Canal system in Scypha
Body of Scypha consists of a complex system of pores and canals. This system is generally referred to as canal system or aquiferous system. Bodywall has cellular layers, outer pinacoderm and inner choanoderm. In between these two layers, there is a non-cellular gelatinous mesenchyme in between. But the bodywall is so folded as to form regularly arranged alternating invaginations and evaginations, establishing the sycon type of canal system. The various components of canal system of Scypha are:
1) Ostia or dermal pores
2) Incurrent canals
3) Prosopyles
4) Radial canals
5) Apopyles
6) Spongocoel
7) Osculum
8) Current of water
1) Ostia or dermal pores:
The external grooves of body surface are stretched over by a thin pore membrane. It bears two or more openings for the ingress of outside water into the body of sponge. These pores are known as Ostia (Latin, Ostium=door) or dermal pores. Because of the presence of contractile cells or myocytes around them, the ostia can reduce in diameter and thus regulate the amount of ingressing (incoming) water.
2) Incurrent canals:
These are the invaginated folds of bodywall and are also called inhalent canals. These communicate with outside through ostia but end blindly at their inner ends. Pinacocytes line these canals throughout.
3) Prosopyles:
Incurrent canals communicate with radial canals through intercellular spaces called prosopyles (Greek. Pros=near, pyle=gate)
4) Radial canals:
Evaginations of bodywall form thimble-shaped chambers lined by flagellated choanocytes. These chambers are called flagellated or radial canals.
Incurrent and radial canals are parallel and alternate with each other, both vertically and radially. The arrangement is such that, in a vertical or tangential section through the wall of a cylinder, each radial canal is surrounded on four sides by incurrent canals, and each incurrent canal is surrounded likewise by four radial canals.
Radial canals end blindly at their outer ends but lead at their inner ends into spongocoel.
5) Apopyles:
Openings of radial canals into spongocoel are called apopyles (Greek apo=away from, pyle=gate) or internal ostia. These are surrounded by contractile myocytes serving as a sphincter.
6) Spongocoel:
It is the large central cavity of the body forming the vertical axis of the cylinder (Greek sponges=sponge+koilos=hollow). In Leucosolenia, spongocoel is lined by flagellated collar cells or choanocytes. In Scypha, the choanocytes line the radial canals, whereas the spongocoel is lined with the epidermal pinacocytes.
7) Osculum:
Spongocoel leads to outside through a terminal opening, the osculum. The oscula are provided with sphincters to regulate the rate of water flow in the body. Sphincters are lined by special contractile pinacocytes.
Sphincters are lined by special contractile pinacocytes, called myocytes (Greek. Myos, muscle+kytos=cell)
8) Current of water
Flow of water in canal system is maintained by continuous beating of flagella of collar cells lining the radial canals. Every beat of a flagellum consists of a normal active stroke and a recovery stroke. Electron microscopy revealed that there is no coordination between the beating of flagella of adjacent cells. The course taken by water current into the canal system is as follows.
Water from outside through dermal ostia Incurrent canals through prosopyles
Radial canals
Through apopyles
to outside Spongocoel Radial canals
Zoology in the Classroom - is a blog for teachers and students of zoology. I have been teaching as Zoology teacher for the last 30 years. I post the notes or handouts that I supply to my students in my classroom. Hope this will benefit Zoology fraternity
Friday, September 23, 2016
Thursday, September 22, 2016
HERDMANIA TYPE STUDY
Affinities of Urochordates .
The typical tunicate looks like a non-chordate animal. If the life history of such animal is studied, the larval form reveals the chordate chara’4ters of that animal.
In 1816, Lamarck and Cuvier placed these animals in one class ‘Tunicata’. Allis described a compound Ascidian “Botryllus’ in 1756.KowalevskSi. Worked on the development ofthe Ascidian and placd them’in tunicates after observing the chordate features
.
Urochordates-Resemblance. With Chordate. :- .
1. Presence of dorsal tubular nerve cord,
2. Presence of Notochord,
3. Well developed pharynx with gill-slits.
4. Presence of endostyle on the ventral side of the pharynx
.5. Presence of atrium around pharynx.
.6. Presence of post - anal tail, with tail fin.
, Because of these chordate features tunicates are included in chordates group.
Urochordates-Resemblance with Amphioxus:
1. Presence of notochord.
2. Presence of dorsal tubular nerve cord.
3. Presence of large pharynx with gill slits.
4. Presence of atrium and atriopore.
5. Presence of muscle band.
Thus, the Urochordates show close relation with Cephalochordates. But the
Urochordates differ with other Chordates because of the following peculiar characters.
1 .Presence of retrogressive metamorphosis.
2. Absence of segmentation.
Because of these characters zoologists included these animals in a
Separate sub-phylum Urochordates.
During recent years many zoologists regarded the tunicates as primitive and ancestral forms to chordates as a whole.
previous topic urochordata classification
Urochordata Classification
Classification of Urochordata
Subphylum Urochordata is divided into three classes.
CLASS 1. ASCIDIACEA CLASS 2. THALIACEA CLASS : 3 . LARVACEA (APPENDICULARIA)
CLASS 1. ASCIDIACEA:
1. These are sedentary tunicates.
2. The body is covered by a test.
3. Pharynx is large and contains gill-slits.
4. Notochord, nerve-cord and tail are absent
5. These are Bisexual animals.
6. Life-history includes a typicalTadpole larva. The class Ascidiacea is divided into two orders.
Order 1. Enterogona
These ascidians bear one gonad in the intestinal loop. Neural gland is ventral to the ganglion. Tadpole larva is seen:
Ex: Ascidia and Ciona.
Order: 1. Pleurogona:
In these ascidians,gonads are paired and are present in the atrial wall. Neural gland is dorsal to the ganglion:
Ex : Herdmania, Botryllus.
CLASS 2. THALIACEA :-
1. These Urochordates are free-swimming and pelagic forms.
2. They are covered by transparent test.
3. The brachial and atrial apertures are placed at anterior and posterior ends.
4. Pharynx is small.
5. Gill-slits number is less.
6. Notochord, nerve-cord and tail are absent in the adult.
7. Asexual reproduction is by budding.
‘8. These are bisexual animals.
9. Tailed larva may be present or absent.
10. Alternation of generations can be seen in the life history.
The class thaliacea is divided into three orders.
1. Doliolida, 2. Pyrosomida and 3. Salpida.
Order 1. Doliolida (Cyclomyarla)
1. Barrel shaped body is completely covered by Muscle bands,
2. Pharynx is small
3. Number of gill slits will be small.
4. Tailed larva is seen
5. Sexual Blasto-zooid and asexual oozooid stages will alternate in the life cycle.
Ex :Doliolum.
Order 2. Pyrosomlda :
1. This order includes colonial forms.
2. Muscle bands are small and present at the ends.
3. Gill-slits are many.
4. Tailed larval stage is absent.
Ex : Pyrosoma (Luminescent colonial form).
Order 3. Salplda (Hemimyaria) :-
1. This order includes organisms whose body is prism like.
2. Muscle bands are complete dorsally and incomplete ventrally.
3. Only one pair of lateral gill slits are present.
4. Tailed larval stage is absent.
5. Life history includes alternation of generations.
Ex: Salpa.
