is called cry-gene)
– In pollution control, microbes have been engineered to break up the crude oil spills.
Dr. Ananda Mohan chakraborthi introduced plasmid from different strains in to single cell of pseudomo-
nas putida. The result was new genetically engineered bacterium which would cleaning oil spills called "Super
bug" (oil eating bug.) He transferred four types of plasmid in bacteria. These are OCT, XYL, CAM & NAH.
(3) Medical Diagnosis of Disease
Recombinant DNA technology is one of the important tool for the diagnosis of several diseases. The diagnostic
technique involves the construction of probes consis ting of short segments of single stranded DNA
attached to a radioactive or fluoroscent marker. Such probes are used to identify infections agents such as
Salmonella (cause food poisoning), Staphylococcus (pus forming bacterium), hepatitis virus, HIV; muscular dys-
trophy, cystic fibrosis and so on. Recombinant DNA technology can also be employed to predict the inheritance
of genetic disorders from carrier parents. The chances of birth of an affected child can be predicted by testing
DNA of repetitive prospective genetic disorder carrier parents.
Applications of Rec ombinant DNA produc ts
M edic ally us eful Ap pl ic at io ns

rec o mbinan t prod uc ts
Human insulin Treatment of insulin - dependant diabetes
Human growth hormone Replacement of missing hormone in short stature people.
Calcitonin Treatment of rickets.
Chorionic gonadotropin Treatment of infertility.
Blood clotting factor VIII/IX Replacement of clotting factor missing in patients with
Haemophilla A/B.
Tissue Plasminogen activator (TPA) Dissolving of blood clots after heart attacks and strokes.
Erythropoetin Stimulation of the formation of erythrocytes (RBCs) for
patients suffering from anaemia during dialysis or side effects
of AIDS patients treated by drugs.
Platelet derived growth factor Stimulation of wound healing
Interferon Treatment of pathogenic viral infections, cancer
Interleukin Enhancement of action of immune system
Vaccines Prevention of infectious diseases such as hepatitis B, herpes,
influenza, pertusis, meningitis, etc.
E 47
Pre-Medical
Applic ation of Genetically Engineered Microbes
M i c ro b e s Ap pl ic at io ns
Escherichia coli (gut bacterium) Production of human insulin, human growth factor
interferons, interleukin and so on.
Bacillus thuringiensis (soil bacterium) Productions of endotoxin (Bt toxin), highly potent, safe
and biodegradable insecticide for plant protection.
Rhizobium meliloti (bacterium) Nitrogen fixation by incorporating "nif" gene in cereal
crops.
Pseudomonas putida (bacterium) Scavenging of oil spills by digesting hydrocarbons of crude
oil.
Bacterial strains capable of Bioremediation (cleaning of pollutants in the enviroment).
accumulating heavy metal
Trichoderma (fungus) Production of enzyme chitinases for biocontrol of fungal
diseases in plants.
Trans genics and t heir potent ial applicat ions
Tra ns g e ni c U s eful appic ations

Bt Cotton Pest resistance, herbicide tolerance, and high yield.
Flavr Savr Tomato Increased shelf-life (delayed ripening) and better nutrient quality
Golden Rice Vitamin A and Fe - rich
Cattles (cow, sheep, goat) Therapeutic human proteins in their milk
Pig Organ transplantation without risk of rejection
POLYMER ASE CHAIN RE ACT ION T ECHNOLOGY (PCR-T ECHNOLOGY)

– This technique was invented by Kary mullis (1983).
– In 1993 Karry Mullis got nobel prize for PCR(for chemistry)
– PCR is a method for amplifying a specific region of DNA molecule without the requirement for time consuming
clonning procedures.
– PCR reaction takes place in Eppendrof tube.
– Using PCR-technique very low content of DNA available from sampels of blood or semen or any other tissue or
hair cell can be amplified many times and analysed. In this technique Taq-Polymerase is used. Taq polymerare
enzyme is used in PCR which is a special type of DNA polymerare enzyme which is resistant to high tempera-
ture.
– Taq Polymerase is isolated from T hermus aquat icus bacterium.
– Some other examples of polymerase which are used in PCR are –
Pflu Polymerase - Isolated from Pyrococus furiosus bacterium.
Vent Polymerase - Isolated from Thermococcus litoralis bacterium.
3 Main steps in PCR :-
(1) Denaturation (2) Annealing (3) Extension
48 E
Biology
Denaturation (94°C)
Primer-1
Annealing (54°C) Primer-2
Extension (72°C)
1. Denaturation :- In this step a double stranded DNA molecule is placed at 94°C. So double stranded DNA
becomes single stranded & each single stranded DNA functions as a template.
2. Annealing :- In this step two primer DNA are attached at 3' end of single stranded DNA.
3. Extension :- In this process taq polymerase enzyme synthesize DNA strain over template.
PCR is automatic process because taq. polymerase enzyme is heat resistant.
DNA FINGER PRINTING / DNA TYPING / DNA PROFILING/ DNA TEST

