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External communication is of two types: 

• Inward communication : When all messages received by the organisation from outside, then it is called inward communication, 

e.g., telephone calls, reports etc. 

• Outward communication: When all messages that go out of an organisation, then it said to be outward communication. It may be in telegram form, letter form etc.

Four types of diagrams are: 

• Pie-diagram 

• Pictogram 

• Histogram 

• Flow-chart 

Four types of graphs are: 

• Line graph 

• Bar graph 

• z-graph 

• Pie-graph

There are various components of communication process: 

• Sender 

• Encoding 

• Message 

• Channel 

• Decoding 

• Receiver 

• Feedback

The term ‘clear days’ implies the number of days between the date of serving notice to the members and scheduled date of the meeting. The member entitled to attend the meeting must get proper ‘clear days’ notice in terms of the provision of the Companies Act, 2013.

Unsolicited applications are those applications applied by the person in search of employment carry their resume and certificates. When suitable jobs are available for them then they may be contacted and considered.

Routine Reports have standard layout which are prepared at regular intervals in the routine course of business. This type of report may be submitted weekly, fortnightly, monthly, quarterly, half-yearly or yearly. 

Examples : Weekly production reports, Monthly reports on sales.

Globalization means integrating the economy of a country with the economics of other countries. It is possible through free flow of trade, capital and movement of persons across borders. Globalization has transformed the manner in which business is conducted. 

Let us have a look on these ways: 

• Due to globalization, it has been possible to invest in newer technologies and production methods which have raised the production standards. Foreign trade has increased in the country. 

• Exchange of technology between countries has been possible. 

• Collaborations with foreign companies has been possible. 

• Due to improved products, it has become possible to compete and survive in international competition. 

• Due to globalization, many of the Indian companies have emerged as multinationals such as Tata Motors and Asian Paints.

Four types of reports are : 

Formal Report: Formal report is prepared in a prescribed format and presented before the management in an established procedure. Reports submitted by officials of Companies are usually formal report. 

Informal Report: 

Informal report is prepared in a format of the convenience of the reporter and presented directly before the required person as and when demanded. An informal report is presented as in the form of letter or memorandum. Generally, it takes the form of a person to person communication. The formal reports are classified into two types. They are statutory report and nonstatutory report. 

Special Report: 

Special report is prepared and presented before the top management on specific requirement. It usually contains the opinions or recommendations of the reporter with the help of facts and arguments. Examples for special report are opening of branch, introducing a new product, improving the quality or changing the shape or size of the product etc. 

Technical Report: 

Technical reports describe the process, progress, or results of technical or scientific research, include in-depth experimental details, data, and results.

Business to Consumer (B2C) : In B2C business model, a website sells its product directly to a customer. A customer can view products shown on the website of business organization. The customer can choose a product and order the same. Website will send a notification to the business organization via E-Mail and organization will dispatch the product/goods to the customer. 

Consumer to Consumer (C2C): The C2C business model helps the consumer to sell their assets like residential property, cars, motorcycles etc. or rent a room by publishing their information on the website. Website may or may not charge the consutiler for its services. Another consumer may opt to buy the product of the first customer by viewing the post/advertisement on the website.

In economics the word ‘land’ is defined not only the surface of the earth but also other free gifts of nature, e.g., mineral resources, forest resources, anything that helps us to carry out the production of good and services but is provided by nature free of cost.

Factors which determine the productivity of land are : 

1. Fertility of Land : The productivity of land is determined by its natural qualities and its fertility. A flat and levelled land is comparatively more productive than an undulating one. The rich soil is more fertile and productive. However, the agricultural productivity can be improved by proper and extensive use of manure and fertilizers along with adoption of mechanized methods.

2. Proper Use of Land : The productivity of ‘land’ is directly related to its proper utilization. 

For example, a piece of land situated in the heart of city is more suitable for construction of a house or a market place. If this piece of land is put for farming or agricultural use, its productivity will almost be negligible. 

3. Location of Land : The location of ‘land’ affects its productivity to a great extent.

For example, the location of land near the market or bus station will result in economy of transportation charges and overall productivity from this point of view will naturally be higher. Similarly, for better agricultural productivity, its location near water resources is desirable.

(i) Capital is regarded as a factor of production as it is a produced means of production. It is first produced by man and then helps in the further production of goods.

