Restriction enzyme : class xii CBSE

What is a resriction enzyme? 
Restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites

What do restriction enzyme do in bacterial cell?
In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. 
A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages, or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the number of strains of bacteriophage that can infect a bacterium.

Why restriction enzymes are important for us?
Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensible tools of recombinant DNA technology (genetic engineering).
Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order to study fragment length differences among individuals. This is referred to as restriction fragment length polymorphism (RFLP). They’re also used for gene cloning.

What are recognition sequences?
Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule—adenine, cytosine, thymine, and guanine). These regions are called recognition sequences and are randomly distributed throughout the DNA. Different bacterial species make restriction enzymes that recognize different nucleotide sequences.

How do restriction enzymes cleave the DNA sequence? 
When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides.

How does Bacteria prevent their own DNA from being degraded by RE?
Bacteria prevent their own DNA from being degraded in this manner by disguising their recognition sequences. Enzymes called methylases add methyl groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease. The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.

What are different types of restriction enzymes? How are they different from each other? 
Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors. 
Types I and III enzymes are similar in that both restriction and methylase activities are carried out by one large enzyme complex, in contrast to the type II system, in which the restriction enzyme is independent of its methylase. 
Type II restriction enzymes also differ from types I and III in that they cleave DNA at specific sites within the recognition site; the others cleave DNA randomly, sometimes hundreds of bases from the recognition sequence. Several thousand type II restriction enzymes have been identified from a variety of bacterial species. These enzymes recognize a few hundred distinct sequences, generally four to eight bases in length. 
Type IV restriction enzymes cleave only methylated DNA and show weak sequence specificity.

When and how were the restriction enzymes discovered? 
Restriction enzymes were discovered and characterized in the late 1960s and early 1970s by molecular biologists Werner Arber, Hamilton O. Smith, and Daniel Nathans. The ability of the enzymes to cut DNA at precise locations enabled researchers to isolate gene-containing fragments and recombine them with other molecules of DNA—i.e., to clone genes.

Explain about nomenclature of RE?
The names of restriction enzymes are derived from the genus, species, and strain designations of the bacteria that produce them; for example, the enzyme EcoRI is produced by Escherichia coli strain RY13.
First restriction enzyme to be discovered was endonuclease-HindII. It was isolated from Haemophilus influenza bacterium and this it’s so named.
The convention for naming these enzymes proceeds in a way that the first letter of the name comes from the genes and the second two letters come from the species of prokaryotic cell, from which they were isolated, e.g., Eco RI comes from E. coli RY13. The letter ‘R’ is derived from the name of strain. Roman numbers following the names, indicate the order in which the enzymes were isolated from that strain of bacteria.
EcoRI →
‘E’ represents Escherichia bacteria,
‘co’ represents coli the species,
‘R’ represents the RY13 strain of the bacteria and
‘I’ it is the 1st to be discovered from that strain.
HindII →
‘H’ represents Haemophilus bacteria,
‘in’ represents influenzae species,
‘d’ represents Rd strain and
‘II’ it is 2nd to be discovered from that strain.

Restricted sites of EcoRI:
5′ GAATTC 3′
3′ CTTAAG 5′

Restricted sites of Hind II:
5′ GTCGAC 3′
3′ CAGCTG 5′

Where do restriction enzymes come from?
Restriction enzymes are found in bacteria. Bacteria use restriction enzymes to kill viruses – the enzymes attack the viral DNA and break it into useless fragments.

When are restriction enzymes used?
Restriction enzymes are a basic tool for biotechnology research. They are used for DNA cloning and DNA fingerprinting.

DNA fragments: Blunt or sticky ends?
DNA consists of two complementary strands of nucleotides that spiral around each other in a double helix. Restriction enzymes cut through both nucleotide strands, breaking the DNA into fragments, but they don’t always do this in the same way.
SmaI is an example of a restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end.
Other restriction enzymes, like EcoRI, cut through the DNA strands at nucleotides that are not exactly opposite each other. This creates DNA fragments with one nucleotide strand that overhangs at the end. This overhanging nucleotide strand is called a sticky end because it can easily bond with complementary DNA fragments.