– It is technique to identify a person on the basis of his/her DNA specificity.
This technique was invented by sir Alec. Jeffery (1984).
– In India DNA Finger printing has been started by Dr. V.K. Kashyap & Dr. Lal Ji Singh.
– DNA of human is almost the same for all individuals but very small amount that differs from person to person
that forensic scientists analyze to identify people.
These differences are called Polymorphism (many forms) and are the key of DNA typing. Polymorphisms are
most useful to forensic scientist. It is consist of variation in the length of DNA at specific loci is called Restricted
fragment. It is most important segment for DNA test made up of short repetitive nucleotide sequences, these
are called VNTRs (variable number of tandem repeat).
VNTR's also called minisatellites were discovered by Alec Jeffery. Restricted fragment consist of hypervariable
repeat region of DNA having a basic repeat sequence of 11-60 bp and flanked on both sites by restriction site.
– The number and position of minisatellites or VNTR in restriction fragmnt is different for each DNA and length
of restricted fragment is depend on number of VNTR.
– Therefore, when the genome of two people are cut using the same restriction enzyme the length of fragments
obtained is different for both the people.
E 49
Pre-Medical
– These variations in length of restricted fragment is called RFLP or Restriction fragment length polymorphism.
– Restriction Fragment Length Polymorphism distributed throughout human genomes are useful for DNA Finger
printing.
– DNA Fingerprint can be prepared from extremely minute amount of blood, semen, hair bulb or any other cell
of the body.
DNA content of 1 - Microgram is sufficient.
Technique of DNA Finger printing involve s the follow ing major stpes.
1. Extraction – DNA extracted from the cell by cell lysis. If the content of DNA is limited then DNA can be
amplified by Polymerase chain reaction (PCR). This process is amplification.
RESTRICTION
ENZYME
DIGESTION
GEL ELECTRO-
PHORESIS
(Agargose polymer
gel)
DENATURATION BY ALKALI
SOLUTION
SOUTHERN BLOTTING
NYLON OR NITROCELLULOSE SHEET
HYBRIDIZA-
TION
DNA PROBES
Steps of DNA Fingerprinting NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2
2. Restriction Enzyme Digestion : Restriction enzyme cuts DNA at specific 4 or 6 base pair sequences called
restriction site.
Hae III (Haemophilus aegyptius) is most commonly used enzyme. It cuts the DNA, every where the bases are
arranged in the sequence GGCC. These restricated fragment transferred to Agarose Polymer gel.
3. Gel Electrophoresis :
– Gel electrophoresis is a method that separates macromolecules-either nucleic acid or proteins-on the basis of
size, electric charge.
– A gel is a colloid in a solid form. The term electrophoresis describes the migration of charged particles under the
influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros,
means "to carry across." Thus, gel electrophoresis refers to the technique in which molecules are forced across
50 E
Biology
a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provides the
driving force. A molecule's properties determine, how rapidly an electric field can move the molecule through a
gelatinous medium.
– Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids
posses ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either
as cation (+) or anions (–). Depending on the nature of the net charge, the charged particles will migrate either
to the cathode or to the anode.
– By the gel electrophoresis these restricted fragments move towards the positive electrode (anode) because DNA
has –ve electric charge (PO4–3).
– Smaller Fragment more move towards the positive pole due to less molecular weight. So after the gel
electrophoresis DNA fragment arranged according to molecular weight.
– These separated fragments can be visualized by staining them with a dye that fluoresces ultraviolet radiation.
4. Southern transfer / Southern blotting :
The gel is fragile. It is necessary to remove the DNA from the gel and permanently attaches it to a solid support.
This is accomplished by the process of Southern blotting. The first step is to denature the DNA in the gel which
means that the double-stranded restriction fragments are chemically separated into the single stranded form.
The DNA then is transferred by the process of blotting to a sheet of nylon. The nylon acts like an ink blotter and
"blots" up the separated DNA fragments, the restriction fragments, invisible at this stage are irreversibly at-
tached to the nylon membrane the "blot".
This process is called Southern blot by the name of Edward Southern (1970).
5. Hybridization : To detect VNTR locus on restricted fragment, we use single stranded Radioactive (P 32 ) DNA
probe which have the base pair sequences complimentary to the DNA sequences at the VNTR locus. Commonly
we use a combination of at least 4 to 6 separate DNA probes.
Labelled Probes are attached with the VNTR loci of restricted DNA Fragments, this process is called Hybridization.
6. Autoradiography : Nylon membrane containing radio active probe exposed to X-ray. Specific bands appear
on X-ray film. These bands are the areas where the radioactive probe bind with the VNTR.
– This appears the specific restricted fragment length pattern. This length pattern is different in different indi-
vidual. This is called Restricted Fragment length Polymorphism (RFLP).
These allow analyzer to identify a particular person DNA, the occurance and frequency of a particular genetic
pattern contained in this X-ray film. These x-ray film called DNA signature of a person which is specific for each
individual.
The probability of two unrelated individual having same pattern of location and repeat number of minisatellite
(VNTR) is one in ten billion (world population 6.1 billion)
In India the centre for DNA finger printing and diagnosis (CDFD - center for DNA finger printing & diagnosis)
located at Hyderabad.
Application of DNA Finger printing
1. Paternity tests. The major application of DNA finger printing is in determining family relationships. For
identifying the true (biological) father, DNA samples of Child, mother and possible fathers are taken and their
DNA finger prints are obtained. The prints of child DNA match to the prints of biological parents.
2. Identification of the criminal. DNA finger printing has now become useful technique in forensic (crime
detecting) science, specially when serious crimes such as murders and rapes are involved. For identifying a
criminal, the DNA fingerprints of the suspects from blood or hair or semen picked up from the scene of crime
are prepared and compared. The DNA fingerprint of the person matching the one obtained from sample
collected from scene of crime can give a clue to the actual criminal.
E 51
Pre-Medical
CLONING
Clone is the exact carbon copy or copies produced by a single parent (mother or father) by non-sexual methods
and are indentical to their parent genetically and morphologically. Clone is a greek word which means twig
(Klon=twig). As all the branches of a tree are similar in morphology and genetical characteristics, in the same
way clones are also similar to one another.
Cloning is the process of producing many identical organisms (clone), generally used to produce new plants with
similar characteristics. Microbes produce clones through asexual reproduction. In higher animals, clones are
produced by nuclear transplantation technique in which the nucleus from a somatic cell is transferred into
an unfertilized enucleated egg. The world's most famous sheep 'Dolly' was a clone produced by this method.
Many plant species show vegetative reproduction. In these plants, the clones produced by a twig (detached
shoot) are similar in their genotype as well as in phenotype (except environmental variations). Scientists have
been much curious to apply this characteristics of plants on animals also to conserve the desired genotypes of
some rare animals by making their clones. In higher animal, showing sexual reproduction, a zygote is formed
after fertilization of the egg by speromatozoan. Zygote differs from its parents in genotype. It was revealed by
the scientists through several experiments that only the egg and/or zygote has the potential to produce a whole
individual from a single cell. J.B. Gurdon (1969) of Oxford University applied this fact while performing an
experiment on frog. He destroyed the nucleus of an unfertilized egg of frog by treating with U.V rays and
transferred the nucleus of intestinal epithelial cell of tadpole into the egg cell . In this experiment a few of the
many transplanted eggs could develop into tadpoles. These developed tadpoles were identical in genotype and
phenotype to their parents. This nuclear transplantation technique devised by Gurdon is still being used in
cloning paractice in some modified manner. A brief introductory history of cloning is given in table.
A n in tr od uc to ry s to ry o f cl on in g
Ye a r Name of the Brief account of the Result
S c i en t is t experiment (s)
March, Roslin Institute Nuclear transplantation from Megan and Moragn sheep
1995 team,Scotland embryo to egg in sheep born normally
Feb, Roslin Institute Nuclear transplantation "Dolly" sheep born