When the percentage increase in demand is equal to the percentage increase in supply then the equilibrium price will remains constant and equilibrium quantity will increases.  

Lacrymal gland :  It is a secretary gland present in top right part of the eye. Its secretion is called tears which lubricated the eye and it contain lysozyme which protects the eye from infection of microbes.

Let the two consecutive natural numbers which are multiples of 3 be 3x and 3(x + 1). 

Now, 3x(3x + 3) = 810 

⇒ x² + x = 90 

⇒ x² + x – 90 = 0 

⇒ (x + 10) (x – 9) = 0 

⇒ x = 9 or x = – 10 

Rejecting negative value of x, because numbers are natural. We have x = 9. 

Hence, the required numbers are 27 and 30.

Total number of cards in the bag = 10 

(i) Total prime numbers = 1 i.e., 2 

∴ Required Probability = 1/10

(ii) Total numbers divisible by 4 = 5 (i.e., 4, 8, 12, 16, 20] 

Required Probability = 5/10 =1/2 

(iii) Total numbers divisible by 6 or multiple of 6 = 3 [i.e., 6, 12, 18] 

∴ Required Probability = 3/10

(iv) Total odd number = 0 

∴ Required Probability = 0/10= 0.

(i) Critical angle is the angle of incidence in the denser medium corresponding to which the angle of refraction in the rarer medium is 90°. 

(ii) aμg = cosec ic where ic in the critical angle. 

(iii) Depth remain the same. Apparent depth = Actual depth.

(a) Explosive: ammonium nitrate

(b) Medicine: ammonium carbonate

(c) Fertilizers: ammonium sulphate

(d) Laboratory reagent: ammonia solution

Disadvantages of ammonia as a refrigerant are as follows:

(i) It is not compatible with copper, so it cannot be used in any system with copper pipes.

(ii) It is poisonous in high concentration although it is easily detectable due to its peculiar smell and since it is less dense than air it goes up in the atmosphere not affecting the life too much on earth.

(i) Malleability 

(ii) Cryolite 

(iii) Zinc blende

(a) 1. Curiosity, temporary escape and mental relaxation. 

2. Inability to adjust to stresses, strains and anxiety. 

3. Differences are shown by members of family and drudgery. 

4. Drugs alter perception, behaviour & consciousness.

Or 

(b) 1. Loss of judgement will power and self-confidence. 

2. Amnesia 

3. Fatty liver syndrome. In advance, stage causes liver cirrhosis. 

4. Effects heart, kidney and stomach.

Gigantism and Acromegaly are due to Hypersecretion of Growth hormone.

(i) Antidiuretic hormone (ADH) or Vasopressin

(ii) Posterior

(iii) Diabetes insipidus

(iv)  Insulin

(v) Diabetes mellitus

(i) Odd term : Styrofoam

Category : Biodegradable materials 

(ii) Odd term : Pepsin

Category : Nitrogenous based of DNA

(iii) Odd term : Iris

Category : Ear ossicles

(iv) Odd term : Cortisone 

Category : Hormones secreted by pituitary gland 

(v) Odd term: Typhoid 

Category: Genetic disorders

Phylogeny deals with the study of evolutionary history of an individual/group of organisms.

(i) Insertional inactivation: Harder problem to solve is to determine which of the transformed colonies comprise cells that contain recombinant DNA molecules, and which contain self- ligated vector molecules. Insertional inactivation is the inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it. Recombinants can, therefore, be identified because the characteristic coded by the inactivated gene is no longer visible. 

pBR322 contains genes which code for ampicillin resistance and tetracycline resistance. BamHl cuts in the middle of the gene which codes for tetracycline resistance. If a gene is inserted here, the plasmid loses it ability to code for
tetracycline resistance. Thus, the plasmid containing the recombinant gene is resistant to ampicillin but sensitive to tetracycline. To screen, we use replica plates. 

(ii) The blue-white screen is a molecular technique that allows for the detection of successful ligation’s in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell (bacteria). In this method, a reporter gene lac Z is inserted in the vector. The lac Z encodes for the enzyme β- galactosidase which breaks a synthetic substrate X-gal (5-bromo-4-chloro-indolyl, (β-D-galacto-pyranoside) into insoluble blue colored product. The competent cells are grown in the presence of X-gal. If the ligation was successful, the bacterial colony will be white because p- galactosidase is not synthesized due to the inactivation of lac Z; if not, the colony will be blue. This technique allows for the quick and easy detection of successful ligation, without the need to individually test each colony. An example of such a vector is the artificially reconstructed plasmid pUC 19.

