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Apomixix in Flowering Plants |

Biology
by Saritha Pujari Reproduction in Plants

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Apomixix in Flowering!
The Apomixis is the formation of new individuals through asexual reproduction
without involving the formation and fusion of gametes.
Amphimixis is the formation of new individuals through the normal process of
sexual reproduction by meiotic formation of gametes and their subsequent
fusion during fertilization.

Thus, apomixis is a form of asexual reproduction that mimics sexual


reproduction. Agamospermy is the formation of new individuals by asexual
reproduction without involving fusion and formation of gametes.
It is of following types:
(a) Adventive embryony:
In this case one or few extra embryos are formed from a diploid cell of
nucellus or integument but never from egg e.g. Citrus, Opuntia, Mango.

(b) Recurrent apomixis (Recurrent agamospermy):


A diploid embryo sac is formed which has a diploid egg of oosphere. The
diploid egg grows parthenogenetically into diploid embryo e.g. Poa, Allium.
(c) Non-recurrent agamospermy:
It is the development of the embryo from haploid female gamete without
fertilization. It is parthenogenesis.
(d) Parthenogenesis:
It is the development of seed/ embryo from a female gamete without
fertilization e.g. grapes.
(e) Parthenogamy:
It is the union of two incompatible gametes i.e. fusion of two female gametes.
(f) Apospory:
It involves the development of gametophyte from sporophyte directly.
(g) Apogamy:
It is the development of sporophyte from gametophyte without the fusion of
gametes.
(h) Diplospory:
In this case diploid embryo sac can develop directly from diploid megaspore
mother cell.
Many flowering plants are reproduced under natural conditions of asexual
reproduction. In asexual reproduction plants are produced without the fusion

of gametes. Sexual reproduction is designed by fusion of gametes and leads


to the production of plants with new characters.

Embryo Development after


Fertilization | Biology
by Saritha Pujari Reproduction in Plants

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Embryo Development after Fertilization!


After fertilization, the fertilized egg is called zygote or oospore. Following a
predetermined mode of development (embryogeny) it gives rise to an embryo,
which has the potentiality to form a complete plant.
Usually the zygote divides immediately after the first division of primary
endosperm nucleus or it divides earlier than the first division of primary
endosperm nucleus. After fertilization the zygote rests for a period that varies
greatly in different taxa-from a few hours to several weeks.
There are no fundamental differences in the early stages of development in
dicotyledonous and monocotyledonous embryos. But later stages of
development do differ as the mature embryos differ considerably.

The first division of the zygote is usually transverse in most of the


angiosperms. However, in some cases first division may be longitudinal or
oblique. From this two celled stage till the differentiation of organs, embryo is
called proembryo.

A. Development of Dicot embryo in Capsella bursa pastories


(Crucifer type):
The first division of the zygote is transverse leading to the formation of basal
cell cb and a terminal cell ca (Fig. 2.30 A, B). Basal cell divides transversely to
form (cm and ci) and latter divides longitudinally resulting in the formation of
reverse-T shaped proembryo (made up of 4 cells) Fig. 2.30 C-E.
Each of the two terminal cells now divides by vertical wall lying at right angles
to the first to form quadrant stage (Fig. 2.30J). The quadrant cells divide by
transverse walls giving rise to octant stage (Fig. 2.30 K, L). Of this octant
lower four cells form stem tip and cotyledons and upper four form hypocotyl.
All the eight cells of undergo periclinal divisions differentiating an outer
dermatogen and inner layer of cells (Fig. 2.30 M, N). The cells of dermatogen
divide anticlinally to give rise to epidermis of embryo, while the inner cells by
further divisions give rise to the ground meristem and procambial system of
the hypocotyl and cotyledons.
By this time two upper cells c i and cm of four-celled proembryo (Fig. 2.30 D)
divide to form a row of 6-10 suspensor cells (Fig. 2.30 F-K) of which the
uppermost cell V becomes swollen and vesicular to form haustorium. The
lower most cell h functions as hypophysis.