CLASS : 3 . LARVACEA (APPENDICULARIA)
1. These are free - swimming, pelagic tunicates.
2. True’ test covering is lacking
3. They show loose gelatinous house.
4. This house is useful for filter feeding.
5. Two gill slits re present.
6. Atrium is absent. ..
7. Notochord and nerve cord are Persistent
8. They show tail throughout their life.
9. Neotenic forms are included.
Ex: Oikopleura. ‘
Urochordata Characters
Urochordata General Characters
The tunicates were first regarded as sponges. Lamark in 1816 placed Tunicata in between the Radiata and Vermes in his system of classification. Later, they were included in Mollusca. In 1866 Kowalevsky kept them in chordates.
Their chordate features are clearly seen in the larval stages. All Urochordates are marine and occur in all the seas. Majority of them are sedentary and some are pelagic.
1. Body shows variation in size and form.
2,. The body is un segmented and has no tail
3. The body is covered by a test. It is formed by tunicine which is
rallied to cellulose. Hence the name Tunicata.
4. Body wall shows one-layered epidermis, dermis is made by connective tissue and muscles, and atrial epithehum.
5.Celome is absent.
6. Atrial cavity surrounds the pharynx, into this cavity the gill slits,anus and genital ducts will open. It opens through atrial aperture.
7. Larva has notochord in the tail. It disappears during metamorphosis.
8. Respiratory system contains gills in the pharyngeal wall.
9. Ciliary mode of feeding is common.
10. Open type of Circulatory system is seen.
11. The heart is ventral and it periodically reverses its function.
12. Nervous system is represented by a single dorsal ganglion in the adult.
13. Excretion is carried on by nephrocytes.
14. Asexual reproduction is by budding.
15. Bisexual animal and cross fertilisation is favored.
16. Fertilization is external.
17. Development includes a minute, free swimming tadpole larva with a tail, a dorsal nerve cord, and a notochord in the tail. In some urochordates retrogressive metamorphosis is seen in the life history.
2 comments:
Herdmania Excretory Organ:
Excretory Organ Of Herdmania:
A neural gland is present above the brain in herdmaina. it Is brown in colour. It is present In the mantle. 1t is 4mm in diameter. It show branching tibuIes. At one end it leads into a short duct which opens by ciliated funnel above the dorsal tubercle. In the blood nephrocytes cell are present. They coiled waste matter. They come to the neural gland from there, they are sent out The neural gland secretes a hormone. It help in metamorphosis. Neural gland is considered homologous to pituitary glands of vertebrates.
Herdmania- Spicules
Herdmania- Spicules in the test:
In the test of Herdmania two types of calcareous spicules are Present. They are:
1) Microscieres : These are 40 to 80 microns in length. They are minute.
2) Megascleres : These are long spicules. They show different shapes. They are 1.5 to 3.5 mm in length. They are two types.
a) Spindle shaped Megascleres. They are 1.5 to 2.5 mm in length.
b) Pipette shaped Megascleres. They are 3.5 mm. in length.
The rnicroscleres are present only in the test. But the megascleres occur in the test, body wall, and viscera.
Herdmania-Spicules Function:
1) They give support to the test
2) They protect the animal from predators.
3) They fix mantle with test.
Herdmania- Nervous System
NERVOUS SYSTEM OF HERDMANIA:
Herdmania shows brain or nerve ganglion. It is 4 mm long. It is present in the mantle in between the two siphons. A neural gland is present above the brain. From the brain three nerves arise, one goes to the bronchial siphon, and two will go to the atrial siphon. The brain represents the degenerated nervous system of the larva.
Receptors:
1. Red pigmented spots on the test are photoreceptors. They are sensitive to light.
2. Sensory cells of the margins of siphons and tentacles are tango receptors. They are sensitive to touch.
3. The cells on the margins of siphons are rheo receptors. They are sensitive to water currents.
4. Cells lining the siphons are thermo receptors. They are sensitive to changes of temperature.
Herdmania Gonads
Gonads of Herdmanla :
Ans: Herdmania is a bisexual animal. It is a protogynous animal. Ovary matures’ first. Hence cross fertilization takes place.
Herdmania contains a pair of gonads. The left gonad lies in the intestinal loop above the heart. Each gonad shows 10 to 25 lobes arranged in two rows. The median lobe is single and large. Other lobes are oval in shape.
Each lobe shows outer large testicular part and inner small ovarian part. The testicular part is brick red in colour and produces sperms. The ovarian part is pink in colour and produce ova. From each testicular part sperm ductule will arise. They open into spermduct. From each ovarian part ovarian ductule will come. They open into the oviduct. Each gonad has an oviduct and spermduct. They run parallelly and open separately into cloaca behind anus.
Herdmania -Retrogressive Metamorphosis
Retrogressive Metmorphosis in herdmania
During metamorphosis the larva will loose all the chordate characters and attains an invertebrate like form. This type of metamorphosis, where highly advanced larval form ends in a lowly organised adult is called retrogressive metamorphosis.
Fixation of the larva: The larva swims for some time without feeding. It is fixed to a sub- stratum with the help of the adhesive papillae. It stands erect with the tail upwards. Then it undergoes retrogressive metamorphosis.
Changes during metamorphosis:
1.Notochord, nerve cord muscles and tail will be reduced. All the above structures will help the larva to swim freely in the water. But they are not useful to the sedentary adult
2.. The alimentary canal becomes complicated. The pharynx en larges ln size. The number of gill slits will increase by divisions. The stomach and intestine will grow.
3. The nervous system is reduced and the anterior part of nervous system is developed into a small neuralganglion attached to it neural gland is present. ;:
4. The atrial cavity enlarges into a sac like structure.
5. The eyespot and statocyst will completely disappear.
6. Gonads develop from mesencyme.
When these changes are taking place, the region between the adhesive papillae and mouth grows very rapidly. At the same time the growth of the dorsal region is stopped. Because of this, the body rotates through 1800 angle and mouth is brought to the top
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HERDMANIA TADPOLE LARVA
ORGANISATION OF HERDMANIA TADPOLE LARVA:
Herdmania is a hermaphrodite animal. The fertilised eggs undergo holoblastic unequal clevage and it develops into blastula. it shows upper micromeres and lower macromeres. By invagination of the macromeres gastrulation takes place and gastrula is formed. This gastrula develops into a tailed larva called Ascidian Tadpole larva. (Herdmanis life history, is not clearly known. Clavilina’s life history is known. It is followed here.
The larva is 3 mm in length. It has short oval body and a long tail.
This larva shows all the chordate features.
1) The body is covered by thin test.
2) The tail is long and shows a tail fin or caudal fin.
3) The tail is supported by notochord. Hence it comes under urochordata.
4) On the dorsal side above the notochord hollow nerve cord is present. This nerve cord is enlarged at the anterior end as a cerebral vesicle. In the cerebral vesicle pigmented eye spot is present. Statocyst is also present. They work as sense organs.
5) On either side of the notochord in the tail region muscles are Present which are helpful in the locomotion.
6) On the trunk region digestive system is present. It shows large pharynx with few gills slits. They open into atrium. On the mid ventral floor of the pharynx an endostyle is present.
7) Atrium opens out through atriopore.
8) Below the pharynx on the ventral side a muscular heart is present.
9) On the anterior end of the trunk three adhesive papillae are present These are very much useful to attach the larva to the substratum
This Herdmania tadpole larva shows all chordate characters. This larvae ‘undergoes retrogressive’ metamorphosis and develops into adult Herdmania.