(i) Agarose gels 

(ii) Polyacrylamide gels.

There are many chemicals which are used to determine the amino acid sequence in protein. Sanger’s reagent.(1-fluoro-2, 4-dinitrobenzene) and dansyl derivatives such as dansyl chloride are used for amino acid sequencing.

Cells in. suspension cultures vary greatly in size, shape of DNA and nuclear content. Moreover, the cell cycle time varies considerably within individual cells. Therefore, cell cultures are mostly asynchronous. This variation complicates studies of biochemical, genetic physiological and other aspects of cell metabolism. A synchronous culture is one in which the majority of cells proceed through each cell cycle phase (G, S,G₂ and M) simultaneously. 

(A) Physical Methods: 

Selection by Volume: Synchronization may be achieved on the basis of selecting the size of cell aggregates present even in the finest possible suspension cultures. Cell fractionation is employed for selection. 

Temperature Shock: Low temperature shocks combined with nutrient starvation are reported to induce — synchronization of suspension culture. 

(B) Chemical Methods: 

Starvation : The principle of starvation is based on depriving suspension cultures of an essential growth compound leading to a stationary growth phase. Resupplying the missing compounds is expected to induce resumption of cell growth synchronously. Growth hormone starvation is also reported to induce synchronization of cell cultures. 

Inhibition : Synchronization is achieved by temporarily blocking the progression of events in the cell cycle and accumulating cells in a specific stage using a biochemical inhibitor. On release the block cells with synchronously enter the next stage. Inhibitors of DNA synthesis (5-aminourail, 5-fluorodexypunne. hydroxyurea or excess thymidine) in cell cultures accumulate cells at the G₁/S boundary.

(i) Glycogen is also known as animal starch and it is the reserve of carbohydrates in animals. It occurs in algae, fungi and yeasts. Glycogen is dextro-rotatory and its hydrolysis yield D- glucose. 

(ii) Chitin : It is hard,tough substance that occurs widely in nature particulary in the shells of arthropods such as crab, insects and spider. The walls of hyphae are composed of slightly different chitin. Chemically, chitin is a polysaccharide drived from glucose.

Automatic DNA Sequencers: Automatic sequencing machines were developed during 1990’s. It is an improvement of Sanger’s method. In this new method, a different fluorescent dye is tagged to the aWNTPs. Using this technique, a DNA sequence containing thousands of nucleotide’s can be determined in a few horns. Each dideoxynucleotide is linked with a fluorescent dye that imparts different colours to all the fragments terminating in that nucleotide. All four labelled ddNTP’s are added to a single capillary tube. It is a refinement of gel electrophoresis which separates fastly. DNA fragments of different colours are separated by their size in a single electrophoretic gel. 

A current is applied to the gel. The negatively charged DNA strands migrate through the pores of gel towards the positive end. The small sized DNA fragments migrate faster and vice-versa. All fragments of a given length migrate in a single peak. The DNA fragments are illuminated with a laser beam. Then the fluorescent dyes are excited and emit light of specific wavelengths which is recorded by a special ‘recorder’. The DNA sequences are read by determining the sequence of the colours emitted from specific peaks as they pass the detector. This information is fed directly to a computer which determines the sequence. A tracing electrogram of emitted light of the four dyes is generated by the computer. Colour of each dya represents the different nucleotides. Computer converts the data of emitted light in the nucleotide sequences.

Embryonic stem cells : are derived from embryos. Most embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocoel, a hollow cavity inside the blastocyst; and the inner cell mass, which is a group of cells at one end of the blastocoel that develop into the embry o proper.

Chief characteristics of stem cells : Embry onic stem cells differ from other kinds of cells in the body. 

Embryonic stem cells have three general properties:

1. they are capable of dividing and renewing themselves for long periods; 

2. they are unspecialized; and 

3. they can give rise to specialized cell types. 

Embryonic stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells-which do not normally replicate themselves- Embiyonic stem cells may replicate many times, or proliferate. Embryonic stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. For example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and it cannot carry oxygen molecules through the bloodstream (like a red blood cell). Embryonic stem cells, can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.