The cell of hypophysis divided to give rise to eight cells. Lower four of these
form root cortex initials. Upper four form root cap and root epidermis. A fully
developed embryo of dicotyledons has an embryonal axis differentiated into
plumule, two cotyledons and radicle.
In the beginning embryo is globular. With the continuous growth the embryo
become heart shaped (cordate) which is made up of two primordial of
cotyledons. The enlarging embryo consists of two cotyledons and embryonal
axis.
The hypocotyl as well as cotyledons soon elongate in size. During further
development, the ovule becomes curved like horse-shoe (Fig. 2.31).

B. Development of Monocot embryo:


In monocots a good deal of variation is found in the stages of development.
However, there is no essential difference between the monocotyledons and
dicotyledons regarding the early cell divisions of proembryo. Here the
embryogeny of a typical type Sagittaria (Family Allismaceae) has been
traced out.

First of all zygote or oospore greatly enlarges in size and divides by


transverse division to form a 3- called proembryo (Fig. 2.33). These are basal
cell, middle cell and terminal cell.

Larger basal cell which lies towards micropylar end does not divide further
and is transformed directly to form large suspensor or vesicular cell. Terminal
cell undergoes number of divisions in various planes and forms a single
cotyledon.
The middle cell undergoes repeated transverse and vertical divisions, thus
differentiating into few suspensor cells, radicle, plumule and hypocotyl. In this
type cotyledon is a terminal structure and plumule is situated laterally in a
depression. In monocots like Colocasia, no suspensor is formed. In
Agapanthus (family Liliaceae); two cotyledons have been reported.

Sexual Reproduction : Definition,


Characteristics, Origin of Sex
by Puja Mondal Biology

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Read this article to learn about the meaning, characteristics, origin of


sex, occurrence, types, advantage and disadvantages in the sexual
reproduction in animals!

Meaning of Sexual Reproduction:


It is the process of development of new individuals through the formation and
fusion of male and female gametes.

Image Courtesy : en.wikipedia.org/wiki/File:HumanEmbryogenesis.svg

Sexual reproduction is also called amphimixis (Gk. amphi both, mixis


union) or syngenesis (Gk. syn together, genesis origin) or amphigony (Gk.
amphi both, gony marriage).
Sexual reproduction involves four processes:
(i) formation of haploid cells, the gametes, by gametogenesis (meiosis)
(ii) fusion of the two gametes forming diploid cells, the zygotes (fertilization)

(iii) repeated mitotic divisions of the zygotes to form embryos (embryogenesis); and
(iv) growth of embryos into new individuals (development). Because there is
fusion of male and female gametes, the offspring produced are not identical to
their parents or fellows.

Characteristics of Sexual Reproduction:


(i) It is usually biparental.
(ii) Gametes are always formed,
(iii) Fertilization takes place,
(iv) It involves both meiosis and mitosis,
(v) Daughter organisms genetically differ from the parents,
(vi) Multiplication is not so rapid as in asexual reproduction.

Origin of Sex:
Sex originated in protistans and simple algae. During favourable conditions,
these organisms multiply asexually but during unfavourable conditions
gametes are formed. The gametes fuse to form zygotes which often develops
a thick wall to become zygospores. The latter are dispersed.
Under favourable conditions zygospore germinates to form new organisms
(e.g., prostist/alga). The zoospores (asexual spores) and gametes are
structurally similar in Chlamydomonas and Ulothrix (both are simple algae).

Early sexual reproduction was isogamous. Later on evolution occurred and


sexual reproduction became anisogamous and oogamous. Similarly sex
organs evolved which differentiated into male and female individuals. Early
evolution of sex organs depended upon environment but later on hormones
started influencing the development of sex organs. In Chlamydomonas sexual
reproduction may be isogamous, anisogamous or oogamous. (To be
described ahead).

Occurrence:
Sexual reproduction occurs almost in all types of plants and animals.

Types:
Sexual reproduction is of two main types; syngamy and conjugation.