1997 team, Scotland udder cell to egg in sheep normally
March, Don Wolf and Nuclear transplantation from Two monkeys "Neti"and
1997 Coworkers, Oregon embryo to egg in monkey "Ditto" born normally
Dec. Roslin Institute Nuclear transplantation from Molly and Polly sheep
1997 team, Scotland embryo to egg in sheep born normally
1999 Kato and Nuclear transplantation from " Geor g e and Cha rlie"
Coworkers adult cell to egg in cow cows born normally
In addition to the fact depicted in table attempts are continuously in progress in this field. In December, 2001
(report published in February, 2002) scientists at University, Texas, successfully produced the first cloned domestic
pet named as copy cat (C.C). Further, in Aug. 2005 Woo-Sukhwang of South Korea produced the clone of an
Afganian hound (Domestic dog used for hunting).
An Italian scientist Dr. S. Antenori is trying hard to produce human clone. It has triggred serious discussions and
debates at global level, focussing on the medical and ethical issues.
Ty pe s of C lo ni ng
Cloning is an extensive technique, which is divided into following types, on the basis of the experimental material
used -
(1) Gene cloning (2) Microbial cloning (3) Cell cloning
(4) Plant cloning and (5) Animal cloning
52 E
Biology
Animal c loning
Embryonic cell in animals, are deprived of their totipotency by the time they enter into gastrula stage. So animal
cloning is some what more difficult than plant cloning. On the basis of aims and end products animal cloning is
of two types (i) Reproductive and (ii) Therapeutic.
(i) Reproductive Cloning :-
A clone of the whole animal is prepared in reproductive cloning. The techniques which are used in such cloning
include blastomere separation, nuclear trans plantation and Honolulu tec hnique.
(a) In blastomere separation, all the eight blastomeres of a 8-celled embryo are separated & cultured
until small embryos are formed which are implanted into the womb of other surrogate mothers.
(b) In nuclear transplantation technique, nucleus is removed from the egg obtained from the female.
After then the nucleus of the desired cell (2n) is transferred into the enucleated egg cell which is then
allowed to develop into an embryo in suitable conditions. The developing embryo is the clone of the donor
cell from which nucleus was obtained for transplantation.
"Dolly" sheep was produced by using nuclear