Uses of Embryonic stem cells: Studies of human embryonic stem cells will yield information about the complex events that occur during human development. A primary goal of this work is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs.

Embryonic stem cells could also be used to test new drugs. 

For example, new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Perhaps the most important potential application of human embryonic stem cells is the generation of cells and tissues that could be used for cell-based therapies.

Messenger RNA contains a triplet sequence of nitrogen bases which code for the specific amino acids, used to make polypeptide chains. Each of the sets of three bases is known as a codon or genetic code. 

Translation starts with a chain initiation codon (start codon). The most common start codon is AUG which is read as methionine or, in bacteria, as formyl methionine. The three stop codons have been given names : UAG is amber, UGA is opal, sometimes also called umber and UAA is ochre. Stop codons are also called “termination” or “non-sense” codons. 

Characteristics of Genetic Code 

• Triplet code : Three adjacent nitrogen bases constitute a codon which specifies the placement of one amino acid in a polypeptide. 

• Start signal: Polypeptide synthesis is signaled by AUG or methionine codon and GUG — Valine codon. They have dual function. 

• Stop signal: Polypeptide chain termination is signaled by three termination codons — UAA, UAG and UGA. They do not specify any amino acid and are hence also called non-sense codon. 

• Universal code : The genetic code is applicable universally i.e., the codon specifies the same amino acid from a virus to a tree or human being. 

• Non-ambiguous codon : One codon specifies only one amino acid and not any other.

Stem cells are the cell which are capable to divided and renew and also produce progeny. These can differentiate into a variety of different cells types, e.g., tissues continuously renew themselves throughout the life. The stem cells have two properties which increases their importance. 

(i) They have potential to form more differentiated cells and 

(ii) these are self-renewing because each division of a stem cells creates at least one stem cell. 

Various types of stem cells : On the basis of development potential, these are divided into following levels: 

• Totipotent Cells : which give rise to entire organism. 

• Pluripotent Cells : w hich form totipotent cells and also give rise to most but not all of cell types that are necessary for foetal development. 

• Multipotent Cells : cells which are formed after further differentiation of pluripotent cells. These can give rise to a limited number of cells types. 

• Unipotent Cells : are also formed from further differentiation of multipotent cells. These give rise to a single cell type.

Two industrial enzymes with uses : 

Amylases (from fungi and plants) : Production of sugars from starch, such as in making high-fructose com syrup. 

Rennin (derived from the stomachs of young ruminant animals) : Manufacture of cheese, used to hydrolyze protein.

The organisms selected for genome, projects were those that are mostly used in genetic and other scientific investigations. Thus, they may be regarded as model organisms. A model organism is an organism about which is a large amount of scientific knowledge is already available. These organisms include both prokaryotic and eukaryotic microorganisms as well as animals and plants. These organisms include E. coli, Bacillus subtilis, Archaeoglobus fulgidus, yeast {Saccharomyces cerevisiae), Arabidopsis thaliana, roundworm (Caenorhabditis elegans) and the fruitfly (Drosophila melanogaster). In addition, human genome project focussed on sequencing of the whole human genome. 

E. coli is without any doubt, the best studied microorganism. Many of the technological tools available today were developed for E. coli genome project. The E. coli genome sequencing was completed in 1997. B. subtislis is a Gram-positive bacterium that colonizes leaf surfaces. 

It is much used in industrial processes for both enzyme production and food supply fermentation. It is generally regarded as safe (GRAS). 

S. cerevisiae is perhaps the most important fungal species used in biotechnological processes. Its name derives from the fact that it can ferment saccharose (sugar). S. cerevisiae genome sequencing project began in 1989, and the sequencing was completed in 1999. Arabidopsis thaliana is a small plant belonging to the Cruciferae
family. It was recognized as a good organism for genetic studies in 1907, and has become a model plant for genetic studies. 

Since it has the smallest genome among higher plants. 

Further, its genome has a low amount of repetitive DNA. Arabidopsis genome sequencing project began in 1990 and was completed in 2000. The fruitfly, D. melanogaster, is fondly regarded the Queen of Genetics. Drosophila genome sequencing was completed in 2000. It has 180 x 106 bp and -16,000 genes. 

Thus, the number of genes in Drosophila is less than four-times as many as in the bacterium E.coli.