A. Syngamy (Gk. syn together, gamos marriage):


It is the complete and permanent fusion of male and female gametes to form
the zygote.
Syngamy is of two types with regard to the source of fusing gametes;
endogamy and exogamy.
1. Endogamy (Self-fertilization):
It involves the fusion of male and female gametes of the same parent. Thus it
is uniparental, e.g., Taenia (tape worm). Taenia is hermaphrodite (=
monoecious or bisexual) worm.

2. Exogamy (Cross-fertilization):
It involves the fusion of two gametes produced by different parents. Thus it is
bi-parental, e.g., Rabbit dioecious or unisexual animal. Cross fertilization
also occurs in many hermaphrodite animals as in earthworm and leech. It is
due to the fact that their male and female reproductive organs mature at
different times.
Syngamy is of following types with regard to the structure of the fusing
gametes; isogamy, anisogamy (heterogamy), oogamy and hologamy.
1. Isogamy (Gk. iso = equal; gamos = marriage):
It involves the fusion of gametes which are similar morphologically but may be
different physiologically. Such gametes are called isogametes. Isogamy takes
place in Chlamydomonas an alga and Monocystis a protozoan.
2. Anisogamy (Heterogamy):
It involves the fusion of gametes which differ in size or motility. Such gametes
are called anisogametes or heterogametes (e.g., microgametes or male
gametes and macrogametes or female gametes. Anisogamy (Gk. n-without,
wo=equal, gamos=marriage) or heterogamy (Gk. hetero=different,
gamos=marriage) occurs in Clamydomonas, some other algae, higher
invertebrates and all vertebrates including human beings.
The common type of anisogamy is oogamy which involves the fusion of a
large non- motile female gamete (egg or ovum) and a small motile male

gamete (antherozoid).Thus in oogamy gametes differ in both properties. It


takes place in Chlamydomonas, red algae, etc.
3. Microgamy:
The fusing gametes or individuals are much smaller than the normal.
Examples certain protistans.
4. Autogamy:
The fusing gametes/nuclei are derived from the same cell. Example
Paramecium.
5. Paedogamy:
It is a type of autogamy where gametes are derived from multiple division of
the nucleus of the single individual. It differs from paedogenesis (reproduction
by larva).
6. Hologamy (Gk. holos = whole, entire, complete, gamos = marriage) or
Macrogamy:
It involves the fusion of two organisms. It means two organisms themselves
act as gametes. It occurs in yeasts.

B. Conjugation:
It involves temporary union of two parents of the same species which
exchange their male pronuclei to form synkaryon and then separate to

produce daughter individuals. It corresponds to cross fertilization of higher


animals. It takes place in Paramecium, Spirogyra, etc.

Advantages of Sexual Reproduction:


(Why is sexual reproduction better than asexual reproduction?)
1. Variations:
Since fusion of gametes from different parents occur during sexual
reproduction, hence genetic recombination takes place causing variations.
2. Evolution:
Variation being a major factor of natural selection, therefore, it plays an
important role in evolution.
3. Adaptation:
The offspring produced due to sexual reproduction adapt better to the
changing environmental conditions.
4. Vigour and Vitality:
Genetic recombination, interaction, etc. during sexual reproduction provide
vigour and vitality to the offspring.

Disadvantages of Sexual Reproduction:


1. Sexual reproduction is usually bi-parental.
2. It is slow process and requires a lot of time.

3. Fertilization has a chance factor.

Types of Gametogenesis:
Spermatogenesis and Oogenesis |
Biology
by Saritha Pujari Human Reproduction

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Gametogenesis is the process of formation and differentiation of haploid


gametes (sperms and ova) from the diploid primary germ cells, gametogonia
(spermatogonia and oogonia) present in primary sex organs called gonads
(testes in male and ovaries in female respectively).
Gametogenesis is of two types:
I. Spermatogenesis and

II. Oogenesis.

I. Spermatogenesis (Figs. 3.13 A and 3.14):


Definition:
It is the formation of haploid, microscopic and functional male gametes,
spermatozoa from the diploid reproductive cells, spermatogonia, present in
the testes of male organism.