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Herdmanla -External characters
External characters of Herdmania
Herdmania is a simple ascidian, In Indian ocean this genus Is represented by 4 species. ,
1. Herdmania pallida, 2. H.ennurensis, 3. H.mauritiana 4. H. ceylonica.
Herdmania belongs to,
Phylum: Chordata,
Sub-phylum: Urochordata,
Class: Ascidiacea,
Order: Pleurogona.
Herdmania is a marine and sedentary animal. It is fixed to rocky substratum by a flat base. When it is disturbed, it suddenly contracts its body, and emits inner contents with force through its apertures. Hence it is called Sea squirt.
External Features :-
It is potato like in shape. It is pink in colour. On the free side, body shows two projections, the brançhial and atrial siphons. The branchial siphon is short. The branchial siphon shows a branchial aperture or the mouth. The atrial siphon is longer. It bears the atrial aperture. Both the openings are bounded by four lips.
Herdmania - external characters.
Test of Herdmania:The body of this animal is covered and protected by test. It Ls a thick, leathery covering of the body.It is secreted by the epidermis of the body wall. It has matrix, corpuscles, fibrils, blood vessels and spicules.
The matrix is composed of tunicin, which is cellulose. The cells in the test are of six types, large eosinophilous cells, amoeboid cells, small eosinophilous cells, vacuolated cells, receptor cells and nerve cells.
Fine fibrils present in the matrix. In the test blood vessels are present. In the test the spicules are calcareous spicules. They are microscleres, and megascleres.
The test protects the body. Anchors the animals to substratum. Its spicules form a supporting frame work.
Herdmania Body wall :
The body-wall of Herdmania is called Mantle. It is thick, and muscular in the antero-dorsal region of the body. It is thin, non-muscular and transparent in the postero ventral region. It shows epidermis, mesenchyme, and inner epidermis.
1.Epidermis : It Is single layer of cells. It covers the bronchial and atrial apertures and siphons. The epidermis is interrupted at places where spicules and blood-vessels pass from the mantle into the test.
. 2. Mesènchyme: It develops from the mesoderm. It has connective tissue containing blood-sinuses, muscle-fibers, nerve fibers and cells. The muscle fibers are long and flat. They contain large nuclei.
3. Inner Epidermis: It is single layer of flat cells. It forms the lining of the atrial cavity. .
‘1.The body-wall protects visceral organs.
2. The outer epidermis secretes the test.
3. The musculature brings contraction of the body and the siphons.
Herdmania Atrium:
In Herdmania coelome is not developed. Atrium is a spacious ectoderm lined cavity. it is covered by the mantle A part of the atrium surrounds the pharynx. The stigmata of the pharynx open into this cavity. Part of the atrium is dorsal to the pharynx. It is very wide and is called cloaca. The rectum and gonoducts open into this. The cloaca opens to the exterior through atrial siphon and trial aperture. The atrial siphon shows a ring of processes called atrial tentacles at its base.
The typical tunicate looks like a non-chordate animal. If the life history of such animal is studied, the larval form reveals the chordate chara’4ters of that animal.
In 1816, Lamarck and Cuvier placed these animals in one class ‘Tunicata’. Allis described a compound Ascidian “Botryllus’ in 1756.KowalevskSi. Worked on the development ofthe Ascidian and placd them’in tunicates after observing the chordate features
.
Urochordates-Resemblance. With Chordate. :- .
1. Presence of dorsal tubular nerve cord,
2. Presence of Notochord,
3. Well developed pharynx with gill-slits.
4. Presence of endostyle on the ventral side of the pharynx
.5. Presence of atrium around pharynx.
.6. Presence of post - anal tail, with tail fin.
, Because of these chordate features tunicates are included in chordates group.
Urochordates-Resemblance with Amphioxus:
1. Presence of notochord.
2. Presence of dorsal tubular nerve cord.
3. Presence of large pharynx with gill slits.
4. Presence of atrium and atriopore.
5. Presence of muscle band.
Thus, the Urochordates show close relation with Cephalochordates. But the
Urochordates differ with other Chordates because of the following peculiar characters.
1 .Presence of retrogressive metamorphosis.
2. Absence of segmentation.
Because of these characters zoologists included these animals in a
Separate sub-phylum Urochordates.
During recent years many zoologists regarded the tunicates as primitive and ancestral forms to chordates as a whole.
previous topic urochordata classification
Urochordata Classification
Classification of Urochordata
Subphylum Urochordata is divided into three classes.
CLASS 1. ASCIDIACEA CLASS 2. THALIACEA CLASS : 3 . LARVACEA (APPENDICULARIA)
CLASS 1. ASCIDIACEA:
1. These are sedentary tunicates.
2. The body is covered by a test.
3. Pharynx is large and contains gill-slits.
4. Notochord, nerve-cord and tail are absent
5. These are Bisexual animals.
6. Life-history includes a typicalTadpole larva. The class Ascidiacea is divided into two orders.
Order 1. Enterogona
These ascidians bear one gonad in the intestinal loop. Neural gland is ventral to the ganglion. Tadpole larva is seen:
Ex: Ascidia and Ciona.
Order: 1. Pleurogona:
In these ascidians,gonads are paired and are present in the atrial wall. Neural gland is dorsal to the ganglion:
Ex : Herdmania, Botryllus.
CLASS 2. THALIACEA :-
1. These Urochordates are free-swimming and pelagic forms.
2. They are covered by transparent test.
3. The brachial and atrial apertures are placed at anterior and posterior ends.
4. Pharynx is small.
5. Gill-slits number is less.
6. Notochord, nerve-cord and tail are absent in the adult.
7. Asexual reproduction is by budding.
‘8. These are bisexual animals.
9. Tailed larva may be present or absent.
10. Alternation of generations can be seen in the life history.
The class thaliacea is divided into three orders.
1. Doliolida, 2. Pyrosomida and 3. Salpida.
Order 1. Doliolida (Cyclomyarla)
1. Barrel shaped body is completely covered by Muscle bands,
2. Pharynx is small
3. Number of gill slits will be small.
4. Tailed larva is seen
5. Sexual Blasto-zooid and asexual oozooid stages will alternate in the life cycle.
Ex :Doliolum.
Order 2. Pyrosomlda :
1. This order includes colonial forms.
2. Muscle bands are small and present at the ends.
3. Gill-slits are many.
4. Tailed larval stage is absent.
Ex : Pyrosoma (Luminescent colonial form).
Order 3. Salplda (Hemimyaria) :-
1. This order includes organisms whose body is prism like.
2. Muscle bands are complete dorsally and incomplete ventrally.
3. Only one pair of lateral gill slits are present.
4. Tailed larval stage is absent.
5. Life history includes alternation of generations.
Ex: Salpa.
CLASS : 3 . LARVACEA (APPENDICULARIA)
1. These are free - swimming, pelagic tunicates.
2. True’ test covering is lacking
3. They show loose gelatinous house.
4. This house is useful for filter feeding.
5. Two gill slits re present.
6. Atrium is absent. ..
7. Notochord and nerve cord are Persistent
8. They show tail throughout their life.
9. Neotenic forms are included.
Ex: Oikopleura. ‘
Urochordata Characters
Urochordata General Characters
The tunicates were first regarded as sponges. Lamark in 1816 placed Tunicata in between the Radiata and Vermes in his system of classification. Later, they were included in Mollusca. In 1866 Kowalevsky kept them in chordates.
Their chordate features are clearly seen in the larval stages. All Urochordates are marine and occur in all the seas. Majority of them are sedentary and some are pelagic.