DNA sequencing : It is the determination of the precise sequence of nucleotide’s in a sample of DNA. 

Sanger dideoxy method: The most popular method for DNA sequencing is called the dideoxy method or Sanger method (named after its inventor, Frederick Sanger, who was awarded the (1980) Nobel prize in chemistry). 

Procedure : The DNA to be sequenced is prepared as a single strand. This template DNA is supplied with 

a mixture of all four normal (deoxy) nucleotides in ample quantities 

• dATP 

• r/GTP 

• c/CTP 

• dT TP 

a mixture of all four dideoxynucleotides, each present in limiting quantities and each labelled with a ” tag that fluoresces a different color: 

• ddATP 

• ddGTP 

• ddc TP 

• ddlTP 

DNA polymerase I
Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines). 

If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process. 

At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.

Embryonic stem cells : These are derived from embryos. Most embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocoel, a hollow cavity inside the blastocyst; and the inner cell mass, which is a group of cells at one end of the blastocoel that develop into the embryo proper. 

Chief characteristics of stem cells : Embryonic stem cells differ from other kinds of cells in the body. Embryonic stem cells have three general properties: 

• they are capable of dividing and renewing themselves for long periods, 

• they are unspecialized; and 

• they can give rise to specialized cell types. 

Embryonic stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells which do not normally replicate themselves; Embryonic stem cells may replicate many times, or proliferate.

Embryonic stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. For example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and it cannot carry’ oxygen molecules through the bloodstream (like a red blood cell). Embryonic stem cells, can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells. Uses of Embryonic stem cells: Studies of human embryonic stem cells will yield information about the complex events that occur during human development A primary goal of this work is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs. 

Embryonic stem cells could also be used to test new drugs. For example, new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Perhaps the most important potential application of human embryonic stem cells is the generation of cells and tissues that could be used for cell-based therapies.

(i) Gene: Gene is the unit of the genome, consisting of a sequence of DNA that occupies a specific position (locus) on a chromosome and determines a particular characteristic in an organism. 

Genome: Genome is the total genetic information or all the genes contained in a haploid set of chromosomes in eukary otes, in a single chromosome in bacteria, or in the DNA or RNA of viruses. 

(ii) Multipotent: These cells have the ability to differen-tiate into many of the various type of specialized cell types and can develop into any cell of a particular group or type. e.g., umbilical cord stem cells. Unipotent: These cells can undergo unlimited reproductive divisions, but can only differentiate into a single type of cell or tissue, e.g., skin cells. 

(iii) Galactose: It is a part of disaccharide that is made- up of two sugars. It is found in milk alongwith glucose. Galactose does not occur freely in nature. It is produced in the body during the digestion of disaccharide lactose. Glycine: Glycine is a neutral amino acid and one of the 20 building blocks of protein. It is a non-essential amino acid, used in purine synthesis, and is a neurotrans¬mitter. 

(iv) Batch culture: It is a type of culture in which nutrients are fed continously depending upon the amount consumed without removing growth products.

Continuous culture: It is a open type of culture in which nutrients are supplied from time to time alongwith removal of product in same volume. 

(v) Coding region: Coding region (exon) is a part of the DNA that actually codes for a protein. 

Non-coding region: Non-coding region (introns) is that part of DNA that does not code directly for a protein.

Bioinformatics is the application of computer technology to the management of biological information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied to gene-based drug discovery and development. Bioinformatics uses many areas of computer science, statistics, mathematics and engineering to process biological data. 

Applications of Bioinformatics: 

• It is used to search the genome of thousands of organisms, containing billions of nucleotides. These programs would compensate for mutations (exchanged, deleted or inserted bases) in the DNA sequence, in order to identify sequences that are related, but not identical.

• It is used for measuring biodiversity. 

• The expression of many genes can be determined by this process. 

• Bioinformatic techniques have been applied to explore various steps in the analysis of regulation. 

• In order to trace the homology of newly identified protein.

(i) Taxonomy browser: This search tool provides taxonomic information on various species. The TAXONOMY database of NCBI has information (including scientific and common names) about all organisms for which some sequence information is available (over 79,000 species). The server provides genetic information and the taxonomic relationship of the species in question. TAXONOMY has links with other servers of NCBI e.g., structure and PubMed. 