Period:
In the seasonally breeding animals, the testes undergo testicular cycle in
which the testes and their spermatogenic tissue become functional only in the
specific breeding season. So in some seasonally breeding mammals like bat,
otter and llama, testes enlarge, become functional and descend into the
scrotum in the breeding season as become heavier due to accumulation of
sperms, while become reduced, non-functional and ascend into the abdomen
in other seasons.
But in human male, lion, bull, horse etc., the testes lie permanently in the
scrotum and spermatogenesis occurs throughout the year. In human male,
testes descend into the respective scrotal sacs during seventh month of
development under the stimulation of FSH of adenohypophysis.
But in some mammals e.g. elephant, echidna, dolphin, whale, seal etc., testes
lie permanently in the abdomen (intra-abdominal) mainly due to presence of
blubber (thick fatty layer beneath the skin). Spermatogenesis is a continuous
process and is completed in about 74 days.

Mechanism:
Spermatogenesis is divided into two parts:
A. Formation of Spermatid:
It is divided into three phases:
1. Multiplicative or Mitotic phase:
It involves the rapid mitotic division of diploid primary or primordial germ cells,
called gonocytes, present in germinal epithelium of the seminiferous tubules
of the testes. These cell are undifferentiated and have large and chromatinrich nucleus.

This forms large number of diploid and rounded sperm mother cells called
spermatogonia (Gr. sperma = seed; gone = offspring). Each spermatogonial
cell is about 12 pm in diameter and has a prominent nucleus. Some
spermatogonia act as stem cells (called Type A spermatogonia) and go on
dividing and adding new cells by repeated mitotic divisions, so forming
spermatogenic lineage, but some spermatogonia move inward and enter
growth phase (called Type B spermatogonia).

2. Growth phase:
It is characterized by spermatocytogenesis in which a diploid spermatogonium
increases in size (about twice) by the accumulation of nutritive materials
(derived from germinal cells and not synthesized) in the cytoplasm and
replication of DNA, and forms diploid primary spermatocyte. Nutritive
materials are derived from germinal cells. During this, the primary
spermatocyte prepares itself to enter meiosis. Growth phase of
spermatogenesis is of much shorter duration than that of oogenesis.

3. Maturation or Meiotic phase:


It is characterised by meiosis. The diploid primary spermatocyte undergoes
meiosis-I (reductional or heterotypical division) and forms two haploid cells
called secondary spermatocytes, each containing 23 chromosomes.
It is immediately followed by meiosis-II (equational or homotypical division) in
each secondary spermatocyte to form two haploid spermatids, each of which
has 23 chromosomes. So each diploid spermatogonium produces 4 haploid
spermatids. Different stages of spermatogenesis are interconnected by
cytoplasmic strands till spermiogenesis when the maturing and interconnected
gametes separate from each other.
B. Spermiogenesis (Fig. 3.15):

The transformation of a non-motile, rounded and haploid spermatid into a


functional and motile spermatozoan is called spermiogenesis or
spermioteliosis. The main aim is to increase the sperm motility by reducing
weight and development of locomotory structure.

It involves the following changes:


1. Nucleus becomes condensed, narrow and anteriorly pointed due to loss of
materials like RNAs, nucleolus and most of acidic proteins.
2. A part of Golgi body of spermatid forms the acrosome, while the lost part of
Golgi body is called Golgi rest.
3. Centrioles of spermatid form the neck of sperm.
4. Distal centriole gives rise to axoneme.
5. Mitochondria form a spiral ring behind the neck around the distal centriole
and proximal part of axoneme. This is called nebenkern.
6. Most of cytoplasm is lost but some cytoplasm forms sheath of tail of sperm.
The spermatids mature into spermatozoa in deep folds of the cytoplasm of the
Sertoli cells (nurse cells) which also provide nourishment to them. Mature
spermatozoa are released in the lumen of seminiferous tubules, called
spermiation. The two testes of young adult form about 120 million sperms
each day.
Changes in spermatid to form sperm during spermiogenesis.