What is the differences between Exonucleases and Endonucleases?
Exonucleases:These nucleases cleave base pairs of DNA at their terminal ends

They act on single – strand of DNA or gaps in double –stranded DNA. They do not cut RNA

Endonucleases:

They cleave DNA at any Pont except the terminal ends

They cleave one strand (figure below) or both strands (figure below) of double – stranded DNA. They may cut RNA

What are molecular scissors? Why are they called so?
Restriction endonucleases are known as “molecular scissors” in genetic engineering.
They are capable of breaking DNA at specific sites called the restricted sites.
What are Palindromic sequences?
Palindromic sequences are sequences that have the same meaning when read from both the sides just like the word ‘MADAM’ & ‘MALAYALAM’.
5‘ → 3‘
GTAC
CATG
3‘ → 5‘

The growth of plant tissues in artificial media is called___________
a) Gene expression
b) Transgenesis
c) Plant tissue culture
d) Cell hybridization

Name the term given to the ability of single cells to divide and produce all the differentiated cell in the organism?
a) Unipotent
b) Pluripotent
c) Multipotent
d) Totipotency

Out of the following, which one is NOT the basic component of culture media used for plant cultivation?
a) Complex mixture of salts
b) Amino acids
c) Serum albumin
d) Sugar/ sucrose

Mark the INCORRECT statement about agar, a gelling agent in plant tissue culture medium?
a) Not digested by plant enzymes
b) It does not use in microprapogation work
c) It does not react with media constituents
d) Remain stable at incubation temperature

Which of the following is NOT a plant growth regulator?
a) Auxin
b) Cytokinins
c) Abcisic acid
d) Polyphenols

Which of the following is the main effect of cytokines in the tissue culture system?
a) Adventitious shoot formation
b) Induction of somatic embryos
c) Adventitious root formation
d) Shoot elongation

Which one of them is NOT the main effect of polyamines in the tissue culture system?
a) Promotion of tuber and bulb formation
b) Adventitious root formation
c) Promotion of shoot formation
d) Somatic embryogenesis

Which of the following plant hormone control fruit ripening?
a) Ethylene
b) Auxin
c) Gibbrellins
d) Abscisis acid

Who is the father of tissue culture?
a) Bonner
b) Haberlandt
c) Laibach
d) Gautheret

Synthetic seed is produced by encapsulating somatic embryo with
a) sodium chloride
b) sodium alginate
c) sodium acetate
d) sodium nitrate

Hormone pair required for a callus to differentiate are
a) auxin and cytokinin
b) auxin and ethylene
c) auxin and absiccic acid
d) cytokinins and gibberllin

DMSO (Dimethyl sulfoxide) is used as
a) Gelling agent
b) alkaylating agent
c) Chelating agent
d) Cryoprotectant

The most widely used chemical for protoplast fusion, as fusogens, is
a) Manitol
b) Sorbitol
c) Mannol
d) Poly ethylene glycol (PEG)

Cybrids are produced by
a) Fusion of two different nuclei from two different species
b) Fusion of two same nuclei from same species
c) Nucleus of one species but cytoplasm from both the parent species
d) None of the above

Callus is
a) Tissue that forms embryo
b) An insoluble carbohydrate
c) Tissue that grows to form embryoid
d) Un organised actively dividing mass of cells maintained in cultured

Part of plant used for culturing is called
a) Scion
b) Explant
c) Stock
d) Callus

Growth hormone producing apical dominance is
a) Auxin
b) Gibberellin
c) Ethylene
d) Cytokinin

A medium which is composed of chemically defined compound is called
a) Natural media
b) Synthetic media
c) Artificial media
d) None of these

To obtain haploid plant, we culture
a) Entire anther
b) Nucleus
c) Embryo
d) Apical bud

Somaclonal variations are the ones
a) Caused by mutagens
b) Produce during tissue culture
c) Caused by gamma rays
d) Induced during sexual embryogeny

Which of the following plant cell will show totipotency?
a) Xylem vessels
b) Sieve tube
c) Meristem
d) Cork cells

What is Plant Tissue Culture?