(ii) BLAST (Basic Local Alignment Search Tool) : BLAST is a family of user-friendly sequence similarity search tools on the web. The BLAST server is supported through NCBI (National Center for Biotechnology Information) U.S.A. This tool is designed to identify potential homologous for a given sequence. It can analyse both DNA and protein sequences. A local alignment finds the optimal alignment between subregions or local regions of specified sequences. 

(iii) ENTREZ: Integrated information database retrieval system of NCBI. Using Entrez system you can access literature, sequences (both proteins and nucleotides) and structures (3D). 

(iv) EMBL (European molecular Biology Laboratory- Nucleic Acid (DNA sequence) databases.

Protein sequencing is a technique to determine the amino acid sequence of a protein as well as which conformation the protein adopts and the extent to which it is complexed with any non-peptide molecules. Discovering the structures and functions of proteins in living organisms is an important tool for understanding cellular processes. It is also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein, if this is known. A protein sequencer is a machine that is used to determine the sequence of amino acids in a protein. They work by tagging and removing one amino acid at a time, which is analysed and identified. This is done repetitively for the whole polypeptide, until the whole sequence is established. 

Determination of amino acid sequences : 

Prior to amino acid sequence determination, it is essential to know the protein’s purity, molecular weight and its amino acid composition. A pure protein, if multimeric must be dissociated into its individual polypeptide chains and the molecular weight of the chains is determined. The molecular weight of a protein roughly tells about the number of amino acid residues contained in it. Each amino acid residue has an average molecular weight of 110 D. So if the molecular weight of a protein is 11000 D, we can predict that 100 amino acid residues are present. The amino acid composition tells the protein chemist about the strategies which could be employed in protein sequencing. 

The following protein sequencing techniques are employed : 

The first protein to be sequenced was the hormone ‘insulin’ whose deficiency leads to the disease diabetes. The Nobel Laureate, Fred Sanger invented a method of protein sequencing using a stepwise release and identification of amino acids starting from the N-terminal amino acid. The reagent employed by Sanger, known as fluoro-dinitro-benzene (Sanger’s reagent) reacted specifically with the free NH₂ group of the N-terminal amino acid and on acid hydrolysis, it yielded a yellow colored dinitro phenyl (DNP) derivative of the original N-terminal amino acid. 

The polypeptide was subsequently, shortened by one amino acid. The DNP-amino acid was identified by comparison with other standard DNP amino acids using chromatography techniques. This procedure was repeated on the shortened polypeptide. In order to complete the sequence of insulin, Sanger used more than a gram of the hormone purified from a large number of pancreatic glands. However
Sanger’s method has become obsolete and historical. The following steps summarise the Sanger’s method for determining the amino acid sequence. 

• Hydrolysis by heating a sample of protein in 6M hydrochloric acid. • Separation of polypeptide chains by ion – exchange chromatography. 

• Fragmentation of the polypeptide chain by 

• Enzymatic methods using proteolytic enzyme, highly selective in hydrolyzing peptide bond, 

• Chemical methods using cyanogen bromide (CNBr) cleaves selectively the peptide bond following a methionine residue. 

• Identification of N-terminal amino acid using Sanger’s reagent (fluoro-dinitrogen-benzene). 

• Identification of DNP amino acid by comparison using chromatography by interpreting chromatogram. 

• Repeating the procedure on the shortened polypeptide to identify rest of amino acids one by one. 

Limitation : The method cannot be used in case the sample material is less in amount.

(i)RNA Polymerase: It is an enzyme found in both prokaryotic and eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of tmRNA.

Taq DNA Polymerase: It is a thermostable DNA polymerase, originally isolated from bacterium, Thermus aquaticus. It is used in polymerase chain reaction. 

(ii) In-situ conservation: It means on-site conservation. It is the process of protecting an endangered plant or animal species in its natural habitat either by protecting or cleaning up the habitat itself, or by defending the species from predators. 

Ex-situ conservation: It means literally, “off-site conservation”. It is the process of protecting an endangered species of plant or animal by removing part of the population from a threatened habitat and placing it in a new location, which may be a wild area or within the care of humans e.g., Zoos, Botanical garden, etc. 

(iii) Micronutrients: Micronutrients are essential elements required by organisms in small quantities. They include microminerals and vitamins. 