Structure of
spermatid

Changes in the sperm

1. Nucleus

Shrinks and elongated.

Changes to acrosome.
Forms axial filament of
2. Golgi complex
3. Distal centriole
4. Mitochondria
5. Cytoplasm

sperm tail.
Form mitochondrial spiral of
sheath called nebenkem.
Generally lost except a thin
sheath called manchette.

Control:
In human male, spermatogenesis starts only at the age of puberty due to
increased secretion of gonadotropin releasing hormone (GnRH) from the
hypothalamus of brain. GnRH stimulates adenohypophysis to secrete two
gonadotropins: FSH and ICSH. ICSH stimulates the Leydigs cells of testis to
secrete male sex hormones, called androgens, most important of which is
testosterone.
Testosterone stimulates the spermatogenesis especially spermiogenesis. FSH
stimulates the Sertoli cells of testis to secrete certain factors which helps in
the process of spermatogenesis. It is called physiological control.

Types:
In man and a large number of other animals having XY mechanism in male,
there are two types of sperms: 50% Gynosperms having X-Chromosome and
50X) Androsperms having Y-Chromosome.

Significance:
(a) Produces haploid sperms.
(b) Crossing over may occur during meiosis-I, so producing variations.
(c) Proves evolutionary relationship.

II. Oogenesis (Fig. 3.13 B):


Definition:
It involves the formation of haploid female gametes called ova, from the
diploid egg mother cells, oogonia, of ovary of female organism. It involves 2
biological processes: Genetical programming and packaging.

Period:
Period of oogenesis is different in different animals. In human female, there
are about 1,700 primary germ cells in the undifferentiated female gonad at
one month of foetal development. These proliferate to form about 600,000
oogonia at two months of gestation period and by its 5th month, the ovaries
contain over 7 million oogonia; however, many undergo atresia (degeneration
of germ cells) before birth. At the time of birth, there are 2 million primary
follicles, but 50% of these are atretic.

Atresia continues and at the time of puberty each ovary contains only 60,00080,000 primary follicles. Oogenesis is completed only after the onset of
puberty and only one out of 500 is stimulated by FSH to mature. So oogenesis
is a discontinuous and wasteful process.

Mechanism:
Like the spermatogenesis, oogenesis is formed of three phases:
1. Multiplicative phase:
In this certain primary germ cells (larger in size and having large nuclei) of
germinal epithelium of ovary undergo rapid mitotic divisions to form groups of
diploid egg mother cells, oogonia. Each group is initially a chord and is called
egg tube of pfluger which later forms a rounded mass, egg nest (Fig. 3.13 B).
2. Growth phase:
Growth phase of oogenesis is of very long duration than that of
spermatogenesis e.g., only three days in Drosophila, 6-14 days in hen, 3
years in frog and many years (12-13 years) in human female. During growth
phase, one oogonium of egg nest is transformed into diploid primary oocyte
while other oogonia of the egg nest form a single-layered nutritive follicular
epithelium around it.
The structure so formed is called primary follicle. Later, each primary follicle
gets surrounded by more layers of granulosal cells and changes into
secondary follicle. Soon secondary follicle develops a fluid-filled antral cavity
called antrum, and is called tertiary follicle. It further changes to form Graafian
follicle. So not all the oogonia develop further.

Growth phase involves:


(a) Increase in size of oocyte (2000 times in frog; 43 times in mouse; 90,000
times in Drosophila; 200 times in hen and about 200 times in human female)
by the formation and accumulation of yolk (vitellogenesis) by a special
mitochondrial cloud lying close to nucleus and called yolk nucleus.
(b) Nucleus becomes bloated with nucleoplasm and is called germinal vesicle.
(c) A thin vitelline membrane is secreted around the oocyte.
(d) Increase in number of mitochondria, amount of ER and Golgi body.
(e) Formation of lampbrush chromosomes in fishes, amphibians, reptiles,
birds, insects, etc. for rapid yolk synthesis.
(f) Gene-amplification or redundancy of r-RNA genes for rapid synthesis of rRNA.
3. Maturation phase:
It is characterized by meiosis. In this, the diploid and fully grown primary
oocyte undergoes meiosis-I (reductional division) to form two unequal haploid
cells. The smaller cell is called first polar body (Polocyte) and has very small
amount of cytoplasm. The larger cell is called secondary oocyte and has bulk
of nutrient-rich cytoplasm. Both of these are haploids and each has 23
chromosomes.
Secondary oocyte undergoes meiosis-II (equational division) to form two
unequal haploid cells. The smaller cell is called second polar body and has