1. Body shows variation in size and form.
2,. The body is un segmented and has no tail
3. The body is covered by a test. It is formed by tunicine which is
rallied to cellulose. Hence the name Tunicata.
4. Body wall shows one-layered epidermis, dermis is made by connective tissue and muscles, and atrial epithehum.
5.Celome is absent.
6. Atrial cavity surrounds the pharynx, into this cavity the gill slits,anus and genital ducts will open. It opens through atrial aperture.
7. Larva has notochord in the tail. It disappears during metamorphosis.
8. Respiratory system contains gills in the pharyngeal wall.
9. Ciliary mode of feeding is common.
10. Open type of Circulatory system is seen.
11. The heart is ventral and it periodically reverses its function.
12. Nervous system is represented by a single dorsal ganglion in the adult.
13. Excretion is carried on by nephrocytes.
14. Asexual reproduction is by budding.
15. Bisexual animal and cross fertilisation is favored.
16. Fertilization is external.
17. Development includes a minute, free swimming tadpole larva with a tail, a dorsal nerve cord, and a notochord in the tail. In some urochordates retrogressive metamorphosis is seen in the life history.
2 comments:
Herdmania Excretory Organ:
Excretory Organ Of Herdmania:
A neural gland is present above the brain in herdmaina. it Is brown in colour. It is present In the mantle. 1t is 4mm in diameter. It show branching tibuIes. At one end it leads into a short duct which opens by ciliated funnel above the dorsal tubercle. In the blood nephrocytes cell are present. They coiled waste matter. They come to the neural gland from there, they are sent out The neural gland secretes a hormone. It help in metamorphosis. Neural gland is considered homologous to pituitary glands of vertebrates.
Herdmania- Spicules
Herdmania- Spicules in the test:
In the test of Herdmania two types of calcareous spicules are Present. They are:
1) Microscieres : These are 40 to 80 microns in length. They are minute.
2) Megascleres : These are long spicules. They show different shapes. They are 1.5 to 3.5 mm in length. They are two types.
a) Spindle shaped Megascleres. They are 1.5 to 2.5 mm in length.
b) Pipette shaped Megascleres. They are 3.5 mm. in length.
The rnicroscleres are present only in the test. But the megascleres occur in the test, body wall, and viscera.
Herdmania-Spicules Function:
1) They give support to the test
2) They protect the animal from predators.
3) They fix mantle with test.
Herdmania- Nervous System
NERVOUS SYSTEM OF HERDMANIA:
Herdmania shows brain or nerve ganglion. It is 4 mm long. It is present in the mantle in between the two siphons. A neural gland is present above the brain. From the brain three nerves arise, one goes to the bronchial siphon, and two will go to the atrial siphon. The brain represents the degenerated nervous system of the larva.
Receptors:
1. Red pigmented spots on the test are photoreceptors. They are sensitive to light.
2. Sensory cells of the margins of siphons and tentacles are tango receptors. They are sensitive to touch.
3. The cells on the margins of siphons are rheo receptors. They are sensitive to water currents.
4. Cells lining the siphons are thermo receptors. They are sensitive to changes of temperature.
Herdmania Gonads
Gonads of Herdmanla :
Ans: Herdmania is a bisexual animal. It is a protogynous animal. Ovary matures’ first. Hence cross fertilization takes place.
Herdmania contains a pair of gonads. The left gonad lies in the intestinal loop above the heart. Each gonad shows 10 to 25 lobes arranged in two rows. The median lobe is single and large. Other lobes are oval in shape.
Each lobe shows outer large testicular part and inner small ovarian part. The testicular part is brick red in colour and produces sperms. The ovarian part is pink in colour and produce ova. From each testicular part sperm ductule will arise. They open into spermduct. From each ovarian part ovarian ductule will come. They open into the oviduct. Each gonad has an oviduct and spermduct. They run parallelly and open separately into cloaca behind anus.
Herdmania -Retrogressive Metamorphosis
Retrogressive Metmorphosis in herdmania
During metamorphosis the larva will loose all the chordate characters and attains an invertebrate like form. This type of metamorphosis, where highly advanced larval form ends in a lowly organised adult is called retrogressive metamorphosis.
Fixation of the larva: The larva swims for some time without feeding. It is fixed to a sub- stratum with the help of the adhesive papillae. It stands erect with the tail upwards. Then it undergoes retrogressive metamorphosis.
Changes during metamorphosis:
1.Notochord, nerve cord muscles and tail will be reduced. All the above structures will help the larva to swim freely in the water. But they are not useful to the sedentary adult
2.. The alimentary canal becomes complicated. The pharynx en larges ln size. The number of gill slits will increase by divisions. The stomach and intestine will grow.
3. The nervous system is reduced and the anterior part of nervous system is developed into a small neuralganglion attached to it neural gland is present. ;:
4. The atrial cavity enlarges into a sac like structure.
5. The eyespot and statocyst will completely disappear.
6. Gonads develop from mesencyme.
When these changes are taking place, the region between the adhesive papillae and mouth grows very rapidly. At the same time the growth of the dorsal region is stopped. Because of this, the body rotates through 1800 angle and mouth is brought to the top
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HERDMANIA TADPOLE LARVA
ORGANISATION OF HERDMANIA TADPOLE LARVA:
Herdmania is a hermaphrodite animal. The fertilised eggs undergo holoblastic unequal clevage and it develops into blastula. it shows upper micromeres and lower macromeres. By invagination of the macromeres gastrulation takes place and gastrula is formed. This gastrula develops into a tailed larva called Ascidian Tadpole larva. (Herdmanis life history, is not clearly known. Clavilina’s life history is known. It is followed here.
The larva is 3 mm in length. It has short oval body and a long tail.
This larva shows all the chordate features.
1) The body is covered by thin test.
2) The tail is long and shows a tail fin or caudal fin.
3) The tail is supported by notochord. Hence it comes under urochordata.
4) On the dorsal side above the notochord hollow nerve cord is present. This nerve cord is enlarged at the anterior end as a cerebral vesicle. In the cerebral vesicle pigmented eye spot is present. Statocyst is also present. They work as sense organs.
5) On either side of the notochord in the tail region muscles are Present which are helpful in the locomotion.
6) On the trunk region digestive system is present. It shows large pharynx with few gills slits. They open into atrium. On the mid ventral floor of the pharynx an endostyle is present.
7) Atrium opens out through atriopore.
8) Below the pharynx on the ventral side a muscular heart is present.
9) On the anterior end of the trunk three adhesive papillae are present These are very much useful to attach the larva to the substratum
This Herdmania tadpole larva shows all chordate characters. This larvae ‘undergoes retrogressive’ metamorphosis and develops into adult Herdmania.
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Herdmanla -External characters
External characters of Herdmania
Herdmania is a simple ascidian, In Indian ocean this genus Is represented by 4 species. ,
1. Herdmania pallida, 2. H.ennurensis, 3. H.mauritiana 4. H. ceylonica.
Herdmania belongs to,
Phylum: Chordata,
Sub-phylum: Urochordata,
Class: Ascidiacea,
Order: Pleurogona.
Herdmania is a marine and sedentary animal. It is fixed to rocky substratum by a flat base. When it is disturbed, it suddenly contracts its body, and emits inner contents with force through its apertures. Hence it is called Sea squirt.