Macronutrients: Macronutrients include carbohydrates, proteins and fats required in larger quantities than micronutrients in the body 

(iv) mRNA: mRNA is present in nucleus and functions in cytoplasm. It carries messages from DNA. 

tRNA: tRNA is an adapter present in cytoplasm. It recognizes and brings amino acids near ribosomes for protein synthesis. 

(v) Essential amino acid: The essential amino acids cannot be synthesized by the human body and are obtained from food. 

Non-essential amino acid: The non-essential amino acids can be synthesized by the human body. They can be produced from other amino acids and substances in the diet and metabolism.

(i) Institute (IARI) New-Delhi 

(ii) National Diary Research Institute (NDRI) Kamal. 

(iii) Indian Veterinary Research Institute (IVRI) Izat Nagar (Near Bareily U P.) 

(iv) Centers for Plant Molecular Biology (CPMB) in various universities such as Delhi University, National Botanical Research Institute Lucknow.

An oil eating bacteria: Pseudomonas capacia, P. putida and other spp. Acinetobacter sp. Efficient degraders have been prepared through genetic
engineering and they need to be established in the environment at a required density.

Production of Human Insulin (Humulin): Human insulin is a dimer comprising one chain of 21 amino acids (A chain) and the other of 30 amino acids (B chain), the C chain that links A and B chains. Chains A and B become linked by two disulphide bridges. This is followed by cleavage of the leader and the C chain sequences leaving the mature insulin molecule. Insulin is the first genetically engineered hormonal drug ever marketed anywhere in the world. It was produced first in 1980 by Eli Lilly (U S A.) with the name Humulin by transferring the insulin gene into E.coli. 

The genes (= DNA sequences) for chains A and B of insulin were synthesized separately as early as 1978. The genes for A and B chains were integrated separetely in pBR322 type vector. The purified chains A and B were then attached to each other by disulphide bonds induced in vitro; this, however turned out to be an inefficient reaction. Subsequently, a gene representing B, C and A chains was synthesized and expressed in E. coli; in this case, the intervening chain is removed proteolytically following spontaneous folding of the proinsulin molecule.

Single cell protein : The SCP is basically a non-pathogenic, fast growing microbial biomass rich in high quality of protein and can be commercially produced throughout the year and independent of climate (except algal process). 

Example : Mushrooms and yeasts are good source of vitamin B complex.

(i) Genomic Library: This is a collection of clones that represent the complete genome of an organism. For construction of a Genomic library the entire genomic DNA is isolated from host cells or tissues, purified and broken randomly into fragments of correct size for cloning into a suitable vector. 

The major use of Genomic library is hierarchichal shotgun sequencing. cDNA Library : The library made from complementary or copy DNA (cDNA) is called cDNA library. The library represents the DNA of only eukaryotic organisms, not the prokaryotic once. 

cDNA library’ is used to express eukaryotic gene in prokaryotes. cDNA libraries are most usefirl in reverse genetics where the additional genomic information is of less use. It is also useful for subsequently isolating the gene that codes for that mRNA.

(ii) Transformation : In the Biotechnology, Transformation means introduction of rDNA molecules into a living cell. It is the method of transfer of recombinants into the host cells. The DNA molecule comes in the contact of the cell surface and then it is taken up by the host cells. 

Transfection : Transfection is the transfer of foreign DNA into cultural host cells mediated through chemicals. This method is used for the transfer of foreign DNA in the host cell. The recipient host cells are overlayed by this mixture. Consequently, the foreign DNA is taken up by the host cell.

(i) PCR: 

• Amplification of desired segment of DNA within hours. 

• No host cell required. 

Gene cloning: 

• Amplification of a desirable gene takes more time under lab condition. 

• Host cell is required.

(ii) Batch culture: 

• In this culture, the same medium and all the cells produced are retained in the culture vessel. Fresh medium is not added. 

• The cell number of biomass exhibits a typical sigmoid curve. 

Continuous culture: 

• In this culture, both cells and used medium are taken out from the continuous culture and replaced by equal volume of fresh medium. 

• Here, the cell population is a maintained in a steady state by regularly replacing a portion of used or spent medium.