very little of cytoplasm, while the larger cell is called ootid. It has almost whole
of cytoplasm and differentiates into an ovum. Meanwhile, first polar body may
divide into two.
So in oogenesis, a diploid oogonium forms one haploid ovum and two or three
polar bodies while in spermatogenesis, a diploid spermatogonium forms four
haploid sperms. The primary function of formation of polar bodies is to bring
haploidy but to retain the whole of the cytoplasm in one ovum to provide food
during the development of zygote to form an embryo. The number of ova is
reduced with the ability of the female to bear and rear them.
In most of organisms including human female, the ovulation occurs at
secondary oocyte stage in which meiosis-I has been completed and first polar
body has been released. Meiosis-II is completed only at the time of spermentry.

Significance:
(a) It produces haploid ovum by releasing 2 or 3 haploid polar bodies.
(b) Most of cytoplasm is retained in functional ovum.
(c) Variations may appear due to crossing over during Meiosis-I.
(d) Proves evolutionary relationship.

Restriction Endonucleases: Types,


Nomenclature, Recognition
Sequences and Cleavage Patterns
by Saritha Pujari DNA

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Restriction Endonucleases: Types, Nomenclature, Recognition


Sequences and Cleavage Patterns!
Endonucleases are enzymes that produce internal cuts, called cleavage, in
DNA molecules. Some endonucleases cleave only one of the two strands of a
DNA duplex, e.g., SI nuclease (from Aspergillus oryzae)-, such cuts are
usually known as nicks.
In contrast many endonuclease cleave both the strands of DNA molecules;
many of such endonucleases cleave DNA molecules at random sites, e.g.,
deoryribonuclease I (DNase I; obtained from cow pancreas). But a class of
endonucleases cleaves DNA only within or near those sites, which have
specific base sequences; such endonucleases are known as restriction
endonucleases, and the sites recognised by them are called recognition
sequences ox recognition sites.
The recognition sequences are different and specific for the different
restriction endonucleases or restriction enzymes. By the year 2000, more than
1,200 different restriction enzymes had been characterized.

Restriction enzymes were discovered due to and named after the


phenomenon of host restriction of bacterial phages. When a phage (say C)
DNA released from one bacterial strain (Say, E. coli strain C; such a X phage
is designated as C) is used to infect another strain (say, E. coli strain K) the
efficiency of phage growth on the latter (strain K) is only a very small fraction
of the efficiency on the former (strain C).
Some phages that survive grow normally on the second strain, and are now
referred to as K; these phage particles infect strain K with the same efficiency
as C does strain C. Thus multiplying a phage on one bacterial strain seems
to restrict the growth of that phage to the strain concerned.
This restriction on phage host range is due to the presence of restriction
enzymes in the host cells, which recognise and cleave foreign DNA introduced
into the cell. The DNA of a cell is protected from its own endonucleases by
methylation (usually of A and C) within their recognition sites. Thus DNA
molecules having the same methylation pattern as that of a bacterial cell itself
will be recognised as own DNA, while those lacking this will be regarded as
foreign DNA.
The presence of restriction enzymes was postulated by W. Arber during
1960s, while the first true restriction endonuclease was isolated in 1970.
Smith, Nathans and Arber were awarded the Nobel Prize for Physiology and
Medicine in 1978 for the discovery of endonucleases.
Restriction endonucleases are indispensable for DNA cloning and
sequencing. They serve as the tools for cutting DNA molecules at

predetermined sites, which is the basic requirement for gene cloning or


recombinant DNA technology.