transfer technique by Dr. lan w ilmut and
his collegues at Roslin Institute of Scotland
in 1997. They used somatic cells from udder
Mother sheep's Host mother sheep
(mammary glands) for forming this clone. One udder
udder cell with its nucleus intact was selected
Micropipette
because this nucleus carried the mother's
Cells in nutrient
genetic information. Meanwhile, and deprived medium
unfertilized egg cell was taken from a Cell fusion by
different sheep. Its nucleus was sucked out electrical stimulation
and an enucleated egg cell was obtained.
After then the udder cell nucleus was fused
with the enucleated egg cell under electrical Embryo
s timulation . Now this egg cell had the

mother's nucleus. At last the fused egg was
implanted into the uterus of surrogate mother,
other than the egg donor where it grew into
Embryo transplanted in
a lamb. Thus the Dolly was born, as a surrogate mother's uterus
genetically identical copy of its mother
(c) In Honolulu technique (1998-Teruhiko wakayama -

cloned mice) there is no fusion of the donor and recipient

Dolly
cells or its nucleus. Instead of ,nucleus from the donor cell
which is in G 0 or G1, stage is substituted into enucleated
egg cell (culture medium or chemical both is used in place
of electric shock to stimulate development).
(i i) Therapeutic cloning :-
Therapeutic cloning technique may prove to be very useful in the field of medical science, particularly when
there is a need for the transplantation to replace some damaged and diseased organ or tissue by a healthy
organ from a suitable donor. In such condition, if the cells from the patient himself are taken and cultured to
form desired organ. The organs developed in such manner( organ cloning) will be easily acceptable by the
patient, and there will be no possibility of its rejection as often occurs otherwise.
E 53
Pre-Medical
A number of diseases like parkinsonia, alzheimer, diabetes and diseases related with kidneys will possibly be
cured in future by the application of cloning technique.
Advantag es of Cloning
1. This technique can be used to improve the breeds of live-stock used in agriculture.
2. Cloning is helpful for providing many useful substances and chemicals required for human body, and also
in the cloning of such animals which can be used as a source of organs for transplantation purpose in
medical practices.
3. British scientists have been successful in obtaining alpha antitrypsin from sheep for curing an incurable
disease emphysema and clotting factor- ix for curing haemophilia. It has been possible by the use of
genetic engineering.
4. Many incurable diseases which are not curable so far, may be cured effectively in near future by applying
the processes relating with cloning and genetic engineering.
5. Cloning is useful to increase the number of individuals of those species which are at the edge of extinction,
thus helpful in conservation of biodiversity.
Disputes Related to Cloning
At present, the issue of cloning has been a matter of discussion and disputes among the scientists and the
sociologists. Many questions are being raised with regards to the ethical, moral, and social aspects of cloning.
Doubts have also been there about the health and ageing of clones and the misuse of cloning.
Cloning has important role in treatment of serious and non-curable diseases, a view that is favoured by most
scientists, however there is no agreement on the issue of human cloning.
GENOMICS
Genomics is the study of genomes and genes based on DNA sequencing. Genome is the total gene complement
of a haploid set of chromosomes and inherited as a unit from one parent through the gamete. A haploid (such
as prokaryotic) cell contains a single genome, a diploid (such as a cell of higher plant or animal) has two
genomes, one paternal and other maternal. Additional DNA is also present in mitochondria which is inherited
from one’s mother.
HUMAN GENOME PROJECT
Genetic make-up of an organism or an individual lies in the DNA sequences. If two individuals differ, then their
DNA sequences should also be different, at least at some places. These assumptions led to the quest of finding
out the complete DNA sequence of human genome. With the establishment of genetic engineering techniques
where it was possible to isolate and clone any piece of DNA and availability of simple and fast techniques for
determining DNA sequences, a very ambitious project of sequencing human genome was launched in the year
1990.
Human Genome Project (HGP) was called a mega project. You can imagine the magnitude and the requirements
for the project if we simply define the aims of the project as follows :
Human genome is said to have approximately 3 × 109 bp, and if the cost of sequencing required is US $ 3 per
bp (the estimated cost in the beginning), the total estimated cost of the project would be aproximately 9 billion