External Features :-
It is potato like in shape. It is pink in colour. On the free side, body shows two projections, the brançhial and atrial siphons. The branchial siphon is short. The branchial siphon shows a branchial aperture or the mouth. The atrial siphon is longer. It bears the atrial aperture. Both the openings are bounded by four lips.
Herdmania - external characters.
Test of Herdmania:The body of this animal is covered and protected by test. It Ls a thick, leathery covering of the body.It is secreted by the epidermis of the body wall. It has matrix, corpuscles, fibrils, blood vessels and spicules.
The matrix is composed of tunicin, which is cellulose. The cells in the test are of six types, large eosinophilous cells, amoeboid cells, small eosinophilous cells, vacuolated cells, receptor cells and nerve cells.
Fine fibrils present in the matrix. In the test blood vessels are present. In the test the spicules are calcareous spicules. They are microscleres, and megascleres.
The test protects the body. Anchors the animals to substratum. Its spicules form a supporting frame work.
Herdmania Body wall :
The body-wall of Herdmania is called Mantle. It is thick, and muscular in the antero-dorsal region of the body. It is thin, non-muscular and transparent in the postero ventral region. It shows epidermis, mesenchyme, and inner epidermis.
1.Epidermis : It Is single layer of cells. It covers the bronchial and atrial apertures and siphons. The epidermis is interrupted at places where spicules and blood-vessels pass from the mantle into the test.
. 2. Mesènchyme: It develops from the mesoderm. It has connective tissue containing blood-sinuses, muscle-fibers, nerve fibers and cells. The muscle fibers are long and flat. They contain large nuclei.
3. Inner Epidermis: It is single layer of flat cells. It forms the lining of the atrial cavity. .
‘1.The body-wall protects visceral organs.
2. The outer epidermis secretes the test.
3. The musculature brings contraction of the body and the siphons.
Herdmania Atrium:
In Herdmania coelome is not developed. Atrium is a spacious ectoderm lined cavity. it is covered by the mantle A part of the atrium surrounds the pharynx. The stigmata of the pharynx open into this cavity. Part of the atrium is dorsal to the pharynx. It is very wide and is called cloaca. The rectum and gonoducts open into this. The cloaca opens to the exterior through atrial siphon and trial aperture. The atrial siphon shows a ring of processes called atrial tentacles at its base.
Thursday, September 15, 2016
Practical Manual B.Voc
Etroplus surantensis
Phylum: Chordata
Class: Actinopterygii
Order: Perciformes
Family: Cichlidae
Genus: Etroplus
Etroplus suratensis is a euryhaline species that inhabits mainly brackish water and river mouths. It is an oval-shaped cichlid with a short snout, small mouth not extending past the front margin of the eye with a greyish-green colouration on the flanks, with 6 to 8 dark bars and a dark spot at base of the pectoral fin. Most scales on the sides are with a pearly spot (Costa 2007). Macrophytic fragments form the most important component of its diet along with molluscs, although detritus, diatoms, and animal matter are also ingested (De Silva et al. 1984). Many aspects of this species was studies in order to assess it's suitability for culture in ponds and tanks by Jayaprakash et al. (1990).Adults engage in altruistic multiple parental care where several adults care for a single brood that presumably were spawned by only two of the adults (Ward and Wyman 1977).
Mugil cephalus
Phylum: Chordata
Class: Actinopterygii
Order: Mugiliformes
Family: Mugilidae
Genus: Mugil
Mugil cephalus is cosmopolitan in the coastal waters of most tropical and subtropical zones. In the western Atlantic Ocean, it is found from Nova Scotia, Canada south to Brazil, including the Gulf of Mexico. It is absent in the Bahamas and the Caribbean Sea. In the eastern Atlantic Ocean, the striped mullet occurs from the Bay of Biscay (France) to South Africa, including the Mediterranean Sea and the Black Sea. The eastern Pacific Ocean range includes southern California south to Chile.
The flathead grey mullet is catadromous, frequently found coastally in estuaries and freshwater environments. Adult mullet have been found in waters ranging from zero salinity to 75‰, while juveniles can only tolerate such wide salinity ranges after they reach lengths of 4–7 cm. Adults form huge schools near the surface over sandy or muddy bottoms and dense vegetation and migrate offshore to spawn in large aggregations. The larvae move inshore to extremely shallow water, which provides cover from predators as well as a rich feeding ground. After reaching 5 cm in length, these young mullet move into slightly deeper waters.
Flathead grey mullet is a diurnal feeder, consuming mainly zooplankton, dead plant matter, and detritus. Mullet have thick-walled gizzard-like segments in their stomach along with a long gastrointestinal tract that enables them to feed on detritus. They are an ecologically important link in the energy flow within estuarine communities. Feeding by sucking up the top layer of sediments, flathead grey mullet remove detritus and microalgae. They also pick up some sediment which functions to grind food in the gizzard-like portion of the stomach. Mullet also graze on epiphytes and epifauna from seagrasses as well as ingest surface scum containing microalgae at the air-water interface. Larval flathead grey mullet feed primarily on microcrustaceans. Copepods, mosquito larvae, and plant debris have been found in the stomach contents of larvae under 35 mm in length. The amount of sand and detritus in the stomach contents increases with length, indicating that more food is ingested from the bottom substrate as the fish matures.
Eleutheronema tetradachylum
Phylum: Chordata
Class: Actinopterygii
Order: Perciformes
Family: Polynemidae
Genus: Eleutheronema
Dorsal spines (total): 9; Dorsal soft rays (total): 13-15; Anal spines: 3; Anal soft rays: 14 - 16. Pectoral filaments 4; fin membranes vivid yellow in life, except in large specimens, > ca 35 cm SL. Vomer with deciduous tooth plates on both sides, except in juveniles. Posterior part of maxilla deep, 3-4% of SL. Short tooth plate extension onto lateral surface of lower jaw. 7-9% SL (Ref. 41639).
Adults occur mainly over shallow muddy bottoms in coastal waters. Also enter rivers (Ref. 3479, 6390, 11230). Juveniles found in estuaries. During winter, adults ascend the rivers. They usually form loose schools, although larger fish are more often observed in pairs or singly (Ref. 6390). Feed on prawns and fish (largely members of Mugilidae, Engraulidae, and Sciaenidae) with occasional polychaetes. Frequency of crustaceans to fish in the diet varies seasonally. Larvae (7-30 mm TL) feed mainly on copepods and mysids but also take shrimps and prawn larvae (Ref. 57343). Juveniles (31-60 TL) feed on prawns shrimps and mysids (Ref. 57343). Protandrous hermaphrodites. Marketed fresh, frozen, and dried or salted.
Phylum: Chordata
Class: Actinopterygii
Order: Perciformes
Family: Cichlidae
Genus: Etroplus
Etroplus suratensis is a euryhaline species that inhabits mainly brackish water and river mouths. It is an oval-shaped cichlid with a short snout, small mouth not extending past the front margin of the eye with a greyish-green colouration on the flanks, with 6 to 8 dark bars and a dark spot at base of the pectoral fin. Most scales on the sides are with a pearly spot (Costa 2007). Macrophytic fragments form the most important component of its diet along with molluscs, although detritus, diatoms, and animal matter are also ingested (De Silva et al. 1984). Many aspects of this species was studies in order to assess it's suitability for culture in ponds and tanks by Jayaprakash et al. (1990).Adults engage in altruistic multiple parental care where several adults care for a single brood that presumably were spawned by only two of the adults (Ward and Wyman 1977).