Just as every person is different and unique, so is each plant. Some have traits like better color, yield, or pest resistance. For years, scientists have looked for methods to allow them to make exact copies of these superior individuals.

Plants usually reproduce by forming seeds through sexual reproduction. That is, egg cells in the flowers are fertilized by pollen from the stamens of the plants. Each of these sexual cells contains genetic material in the form of DNA. During sexual reproduction, DNA from both parents is combined in new and unpredictable ways, creating unique plants.

This unpredictability is a problem for plant breeders as it can take several years of careful greenhouse work to breed a plant with desirable characteristics. Many of us think that all plants grow from seeds. However, researchers have now developed several methods of growing exact copies of plants without seeds. And they are now doing this through a method called “tissue culture”.

Tissue culture (TC) is the cultivation of plant cells, tissues, or organs on specially formulated nutrient media. Under the right conditions, an entire plant can be regenerated from a single cell. Plant tissue culture is a technique that has been around for more than 30 years. Tissue culture is seen as an important technology for developing countries for the production of disease-free, high quality planting material and the rapid production of many uniform plants.

Micropropagation, which is a form of tissue culture, increases the amount of planting material to facilitate distribution and large scale planting. In this way, thousands of copies of a plant can be produced in a short time. Micropropagated plants are observed to establish more quickly, grow more vigorously and are taller, have a shorter and more uniform production cycle, and produce higher yields than conventional propagules.

Tissue culture technique

Tissue culture is a process that involves exposing plant tissue to a specific regimen of nutrients, hormones, and light under sterile, in vitro conditions to produce many new plants, each a clone of the original mother plant, over a very short period of time.

There are three main steps to the tissue culture process. 

STAGE I is the initiation phase. It concerns the establishment of plant tissue in vitro by sterilizing the material and initiating it into culture.

STAGE II is the multiplication phase. At this stage, the in vitro plant material is re-divided and placed in a medium with plant growth regulators that induce the proliferation of multiple shoots. This process is repeated many times until the number of plants desired is reached.

STAGE III is the root formation phase. It involves the introduction of hormones to induce rooting and the formation of complete plantlets

Following these three stages, the plants are then moved from the laboratory to the greenhouses for acclimatisation and further development.

Need:

• When large-scale propagation of new or superior plant varieties is required for early introduction to market
• When mass multiplication is needed for varieties which are difficult to regenerate by conventional methods of propagation

• When disease-free plant propagation is important
• AgriForest’s tissue culture plants are characterised by disease free growth, a more fibrous, healthier root system, a bushier branching habit, and a higher survival rate.

Advantage:

Video links

• https://youtu.be/TORRxwbz7aY

• https://youtu.be/7ba-hqLrgf8

1. (a) Describe vector – mediated and vector-less gene transfer in plants.

            (b) Why is Agrobacterium tumefaciens regarded as nature’s genetic engineer?

2. (i) What is a ‘crown gall’? Name the organism which causes this phenotype in plants.

            (ii) What are the essential steps in regeneration of whole plants using tissue culture techniques? Depict diagrammatically only.

3. What are the use of:

            a. Haploid plants

            b. Triploid plants.