Types of Restriction Endonucleases:


There are the following four distinct types of restriction endonucleases: (i)
Type I, (ii) Type II and (iii) Type III and (iv) Type IIs restriction endo-nucleases.
Type I restriction endonucleases are complex endonucleases, and have
recognition sequences of about 15 bp; they cleave the DNA about 1000 bp
away from the 5-end of the sequence TCA located within the recognition
site, e.g., Eco K, Eco B, etc.
Type II restriction endonucleases are remarkably stable and induce cleavage
either, in most cases, within or immediately outside their recognition
sequences, which are symmetrical. More than 350 different type II
endonucleases with over 100 different recognition sequences are known.
They require Mg2+ ions for cleavage. The first type II enzyme to be isolated
was Hindll in 1970. Only type II restriction endonucleases are used for
restriction mapping and gene cloning in view of their cleavage only at specific
sites.
Type III restriction endonucleases are intermediate between the type I and
type II enzymes; they cleave DNA in the immediate vicinity of their recognition
sites, e.g., EcoP1, EcoP15, HinfllI, etc. Type 1 and Type III restriction
enzymes are not used in gene cloning. The Type III enzymes recognize
asymmetric target sites, and cleave the DNA duplex on one side of the
recognition sequence up to 20 bp away.

Nomenclature:
The nomenclature of restriction endonucleases follows a general
pattern:
(1) The first letter of the name of genus in which a given enzyme is discovered
is written in capital.
(2) This is followed by the first two letters of species name of the organism.
These three letters are generally written in italics. e.g., Eco from Escherichia
Coli, Hin from Haemophilus influenzae, Hpa from Haemophilus
parainfluenzae, etc.
(3) Strain or type identification is depicted as subscript, e.g., EcoK; if the
enzyme is encoded by a plasmid, the plasmid name is written as a subscript,
e.g., EcoRI.
(4) When an organism produces more than one enzyme, they are identified by
sequential Roman numerals, e.g., the different enzymes produced by H.
influenzae strain Rd are named Hindll, HindIII, etc.
(5) All restriction enzymes are designated by the general symbol R, which is
prefixed to their names, e.g., RECORI RHindIII, RBamHl, etc. (this is to
distinguish them from the corresponding methylases isolated from the same
strains; the methylases are prefixed by M).
However, in practice, the following simplifications are used:
(1) The subscripts (due to strain or plasmid names) are ordinarily written on
line, e.g., REcoRI, RHind III, etc., and

(2) The prefix R is not used where the context makes it clear that the enzyme
under reference is a restriction endonuclease (Table 2.1).

Recognition Sequences:
The recognition sequences for Type 11 endonucleases form palindromes with
rotational symmetry (Table 2.1). In a palindrome, the base sequence in the
second half of a DNA strand is the mirror image of the sequence in its first
half; consequently, the complementary DNA strand of a double helix also
shows the same situation (Fig. 2.1).
But in a palindrome with rotational symmetry, the base sequence in the first
halt of one strand of a DNA double helix is the mirror image of the second half
of its complementary strand (Fig. 2.2). Thus in such palindromes, the base
sequence in both the strands of a DNA duplex reads the same when read
from the same end (either 5 or 3) of both the strands.

Most of the type II restriction endonucleases have recognition sites of 4, 5 or 6


bp (base pairs), which are predominantly GC-rich. Longer palindromic target
sequences are also known, and so are nonpalindromic ones (specific for
some enzymes). Some restriction enzymes have ambiguities in their

recognition sites, e.g., Hi nil, EcoRII, etc., so that they may recognise upto 4
or even more, e.g., Sfil, different target sequences. (Table 2.1).

Cleavage Patterns:
Most type II restriction endonucleases cleave the DNA molecules within their
specific recognition sequences, but some produce cuts immediately outside
the target sequence, e.g., Nlalll, Sau3A, etc. (Table 2.1). These cuts are either
(1) staggered or (2) even, depending on the enzyme.