Mugil cephalus
Phylum: Chordata
Class: Actinopterygii
Order: Mugiliformes
Family: Mugilidae
Genus: Mugil
Mugil cephalus is cosmopolitan in the coastal waters of most tropical and subtropical zones. In the western Atlantic Ocean, it is found from Nova Scotia, Canada south to Brazil, including the Gulf of Mexico. It is absent in the Bahamas and the Caribbean Sea. In the eastern Atlantic Ocean, the striped mullet occurs from the Bay of Biscay (France) to South Africa, including the Mediterranean Sea and the Black Sea. The eastern Pacific Ocean range includes southern California south to Chile.
The flathead grey mullet is catadromous, frequently found coastally in estuaries and freshwater environments. Adult mullet have been found in waters ranging from zero salinity to 75‰, while juveniles can only tolerate such wide salinity ranges after they reach lengths of 4–7 cm. Adults form huge schools near the surface over sandy or muddy bottoms and dense vegetation and migrate offshore to spawn in large aggregations. The larvae move inshore to extremely shallow water, which provides cover from predators as well as a rich feeding ground. After reaching 5 cm in length, these young mullet move into slightly deeper waters.
Flathead grey mullet is a diurnal feeder, consuming mainly zooplankton, dead plant matter, and detritus. Mullet have thick-walled gizzard-like segments in their stomach along with a long gastrointestinal tract that enables them to feed on detritus. They are an ecologically important link in the energy flow within estuarine communities. Feeding by sucking up the top layer of sediments, flathead grey mullet remove detritus and microalgae. They also pick up some sediment which functions to grind food in the gizzard-like portion of the stomach. Mullet also graze on epiphytes and epifauna from seagrasses as well as ingest surface scum containing microalgae at the air-water interface. Larval flathead grey mullet feed primarily on microcrustaceans. Copepods, mosquito larvae, and plant debris have been found in the stomach contents of larvae under 35 mm in length. The amount of sand and detritus in the stomach contents increases with length, indicating that more food is ingested from the bottom substrate as the fish matures.
Eleutheronema tetradachylum
Phylum: Chordata
Class: Actinopterygii
Order: Perciformes
Family: Polynemidae
Genus: Eleutheronema
Dorsal spines (total): 9; Dorsal soft rays (total): 13-15; Anal spines: 3; Anal soft rays: 14 - 16. Pectoral filaments 4; fin membranes vivid yellow in life, except in large specimens, > ca 35 cm SL. Vomer with deciduous tooth plates on both sides, except in juveniles. Posterior part of maxilla deep, 3-4% of SL. Short tooth plate extension onto lateral surface of lower jaw. 7-9% SL (Ref. 41639).
Adults occur mainly over shallow muddy bottoms in coastal waters. Also enter rivers (Ref. 3479, 6390, 11230). Juveniles found in estuaries. During winter, adults ascend the rivers. They usually form loose schools, although larger fish are more often observed in pairs or singly (Ref. 6390). Feed on prawns and fish (largely members of Mugilidae, Engraulidae, and Sciaenidae) with occasional polychaetes. Frequency of crustaceans to fish in the diet varies seasonally. Larvae (7-30 mm TL) feed mainly on copepods and mysids but also take shrimps and prawn larvae (Ref. 57343). Juveniles (31-60 TL) feed on prawns shrimps and mysids (Ref. 57343). Protandrous hermaphrodites. Marketed fresh, frozen, and dried or salted.
Thursday, September 8, 2016
Gene Cloning
Gene Cloning – Enzymatic cleavage of DNA, Restriction enzymes (Endonucleases) and Ligation.
Gene Cloning
To clone means to make identical copies. Gene cloning means production of a number of similar copies of a required gene. DNA cloning involves separating a specific gene or DNA segment from a larger chromosome, attaching it to a small carrier DNA. The resultant hybrid DNA is called recombinant DNA, which is transferred to a proper host (bacteria, virus or yeast) and replicated to make multiple copies of the selected gene.
This technology has made it possible to isolate, clone and produce DNA for all the genes in appropriate quantity so that they can be sequenced and characterized. Similarly, some of the genes which are expressed at very low level, can be cloned and desired amount of recombinant proteins can be produced.
Gene cloning involves the following steps
1. Cutting the DNA to be cloned from the chromosomal using sequence specific Restriction Endonuclease.
2. Selecting a cloning vector (a small molecule capable of self-replicating inside host cells), and cutting the cloning vector with the same restriction endonuclease to produce sticky ends.
3. Incubating the vector and subject DNA to join together DNA ligase. The resultant DNA is called recombinant DNA.
4. Transferring the reconbinant DNA to an appropriate host such as bacteria, virus or yeast which will provide necessory biomachinary for DNA replication.
5. Identifying the host cells that contain the recombinant DNA.
Enzymatic Cleavage of DNA
To cut the DNA at specific sites Restriction endonucleases are used. (RENs).
Restrictions Endonucleases:
A Restriction Endonuclease is an enzyme that cuts DNA at specific recognition points known as Restriction sites. These are most important groups of enzymes for manipulation of DNA. These enzymes were discovered in Escherichia coli. In bacteriophages these enzymes restrict the replication of viral DNA. Many types of restriction endonucleases were isolated. RENs were named based on the bacterial from which they were isolated.
The first letter of the enzyme indicates the genus name and next two letters the species name, followed by strain name and finally a number indicating the order of discovery. Eg. EcoRI: here E represents Escherichia, co represents coli, R represents the strain and I represents the first endonuclease.
Types of Endonucleases: over 3000 RENs have been studied in detail and more than 600 are available commercially. Naturally occurring Endonucleases are categorized in to four groups namely type I, II, III and IV based on their nature of their restriction sites. All types of enzymes recognize a specific DNA sequence and cleave that DNA at that point. They differ in their recognition sequence and cofactor requirements.
Type I RENs cuts DNA at random location as far as 1000 or more Basepairs from the recognition site. Type II cuts approximately 25 base pairs from the site. Type I and Type III require ATP. They are large enzymes with multiple subunits.
Type II RENs cut the DNA with in the recognized sequence without the need of ATP. They are smaller and simpler. Hence they are predominantly used in biotechnology.
Most of the Type II RENs generate Sticky ends. The open ends of the DNA molecule after the cut are called as Sticky ends.
The Length of restriction recognition sites varies. The Enzyme EcoRI, SacI etc recognize 6 basepair sequence of DNA. Most of the recognition sequences are palindromes – they read the same forward and backward.
Some of the important RENS and their restriction sites.
Enzyme Source Recognition Sequence Cut
EcoRI
Escherichia coli
5'GAATTC
3'CTTAAG 5'---G AATTC---3'
3'---CTTAA G---5'
EcoRII
Escherichia coli
5'CCWGG
3'GGWCC 5'--- CCWGG---3'
3'---GGWCC ---5'
BamHI
Bacillus amyloliquefaciens
5'GGATCC
3'CCTAGG 5'---G GATCC---3'
3'---CCTAG G---5'
HindIII
Haemophilus influenzae
5'AAGCTT
3'TTCGAA 5'---A AGCTT---3'
3'---TTCGA A---5'
TaqI
Thermus aquaticus
5'TCGA
3'AGCT 5'---T CGA---3'
3'---AGC T---5'
For convenience it is usual practice to simplifly the description of recognition sequences by showing only one strand of DNA, which runs in the 5’ to 3’ direction. Thus the EcoRI recognition sequence would be shown as G\AATTC.