US dollars. Further, if the obtained sequences were to be stored in typed form in books, and if each page of the
54 E
Biology
book contained 1000 letters and each book contained 1000 pages, then 3300 such books would be required
to store the information of DNA sequence from a single human cell. HGP was closely associated with the rapid
development of a new area in biology called as Bioinformatics.
Goals of HGP
Some of the important goals of HGP are as follows :
(i) Identify all the genes in human DNA.
(ii) Determine the sequences of the 3 billion chemical base pairs that make up human DNA.
(iii) Store this information in databases.
(iv) Improve tools for data analysis.
(v) Transfer related technologies to other sectors, such as industries.
(vi) Address the ethical, legal, and social issues (ELSI) that may arise from the project.
The project was completed in 2003. Knowledge about the effects of DNA variations among individuals can lead
to revolutionary new ways to diagnose, treat and someday prevent the thousands of disorders that affect human
beings. Besides providing clues to understanding human biology, learning about non-human organisms, DNA
sequences can lead to an understanding of their natural capabilities that can be applied toward solving challenges
in health care, agriculture, energy production, environmental remediation. Many non-human model organisms,
such as bacteria, yeast, Caenorhabditis elegans (a freeliving non-pathogenic nematode), Drosophila (the fruit
fly), plants (rice and Arabidopsis), etc., have also been sequenced.
Methodologie s : The methods involved t wo major approaches. (1) Expre ssed S equence Tags (ESTs) -
Identifying all the genes that expressed as RNA. (2) Sequence Annotation - The blind approach of simply
sequencing the whole set of genome that contained all the coding and non-coding sequence, and later assigning
different regions in the sequence with functions. For sequencing, the total DNA from a cell is isolated and
converted into random fragments of relatively smaller sizes (recall DNA is a very long polymer, and there are
technical limitations in sequencing very long pieces of DNA) and cloned in suitable host using specialised vectors.
The cloning resulted into amplification of each piece of DNA fragment so, that is subsequently could be sequenced
with ease. The commonly used hosts were bacteria and yeast, and the vectors were called as BAC (bacterial
artificial chromosomes), and YAC (yeast artificial chromosomes).
The fragments were sequenced using automated DNA sequencers that worked on the principle of a method
developed by Frederick Sanger. (Remember, Sanger is also credited for developing method for determination
of amino acid sequences in proteins). These sequences were then arranged based on some overlapping regions
present in them. This required generation of overlapping fragments for sequencing. Alignment of these sequences
was humanly not possible. Therefore, specialised computer based programmes were developed. These sequences
were subsequently annotated and were assigned to each chromosome. The sequence of chromosome I was
completed only in May 2006 (this was the last of the 24 human chromosomes -22 autosomes and X and Y- to
be sequenced). Another challenging task was assigning the genetic and physical maps on the genome. This was
generated using information on polymorphism of restriction endonuclease recognition sites, and some repetitive
DNA sequences known as microsatellites.
Salient Features of Human Genome –
Some of the salient observations drawn from human genome project are as follows :
(i) The human genome contains 3164.7 million nucleotide bases.
(ii) The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene
being dystrophin at 2.4 million bases.
(iii) The total number of genes is estimated at 30,000-much lower than previous estimates of 80,000 to
1,40,000 genes. Almost all (99.9 per cent) nucleotide bases are exactly the same in all people.
(iv) The functions are unknown for over 50 per cent of discovered genes.
E 55
Pre-Medical
(v) Less than 2 per cent of the genome codes for proteins.
(vi) Repeated sequences make up very large portion of the human genome.
(vii) Repetitive sequences are stretches of DNA sequences that are repeated many times, sometimes hundred
to thousand times. They are thought to have no direct coding functions, but they shed light on chromosome
structure, dynamics and evolution.
(viii) Chromosome 1 has most genes (2968). and the Y has the fewest (231).
(ix) Scientists have identified about 1.4 million locations where single-base DNA differences (SNPs- single
nucleotide polymorphism, pronounced as 'snips') occur in humans, This information promises to
revolutionise the processes of finding chromosomal locations for disease-associated sequences and tracing
human history.
Org an i sm s Bas e pair Gene No.
Bateriophage 10,000 ------/-----

