Genetic Engineering -For Various Copetitive Exams

Genetic Engineering, Cloning and Genomics
1. Introduction

1. Genetic engineering involves transfer or replacement of genes, so also known as recombination DNA technology or gene splicing.

2. Genetic engineering aims at adding, removing or repairing a part of genetic material.

3. Gene splicing is the cutting of DNA molecules at precise points with the help of enzymes.

4. Genetic engineering helps in the rectification of genetic errors.

5. Har Goving Khorana of India is associated with genetic engineering. He synthesized 'gene' artificially in a test tube (1969).

6. First protoplast fusion was done by Harris and Watkins of Oxford using somatic cells of mouse and man.

7. Hybridization by protoplast fusion is called parasexual hybridization.



Recombinant DNA Technology:-

1. The term recombinant DNA refers to the creation of a new association between DNA molecules or segments of DNA molecules from different biological sources.

2. Genetic engineering means manipulation of genes and it depends upon recombinant DNA technology.

3. Genetic engineering has revolutionized genetic studies in higher organisms and is a powerful tool in basic research on microbial genetics.

4. Genetic engineering is providing vast opportunities for improving processes of economic value and treating diseases.



Tools of Genetic Engineering:-

8. Nuclease are enzymes used in genetic engineering that cut, shorten or degrade e.g. restriction endonuclease and restriction exonuclease.

6. Restriction endonuclease is used to cut the plasmid as well as the foreign DNA molecules at specific points.

7. Ligase, another enzyme is used to seal gaps or to join pieces of DNA.



Restriction Endonucleases:-

8. The ability to clone and sequence essentially any gene or other DNA sequence of interest from any species depends on a special class of enzymes called restriction endonucleases (from the Greek term endon meaning "within";

9. Endonucleases make internal cuts in DNA molecules).

10. Restriction endonucleases are also called as 'molecular scissors' or 'chemical scalpels'.

11. Many endonucleases make random cuts in DNA, but the restriction endonucleases are site-specific.

12. Restriction endonucleases cleave DNA molecules only at specific nucleotide sequences called restriction sites.

13. Different restriction endonucleases are produced by different microorganisms and recognize different nucleotide sequence in DNA.

14. The restriction endoculeases are named by using the first letter of the genus and the first two letters of the species the produces the enzyme.

15. If an enzyme is produced only by a specific strain, a letter designating the strain is appended to the name.

16. The first restriction enzyme identified from a bacterial strain is designated I, the second II, and so on.

17. Thus, restriction endonuclease EcoRI is produced by Escherichia coli strain R Y I3.

18. Restriction enzyme called Eco RI recognizes the sequence

GAATTC

CTTAAG

and cleaves the DNA between G and A on both strands.

19. Restriction endonucleases were discovered in 1970 in Bacteria by Hamilton Smith and Daniel Nathans.

20. Nobel Prize of 1978 for restriction endonuclease technology and their role in genetic engineering was given to Daniel Nathans (USA), Hamilton Smith (USA) and Werner Arber (Switzerland) (BHU 1989).























21. The biological function of restriction endonucleases is to protect the genetic material of bacteria from "invasion" by foreign DNAs, such as DNA molecules from another species or viral DNAs.

22. As a result, restriction endonucleases are sometimes referred to as the immune systems of prokaryotes.

Characteristic of Some Restriction Endonucleases



Enzyme Name

Pronunciation

Organism in which enzyme is found

Recognition sequence and position of cut

Bam HI

"bam-H-one"

Bacillus amyloliquefaciens H

5' GGATC C 3'

3' C CTAGG 5'

Bgl II

"bagel-two"

Bacillus globigi

AG A T C T

T C T A G A

Eco RI

"echo-R-one"

E. coli RY 13

GA A T T C

C T T A A G

Hind III

"hin-D-three"

Haemophilus influenzae Rd

AA G C T T

T T C G A A

Pst I

"P-S-T-one"

Providencia stuartii

C T G C A G

G A C G T C

Sma I

"sma-one"

Serratia marcescens

C C C  G G G

G G G  C C C

Hae III

"hay-three"

Haemophilus hemolyticus

G G CC

C C  G G

Hha I

"ha-ha-one"

Haemophilus hemolyticus

G C GC

CG C G

Hpa II

"heap-two"

Haemophilus parainfluenzae

CC G G

G G CC

23. All cleavage sites in the DNA of an organism must be protected form cleavage by the organism's own restriction endonucleases; otherwise the organism would commit 'suicide' by degrading its own DNA.

24. In many cases, this protection of endogenous cleavage sites is accomplished by methylation of one or more nucleotides in each nucleotide sequence that is recognized by the organism's own restriction endonuclease.