Restriction enzymes with same sequence specificity and cut site are known as isochizomers. Enzymes that recognize the same sequences but cleave at different points are known as Neochizomers. Under extreme conditions like rise in pH, Low ionic strength) RENs are capable of cleaving sequences which are similar but not identical to their definied recognition sequences.
Applications of Restriction Endonucleases:
1. RENs formed in different bacteria can be used to break the DNA of any organism and the required DNA segment can be introduced in the DNA of another organism to produce Recombinant DNA
2. Restriction enzymes are highly useful to get desirable DNA segments. This is because these enzymes break the DNA at specific sites.
3. The DNAsegment isolated with the help of restriction enzymes can be used in DNA probe, genomic libraries and cDNA libraries.
4. With the isolated DNA segments, mRNA can be transcribed with which required proteins can be synthesized.
5. In restriction Fragment Length Polymorphism (RFLP) also restriction enzymes are very useful.
Enzymatic ligation of DNA
To hybridize (attach) the DNA fragments formed by the RENs to a plasmid DNA, the ends of both DNA are to be attached by forming hydrogen bonds and diester bonds. This process of attachment of the DNA fragments is known as Ligation. It is done by certain specialized enzymes called Ligases.
Ligase: (ligare = to glue together) is a special type of enzyme that can link together two DNA strands that have double strand break.
Mertz and Davis, 1972 first succeeded in producing rDNA in Escherichia coli by ligation of the sticky ends of DNA with ligase.
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3’hydroxyl ends of one nucleotide with the 5’ phosphate end of another. ATP is required for ligation. Ligase will also work with blunt ends, but neets high concentrations of enzymes.
In mammals there are four specific types of ligases.ie. DNA ligases, I, II, III and IV. LIgases can be classified into two groups on their requirement for ATP and NAD+ as co factors. All eukaryotic enzymes are ATP dependent, whereas most prokaryotic enzymes require NAD+ for their activity.
Most experiments use T4 DNA ligase (isolated from bacteriophase T4) which is most active at 25oC. High temperatures disrupts hydrogen bonding. The commonly available DNA ligases were originally discovered in bacteriophase T4, E.coli and other bacteria.
Applications of Ligases
DNA ligases are essential tools in modern molecular biology for generating rDNA sequences such as
1. Joining double stranded DNA with cohesive or blunt ends
2. Joining of oligonucleotide linkers or adaptors to blunt ended DNA
3. Repairing in duplex DNA, RNA or DNA-RNA hybrids
4. DNA ligases are used with restriction enzyes to insert DNA fragments often genes into plasmids.
Transgenesis and Production of transgenic animals (Fish and Goat).
Transgenesis is the process of introducing an exogenous gene – called transgene- into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. A transgene is the name given to the introduced DNA. The term transgenesis was coined by Gordon and Ruddle in 1981.
Animals produced through transgenesis are called Transgenic animals. Transgenic animals are genetically modified organisms with a new hereditary character.
Transgenic animals can be used to produce valuable products. Many proteins produced by transgenic animals are important for medical applications. For example, a transgenic pig has been produced with the ability to synthesize human hemoglobin for use as a blood substitute. Transgenic goat has been developed to produce a protein needed by the patients suffering from cystic fibrosis. Transgenisis is essential for improving the quality and quantity of the eggs, meat, milk etc, in addition to drug resistant animals.
The Mouse is the first animal used for transgenesis. RD Palmiter and RL Brinter (1982) isolated gene for growth hormone in human being. This gene was ligated with plasmid pBR322 to produce rDNA. It was transferred to the zygote of a mouse invitro. The embryo was implanted in the uterus of a foster mouse. Then the new born mouse was found to be transgenic which contained a gene from humans.
Methods of creation of transgenic Animals
There are three methods used for creations of transgenic animals are DNA micro injection, Embryonic stemcell-mediated gene transfer and Retrovirus mediated gene transfer.
DNA micro injection
Introduction of transgene by microinjection involves the following procedure. A young female mouse is given the FSH (follicle stimulating hormone) and HCG (human chorionic gonadotropin). Thus the mouse produces 30-35 ova. It was allowed to mate with a male. Then the fertilized ova are collected from the fallopian tubes. The transgene is introduced into the male pronucleus by using
micro injection needle. After amphimixis the embryo is allowed to devide. Then the embryo was implanted into the uterus of a foster mother. The new borns are called transgenic mice.
Hence DNA micro injection method is a random method. Success rate is very poor. This method has many disadvantages. The introduced DNA may not insert into the genome of the host. The foster mother may not accept the introduced fertilized ovum for further development. The introduced DNA may not express the desired trait. . A major advantage of this method is its applicability to a wide variety of species..
Embryonic stemcell-mediated gene transfer
The Recombinant DNA is transferred into embryo stem cells (ES). The cells are then cultured in the laboratory and those expressing the desired protein are selected. These modified ES cells are incorporated into the cavity of the embryo. This embryo is raised in a foster mother. The resulting transgenic animal will be a mosaic, because only a small proportion of the cells in its body will be expressing the protein.
Through this method transgenic goats and cows can now be designed to produce human proteins like blood clotting factors intheir milk.
Retroviral vector method:
Small fragments of DNA (8kb) can be effectively transferred by retrovirus. This method is not suitable for the transfer of large fragments of DNA. The main drawback in this method is the risk of retroviral contamination in products (such as human food) produced by transgenic animals. Hence this method is not popular in transgenesis.
Gene Cloning
To clone means to make identical copies. Gene cloning means production of a number of similar copies of a required gene. DNA cloning involves separating a specific gene or DNA segment from a larger chromosome, attaching it to a small carrier DNA. The resultant hybrid DNA is called recombinant DNA, which is transferred to a proper host (bacteria, virus or yeast) and replicated to make multiple copies of the selected gene.
This technology has made it possible to isolate, clone and produce DNA for all the genes in appropriate quantity so that they can be sequenced and characterized. Similarly, some of the genes which are expressed at very low level, can be cloned and desired amount of recombinant proteins can be produced.
Gene cloning involves the following steps
1. Cutting the DNA to be cloned from the chromosomal using sequence specific Restriction Endonuclease.
2. Selecting a cloning vector (a small molecule capable of self-replicating inside host cells), and cutting the cloning vector with the same restriction endonuclease to produce sticky ends.
3. Incubating the vector and subject DNA to join together DNA ligase. The resultant DNA is called recombinant DNA.
4. Transferring the reconbinant DNA to an appropriate host such as bacteria, virus or yeast which will provide necessory biomachinary for DNA replication.
5. Identifying the host cells that contain the recombinant DNA.
Enzymatic Cleavage of DNA
To cut the DNA at specific sites Restriction endonucleases are used. (RENs).
Restrictions Endonucleases:
A Restriction Endonuclease is an enzyme that cuts DNA at specific recognition points known as Restriction sites. These are most important groups of enzymes for manipulation of DNA. These enzymes were discovered in Escherichia coli. In bacteriophages these enzymes restrict the replication of viral DNA. Many types of restriction endonucleases were isolated. RENs were named based on the bacterial from which they were isolated.
The first letter of the enzyme indicates the genus name and next two letters the species name, followed by strain name and finally a number indicating the order of discovery. Eg. EcoRI: here E represents Escherichia, co represents coli, R represents the strain and I represents the first endonuclease.