Lily 106 Billion B.P. ------
E.coli 4.7 million B.P. 4,000
S. cerviceae 12 Million B.P. 6,000
D. melangaster 180 Million B.P. 13,000
Caenorhabditis elegans 97 Million B.P. 18,000
Human 3 Billion B.P. 30,000
(a) First prokaryotes in which complete genome was sequenced is Haemophilus influenzae.
(b) First Eukaryote in which complete genome was sequenced is Saccharomyce s cerviceae (Yeast).
(c) First plant in which complete genome was sequenced is Arabidops is thaliana (Small mustard
plant).
(d) First animal in which complete genome was sequenced is Caenorhabditis elegans (Nematode).
–  – globin and insulin gene are less than 10 kilo base pair T.D.F. gene is the smallest gene (14 base pair)
and Duchenne muscular Dystrophy gene is made up of 2400 kilo base pair.(Longest gene)
GENE LIBR ARY (GENOMIC DNA LIBR ARY) AND GENE BANKS
A gene library is a collection of many of the desired genes of DNA fragments maintained in clones of bacterial
or some other cells. It is prepared by the following method.
DNA fragments containing one or few desired genes are obtained with the help of specific restriction endonucleases.
Each fragment is joined to a suitable vehicle DNA to form recombinant DNAs of different nature. These are
then introduced into host (bacterial, yeast, plant or animal) cells. The cells containing recombinant DNAs are
allowed to multiply in cultures. This will produce clones of cells where the daughter cells carry the same genes
which are identical to those of parent cells. A collection of clones with recombinant DNA containing desired
genes is a gene library. Gene libraries are maintained through special techniques.
A gene bank is a store house of clones of known DNA fragments, genes, gene maps, seeds, spores, frozen
sperms or eggs or embryos. These are stored for possible use in genetic engineering and breeding experiments
where species have become extinct. The need of gene banks is being increasingly felt as the rate of extinction
is increasing day by day. The human genome project is the most remarkable contribution in this field.
56 E
Biology
BIOINFORM AT ICS
Definition : Bioinformatics is application of computer technology to the management of biological information.
Computer techniques are used togather, store, analyze and integrate biological and genetic information which can
be applied to gene based drug discovery and development.
Dr ug d es ign b as ed o n bi o in fo rm atic s
Bioinformatics is a new approach for drug designing. In this procedure all the knowledge about the disease is collected,
analysed and find out a 'target molecules' that cause the disease. Structure of these molecules is analysed by X-
ray or N.M.R. technique, then drugs are developed which can bind and block the activity of these molecules.
Bi ol ogic al da ta b as e
Biological data base is collection of genomic, proteomic and metabolic data assosiated with computer software, which
include nucleotide sequence of gene or amino acid sequence of protein, information about structure, function, location
of genes and protein.
Importance –
(1) Easy asses to the information.
(2) A method for extracting only required information.