(i) Dolly – The first cloned animal Using nuclear transfer technique, the world’s first mammalian clone – Dolly, was born in February 1996. In 1995, Ian Wilmut and his research group (Scotland) took out udder from a six year old sheep A called clone mother, and put in a special solution. Nucleus of udder cell was taken out and put in a solution. At the same time an unfertilized egg was taken out from another sheep B called egg mother. Nucleus of the egg was removed and nucleated egg was put in a culture medium. The nucleus of udder cell and nucleated egg cell were put together followed by mild electric shock. Consequently nucleus was taken up by the nucleated cell. This cell was incubated onto growth medium then transferred into a surrogate mother. A little lamb Dolly was born in February, 1996. 

(ii) It is true that proteins (vaccines) stimulate immune system and cause to secrete specific antibodies. Such specific amino acid sequences in the protein that stimulate immune response are called epitopes. Based on selected epitopes recombinant vaccines may be produced on commercial level which can prove more effective and safer than the conventional vaccines. 

Working on these lines, a recombinant Hepatitis B vaccine was produced by cloning the synthetic gene (for the surface antigen of the virus) in yeast cells. This gene expressed well in yeast cells and produced 22 nm particles of hepatitis B virus (HBV) surface antigen (as produced in patients) infected with hepatitis B virus. The recombinant vaccine has high immunogenecity. This product has been marketed as a vaccine for protection against HBV infection.

Purines and pyrimidines are two of the basic units of the nucleic acids. They are found in a DNA and RNA in a cell Purines is a large sized double ring structure. It contains two bases, i.e., adenine and guanine, Pyrimidines are small sized, single ring structures. It contains three type of bases i.e., thymine, cytosine and uracil.

(i) Recombinant vaccines : Vaccine is produced using recombinant DNA technology. A recombinant vaccine contains protein or a gene encoding a protein of a pathogen origin that is immunogenic A gene coding an immunogenic protein from the pathogen, is isolated, cloned and used for vaccine production The vaccines based on recombinant proteins are also called Sub-Unit Vaccines. 

Whole protein vaccine : Hepatitis B vaccine is produced from surface antigens of transgenic yeast by r-DNA technology They can also be produced in genetically engineered microbes, cultured animal cells, possibly in insects and plants. 

Recombinant – polypeptide vaccines : In some cases, the immunogenic portion of the protein- recombinant polypeptide is used as vaccine e.g., gene encoding B polypeptide (part of cholera enterotoxin – Ab A₂ and B polypeptide) has been cloned and the recombinant B polypeptide produced is being used, in combination with inactivated cholera cells, as an oral vaccine in place of conventional injectable cholera vaccine. Immunogenicity of foot and mouth disease virus coat protein is due to its amino acids 114-160 and also 201 -213. They induce antibodies which neutralize the virus and thereby provide protection against the foot and mouth disease. 

Live recombinant vaccine : The most advanced and promising approach in which concerned pathogen gene is introduced into the genome of selected viral / bacterial vector which is suitably attenuated and the live microorganisms are used for vaccination. Vaccinia virus appears to be more promising vector. 

DNA vaccines: Recently vaccines based on pathogen naked DNA are being developed. Tue various approaches for DNA vaccines are as follow 

• injection of pure DNA (or RNA) preparation into muscles 

• reimplantation of autologus cells (cells of the individual to be vaccinated) into which the gene has been transferred and 

• particle gun delivery of plasmid DNA which contains the gene in an expression casette e.g., skin cells They elicit humoral immune response and are usually shed off in a few days preventing long term persistence modified cells. 

(ii) Delayed fruit ripening 

A major problem in fruit marketing is the pre-mature ripening and softening dining transport of fruits. Consequently shelf-life of fruit remains short in the market. During ripening, genes encode the enzyme cellulase and polygalacturonase. Therefore, ripening process can be delayed by interfering the expression of these genes. In the U.S.A., a transgenic tomato named FlavrSavr (flavour saver) was produced where ripening is delayed by , lowering polygalacturonase activity. 

A plant growth hormone ethylene is produced during fruit ripening and senescence. It is synthesised from S-adenosylmethionine through an intermediate compound 1-aminocyclopropane-l-carboxylic acid (ACC). There is a large number of bacteria that can degrade ACC. Therefore, bacterial gene (for ACC) deaminase associated with ACC degradation was isolated and introduced into tomato. In transgenic tomato, fruit ripening was delayed because it synthesised lower amount of ethylene (due to inhibition in ACC synthesis) than the normal tomatoes. Such tomatoes and other fruits can be transported to a longer distance without spoilage.