Types of Endonucleases: over 3000 RENs have been studied in detail and more than 600 are available commercially. Naturally occurring Endonucleases are categorized in to four groups namely type I, II, III and IV based on their nature of their restriction sites. All types of enzymes recognize a specific DNA sequence and cleave that DNA at that point. They differ in their recognition sequence and cofactor requirements.
Type I RENs cuts DNA at random location as far as 1000 or more Basepairs from the recognition site. Type II cuts approximately 25 base pairs from the site. Type I and Type III require ATP. They are large enzymes with multiple subunits.
Type II RENs cut the DNA with in the recognized sequence without the need of ATP. They are smaller and simpler. Hence they are predominantly used in biotechnology.
Most of the Type II RENs generate Sticky ends. The open ends of the DNA molecule after the cut are called as Sticky ends.
The Length of restriction recognition sites varies. The Enzyme EcoRI, SacI etc recognize 6 basepair sequence of DNA. Most of the recognition sequences are palindromes – they read the same forward and backward.
Some of the important RENS and their restriction sites.
Enzyme Source Recognition Sequence Cut
EcoRI
Escherichia coli
5'GAATTC
3'CTTAAG 5'---G AATTC---3'
3'---CTTAA G---5'
EcoRII
Escherichia coli
5'CCWGG
3'GGWCC 5'--- CCWGG---3'
3'---GGWCC ---5'
BamHI
Bacillus amyloliquefaciens
5'GGATCC
3'CCTAGG 5'---G GATCC---3'
3'---CCTAG G---5'
HindIII
Haemophilus influenzae
5'AAGCTT
3'TTCGAA 5'---A AGCTT---3'
3'---TTCGA A---5'
TaqI
Thermus aquaticus
5'TCGA
3'AGCT 5'---T CGA---3'
3'---AGC T---5'
For convenience it is usual practice to simplifly the description of recognition sequences by showing only one strand of DNA, which runs in the 5’ to 3’ direction. Thus the EcoRI recognition sequence would be shown as G\AATTC.
Restriction enzymes with same sequence specificity and cut site are known as isochizomers. Enzymes that recognize the same sequences but cleave at different points are known as Neochizomers. Under extreme conditions like rise in pH, Low ionic strength) RENs are capable of cleaving sequences which are similar but not identical to their definied recognition sequences.
Applications of Restriction Endonucleases:
1. RENs formed in different bacteria can be used to break the DNA of any organism and the required DNA segment can be introduced in the DNA of another organism to produce Recombinant DNA
2. Restriction enzymes are highly useful to get desirable DNA segments. This is because these enzymes break the DNA at specific sites.
3. The DNAsegment isolated with the help of restriction enzymes can be used in DNA probe, genomic libraries and cDNA libraries.
4. With the isolated DNA segments, mRNA can be transcribed with which required proteins can be synthesized.
5. In restriction Fragment Length Polymorphism (RFLP) also restriction enzymes are very useful.
Enzymatic ligation of DNA
To hybridize (attach) the DNA fragments formed by the RENs to a plasmid DNA, the ends of both DNA are to be attached by forming hydrogen bonds and diester bonds. This process of attachment of the DNA fragments is known as Ligation. It is done by certain specialized enzymes called Ligases.
Ligase: (ligare = to glue together) is a special type of enzyme that can link together two DNA strands that have double strand break.
Mertz and Davis, 1972 first succeeded in producing rDNA in Escherichia coli by ligation of the sticky ends of DNA with ligase.
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3’hydroxyl ends of one nucleotide with the 5’ phosphate end of another. ATP is required for ligation. Ligase will also work with blunt ends, but neets high concentrations of enzymes.
In mammals there are four specific types of ligases.ie. DNA ligases, I, II, III and IV. LIgases can be classified into two groups on their requirement for ATP and NAD+ as co factors. All eukaryotic enzymes are ATP dependent, whereas most prokaryotic enzymes require NAD+ for their activity.
Most experiments use T4 DNA ligase (isolated from bacteriophase T4) which is most active at 25oC. High temperatures disrupts hydrogen bonding. The commonly available DNA ligases were originally discovered in bacteriophase T4, E.coli and other bacteria.
Applications of Ligases
DNA ligases are essential tools in modern molecular biology for generating rDNA sequences such as
1. Joining double stranded DNA with cohesive or blunt ends
2. Joining of oligonucleotide linkers or adaptors to blunt ended DNA
3. Repairing in duplex DNA, RNA or DNA-RNA hybrids
4. DNA ligases are used with restriction enzyes to insert DNA fragments often genes into plasmids.
Transgenesis and Production of transgenic animals (Fish and Goat).
Transgenesis is the process of introducing an exogenous gene – called transgene- into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. A transgene is the name given to the introduced DNA. The term transgenesis was coined by Gordon and Ruddle in 1981.
Animals produced through transgenesis are called Transgenic animals. Transgenic animals are genetically modified organisms with a new hereditary character.
Transgenic animals can be used to produce valuable products. Many proteins produced by transgenic animals are important for medical applications. For example, a transgenic pig has been produced with the ability to synthesize human hemoglobin for use as a blood substitute. Transgenic goat has been developed to produce a protein needed by the patients suffering from cystic fibrosis. Transgenisis is essential for improving the quality and quantity of the eggs, meat, milk etc, in addition to drug resistant animals.
The Mouse is the first animal used for transgenesis. RD Palmiter and RL Brinter (1982) isolated gene for growth hormone in human being. This gene was ligated with plasmid pBR322 to produce rDNA. It was transferred to the zygote of a mouse invitro. The embryo was implanted in the uterus of a foster mouse. Then the new born mouse was found to be transgenic which contained a gene from humans.
Methods of creation of transgenic Animals
There are three methods used for creations of transgenic animals are DNA micro injection, Embryonic stemcell-mediated gene transfer and Retrovirus mediated gene transfer.
DNA micro injection
Introduction of transgene by microinjection involves the following procedure. A young female mouse is given the FSH (follicle stimulating hormone) and HCG (human chorionic gonadotropin). Thus the mouse produces 30-35 ova. It was allowed to mate with a male. Then the fertilized ova are collected from the fallopian tubes. The transgene is introduced into the male pronucleus by using
micro injection needle. After amphimixis the embryo is allowed to devide. Then the embryo was implanted into the uterus of a foster mother. The new borns are called transgenic mice.
Hence DNA micro injection method is a random method. Success rate is very poor. This method has many disadvantages. The introduced DNA may not insert into the genome of the host. The foster mother may not accept the introduced fertilized ovum for further development. The introduced DNA may not express the desired trait. . A major advantage of this method is its applicability to a wide variety of species..
Embryonic stemcell-mediated gene transfer
The Recombinant DNA is transferred into embryo stem cells (ES). The cells are then cultured in the laboratory and those expressing the desired protein are selected. These modified ES cells are incorporated into the cavity of the embryo. This embryo is raised in a foster mother. The resulting transgenic animal will be a mosaic, because only a small proportion of the cells in its body will be expressing the protein.
Through this method transgenic goats and cows can now be designed to produce human proteins like blood clotting factors intheir milk.
Retroviral vector method:
Small fragments of DNA (8kb) can be effectively transferred by retrovirus. This method is not suitable for the transfer of large fragments of DNA. The main drawback in this method is the risk of retroviral contamination in products (such as human food) produced by transgenic animals. Hence this method is not popular in transgenesis.
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