E 57

Pre-Medical Sex Determination

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Pre-Medical Sex Determination

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Pre-Medical

SEX D ET ERMINAT ION

– Male is heterogametic (male produces two types of gamete)

In male X-chromosome containing gametes is called "Gynosperm" and Y- chromosome containing

Haploid - diploid mechanism –

Haploid (One set)  Male

Feeds on Royal jelly

[4] Sex determination by Hormone –

this X chromosome will be lost

Sex determination in human –

Sex determination in plant –

PHENOT YPIC EXPRESSION IN HAPLOID ORGANISMS

organisms, the genetics of haploid organisms exhibit the following features:

Diploid zygote Aa AaBb

Linkage And Rec ombination in Neuros pora (Drosophila of plant kindgom)

Tetrad Analysis in Ordered Tetrads –

Meiosis - I Meiosis - II Mitosis

1. Firs t Divis ion Seg regation Between Centromere and gene-a.

2. Second Division Segregation Between Centromere and Gene-a .

(Out of 40 only 20 will be the recombinant type)

Effect of medium supplements on the growth of mutants Neurospora c rassa

encoded enzyme Enzyme A Enzyme B Enzyme C

Location of gene Gene A Gene B Gene C

In Drosophila allele W+c W +s W+g  produce red eye colour

REGUL AT ION OF GENE EXPRERSION

[OPERON – ON] E1 E2 E3 Enzymes

Differenc es between Induc tion and R epression

Induc tion Repression

is normally in switched-off state. is normally in switched-on state: NODE2\E:\DATA\2014\SMP\BIO\SET-03\16-GENETICS\ENG\02-GENE-2

2. It starts transcription and translation. 2. It stops transcription and translation.

3. It is caused by a new metabolite which 3. It is caused by increased formation

needs enzymes to get metabolised. or availability of a metabolite.

4. It generally operates in a catabolic 4. It generally operates in an

pathway. anabolic pathway.

GENE EXPRESSION IN EUK ARYOT ES

Activator RNA mRNA

regulator gene) (Operater gene) (Structural gene)

Sensor site Integrator gene Receptor site Producer gene

Britten-Davids on model or Gene-battery model

Cha racter Domina nt R e c e s s iv e

Eye colour Brown / Black Blue

Eye size Large Small

Ear lobes Free Fused

Hair Curly hair Straight

Cheek Dimple cheek Normal

Hand Right Handed Left handed

Rolling of tongue Rollar Non Rollar

PTC (Phenyl thiocarbamide) taster Taster Non Taster

Skin pigmentation Normal Albino

4. The siblings are indicated in chronological order of birth

7. , Affected male and female individual

8. , Heterozygous for autosomal recessive

9. Carrier female of sex linked recessive character or disease

10. Death of individual

11. Abortion or still birth (sex unspecified)

12. Consanguineous marriage

13. Parent with male child affected with disease

14. 5 – Five unaffected offsprings

POPUL AT ION GENET ICS

Study of gene frequency in a population is called population genetics.

Hardy Weinberg theorem or Hardy weinbergh law

Factors affecting gene frequency :

population  sampling error 