Most enzymes produce staggered cuts in which the two strands of a DNA
double helix are cleaved at different locations; this generates protruding (3 or

5) ends (Fig. 2.3), i.e., one strand of the double helix extends some bases
beyond the other. Due to the palindromic (symmetrical) nature of the target
sites, the two protruding ends generated by such a cleavage by a given
enzyme have complementary base sequence.
As a result, they readily pair with each other under annealing conditions; such
ends are called cohesive or sticky ends. An important consequence of this fact
is that when fragments generated by a single restriction enzyme from different
DNAs are mixed, they join together due to their sticky ends (Fig. 2.4).
Therefore, this property of the restriction enzymes is of great value for the
construction of recombinant DNAs.

FIG. 2.4. Two distinct samples (A and B) of DNA are cleaved with the same
restriction enzyme (EcoRl). The fragments from sample A readily join with
those from sample B due to their cohesive (or sticky = complementary)
protruding ends. The dotted line within the recognition sequences separates
the base sequences belonging to the different fragments.
In addition, two or more restriction enzymes having different recognition sites
generate the same sticky end as is depicted in Fig 2.5 for BamHl, Bglll and
Sau3A; they all produce GATC sticky ends.

FIG. 2.5. Different restriction enzymes may generate the same sticky end
e.g. 5GATC3 in this case.
Therefore, the sticky ends produced by these three enzymes will be
complementary with each other, and can be regarded as having been
produced by a single enzyme for the purposes of joining together of the
different DNA fragments. The same will be true for any other group of such
enzymes that generate the same sticky ends.
There are cases where two different restriction enzymes recognize the same
target sequence, but one of them is able to recognize both methylated as well
non-methylated target sequences, while the other enzyme can recognize only
the non-methylated target sequence; such enzymes are known as
isoschizomers.
For example, restriction enzymes Hpall and MspI are isoschizomers; they
both recognize the sequence 5CCGG3 when it is unmethylated. But when
the second C of the sequence is methylated, Hpall can no longer recognize it,
while MspI recognizes it just as well as it does the unmethylated sequence.
Isoschizomers are very useful in determining the state of methylation of a
DNA molecule.
Some restriction enzymes, on the other hand, cut both the strands of a DNA
molecule at the same site so that the resulting termini or ends have blunt or

flush ends in which the two strands end at the same point (Fig. 2.3). The blunt
cut ends also can be effectively utilized for construction of recombinant DNAs
following one of several strategies.

Frequency of Recognition Sites:


The frequency of recognition sites for a restriction enzyme likely to be present
in a DNA molecule can be estimated theoretically if we make the following two
assumptions: (1) the frequencies of A, T. G and C in the molecule are equal,
i.e., it has a G: C content of 50%, and (2) the bases are present in a random
order.
In such a case, any given base, say, A, is expected to occur on, an overage,
once every 4 bases (=41), a sequence of two bases, e.g., AT, would occur
every 16 bases (=42) and so on. Therefore, a recognition sequence of 4
bases, e.g., 5CCGG3 (for Hpall), is expected to occur, on an average, once
every 44 (= 256) bp along a DNA molecule. In contrast, a 6 base recognition
sequence, e.g., 5GAATTC3 (for EcoRI) should be much less frequent and
occur once every 46 bp (= 4,096 bp).
The above estimates are, however, theoretical estimates, and the actual
situation may differ remarkably from the expectation. For example, X phage
genome is 49 kb long; therefore, it is expected to contain about 12 (=
49,000/4,096) recognition sites for a restriction enzyme having recognition
sequence of 6 bp. But the actual number of sites present for BamHl, Bglll and
Sail is only 5, 6 and 2, respectively, which is a reflection of the less than 50%
G: C content of DNA.

In addition, the restriction sites are not evenly distributed along a DNA
molecule; this fact is clearly demonstrated by the sizes of different fragments
obtained following restriction digestion of , genome with BamHl, Bglll and Sail
(Table 2.2). But it would also be noted that distribution of the sites is more
even for some enzymes than for others.