            c. Protoplast Culture

            d. Callus Culture

            e. Artificial Seeds

4. What are the genetic engineering strategies to create the following traits in transgenic crops:

a. Hebicide tolerance

b. Abiotic stress tolerance

c. Insect resistance

d. Virus resistance

e. Disease resistance

5. Write a descriptive account on the methodology of transforming plant with its natural genetic engineer.

1. Parth has identified luciferase gene from firefly. Construct a flowchart of the steps that you would follow to transfer the gene to a plant.
2. Depict the induction of crowngall on a stem of a plant by Agrobacterium tumefaciens with the help of a schematic diagram
3. Outline various steps involved in the regeneration of whole plants using culture techniques.
4. As a GM crop breeder you had learnt the various constraints that are associated with public acceptance of transgenic crops. Discuss
5. What is germ plasm? What is germplasm conservation? What are the benefits of non-conventional gene banks over conventional gene banks?
6. What is the principal of barnase-barstar gene system? How have plant breeders exploited this system?
7. What are secondary metabolites? Suggest any four secondary metabolites which are useful in medicine.
8. Explain the saying: “Transgenic plants as bioreactors”.
9. Describe how Agrobacterium tumefaciens can be used to introduce foreign gene into plants.
10. What are edible vaccines? How are they better than conventional vaccines?
11. Though a genetically engineered crop is herbicide and pesticide resistant, it still requires use of agrochemicals. Mention atleast three facts to justify the statement.
12. Somatic hybrids and cybrids are produced by a technique of plant biotechnology. Identify this technique and explain by citing two examples of plants.
13. What are molecular markers? Where they are used? What are there advantages over other markers?
14. Mention one advantage of obtaining male sterile plants. How can one express barnase specifically in tapetal cells of an anther to obtain such plants? Which other enzymes can be used to restore its fertility?
15. Biotechnology strategies can be used to overcome limitations caused by various abiotic stresses. Explain the above said statement?
16. Explain why Bt cotton flowers undergo pollination by butterflies and bees in spite of being insect/pest resistant?
17. Write various markers used in selection/screening of transgenic plants.
18. Write on the bioethics of plant genetic engineering.
19. (a) How the unnecessary pollination can be prevented using transgenic plant? (b) How the insect resistant plants can be produced?   (c) Define biolistic
20. What are the proposed benefits of genetic engineering in crop improvement?

                        (c) Define biolistic

1. Execute the engineered biosynthetic pathway which leads to the production of valuable secondary metabolites for their overproduction.
2. Weeds compete with crop plants for nutrients, water etc. and are responsible for significant reduction in crop yields. As a biotechnologist, suggest a way to overcome this problem.
3. How is callus tissue generated? How is this tissue useful?
4. Differentiate between in-vivo and in-vitro gene banks.
5. Differentiate between somaclonal variations and gametoclonal variations.
6. What is an artificial seed?
7. What are round up ready crops? how they are made?
8. A farmer wants to produce hybrid of mustard plants in his field. As a biotechnologist, what would you suggest to him to ensure successful pollination or fertilization?
9. What are edible vaccines? How are they better than conventional vaccines?
10. Suggest any two applications of plant cell culture technique?
11. Somatic hybrids and cybrids are produced by a technique of plant biotechnology. Identify this technique and explain by citing two examples of plants.
12. Interspecific cross leads to formation of sterile seeds. What could be the reasons for the same and how can the embryo rescue be achieved?
13. Which future vaccine holds promise of bypassing the need to visit the doctor regularly for childhood immunization? How they works?
14. It is difficult to raise hybrids which are interspecific and intergeneric. Why? How can these types of hybrids be obtained?
15. Environmentalists advocate the disuse of chemical insecticides. Suggest an alternative way to improve crop yields.
16. Differentiate between primary metabolite and secondary metabolite . Name two secondary metabolite obtained through tissue culture and their application.
17. Mention one advantage of obtaining male sterile plants. How can one express barnase specifically in tapetal cells of an anther to obtain such plants? Which other enzymes can be used to restore its fertility?
18. How can you obtain virus free sugarcane plants from virus infected plants? Are these plants virus resistant? Why or why not?
19. Name the plant variations developed by long term callus and suspension culture in plants. How can such variations be used in crop improvement?
20. What is the principal of barnase barstar gene system?
21. (a) What are the benefits of developing insect resistant transgenic plants? (b)Which future vaccine holds promise of by passing the need to visit the doctor regularly for childhood immunization? What are its advantages?
22. The ‘in-vitro’ cultuires of plant cells require several nutrients unlike intact plants. Why? Name two of these nutrients.
23. Why plants produce secondary metabolites?
24. Explain why Bt cotton flowers undergo pollination by butterflies and bees in spite of being insect/pest resistant?
25. What are the uses of callus and protoplast culture?
26. Write various markers used in selection/screening of transgenic plants.
27. Artificial seeds for rapid and mass propagation of elite plant species (as well as hybrid varieties) are essentially the embryo encapsulated in protective chemical which prevents them from desiccating.  Name the protective chemical used to make artificial seeds.
28. When substance X is added to a plant tissue culture medium, it promoted rooting whereas substance Y is added, it promoted shooting. Identify substances X and Y?