25. Methylation occurs rapidly after replication, catalyzed by site-specific methylases produced by the organism.

26. Each restriction endonuclease will cleave a foreign DNA molecule into a fixed number of fragments, the number depending on the number of restriction sites in the particular DNA molecule.

27. An interesting feature of restriction endonucleases is that they commonly recognize DNA sequences that are palindromes that is, nucleotide-pair sequences that read the same forward or backward form a central axis of symmetry.

28. In addition, a useful feature of many restriction nucleases is that they make staggered cuts; that is, they cleave the two strands of a double helix at different points.

29. Other restriction endonucleases cut both strands at the same place and produce blunt ended fragments.

30. Restriction endonuclease Sma I is produced by Serratia marcescens recognizes the sequence

C C C  G G G

G G G  C C C

producing blunt ends.

31. Because of the palindromic nature of the restriction sites, the staggered cuts produce segments of DNA with complementary single stranded ends.

32. DNA fragments can be rejoined under the appropriate renaturation condition by using the enzyme DNA ligase to reform the missing phosphodiester linkages in each strand.

33. Thus, DNA molecules can be cut into pieces, called restriction fragments and the pieces can be joined together again with DNA ligase.

34. Exonucleas, which remove nucleotide one at a time form the end of the a DNA molecules. These enzymes are less useful in genetic engineering.

35. Polymereses make copies of molecules.

36. Topoisomerases introduce or remove super coils form covalently closed circular DNA.



c-DNA:-

1. c-DNA stands for complementary DNA or copy DNA.

2. c-DNA is a DNA copy of an RNA and is made from m-RNA that codes for a known protein by reverse transcription or Teminism.

3. c-DNA can be formed by RNA dependent DNA polymerase or Reverse Transcriptase.

4. c-DNA is used in a gene cloning technology.

5. c-DNA is also made to use PCR (Polymerase Chain Reaction) to amplify an RNA.

6. PCR does not work on RNA, so one can copy it to DNA using reverse transcriptase and then PCR amplify the c-DNA; this is called RT-PCR (reverse transcriptase PCR).



Vector:-

1. Vector in genetic engineering is usually a DNA segment used as a carrier for transferring selected DNA into living cells.

2. A vector may be used to construct recombinant DNA molecules. Some of the example are:

Plasmid vectors

Bacteriophage vectors

Cosmid vectors

BACs (Bacterial Artificial Chromosomes)

YACs (Yeast Artificial Chromosomes) and

MACs (Mammalian Artificial Chromosomes)

Phase P-1 derived artificially chromosome (PACs) are another relatively recently developed class of vector for use in E. coli.

3. Plasmids are extrachromosomal, covalently closed circular double stranded molecules of DNA present in most prokaryotes.

4. Bacteria contain one or more different plasmids; their functions are often unknown.

5. Many plasmids are without vital genes but carry genes for sexuality, antibiotic resistance, virulence etc., bacteria can survive without plasmids.



Plasmid Classification:-

1. Fertility or F plasmids carry tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids e.g. F plasmid of Ecoli.

2. Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents such as ampicillin, chloramphenicol, Hg e.g. RP4, commonly found in Pseudomonas.

3. Col plasmids code for colicin – protein that kill other bacteria e.g. col E1 of Ecoli

4. Degradative plasmids allow the host bacterium to metabolized unusually molecules such as Toluene and salicylic acid. E.g. TOL of Pseudomons putida

5. Virulence plasmids confers pethogenicity on the host bacterium e.g. Ti plasmids of Agro bacterium tumefaciens which induce crown gall decease in dicot plants, Ri plasmids of Agro bacterium rhizogens which causes hairy root disease in plants.



Some Plasmid-Coded Traits



Trait

Organisms in which Traits is found

Antibiotic resistance

Escherichia coli

Insect toxin synthesis

Bacillus thuringiensis

Nitrogen fixation

Rhizobium sp.

Oil degradation

Pseudomonas sp.

Toxin production

Bacillus anthracis

Tumour formation in plants

Agro-bacterium tumefaciens



6. All plasmids carry replicons pieces of DNA or ORi that have the genetic information required to replicate.

7. Plasmids vary in size from few genes to several hundred.

8. Plasmids range from about 1 kb (kilobase = 100 base pairs) to over 200 kb in size and replicate autonomously.

9. Plasmid pBR 322 was one of the first widely used cloning vectors, it contain both ampicillin and tetracycline resistance genes and having 4363 base pairs it was discovered by Boliver and Rodriguez.

10. Most bacteriophage cloning vectors have been constructed from the phage  chromosome.

11. The hybrids between plasmids and the phage  chromosome give rise to cosmid (for  cos site and plasmid) vectors.

12. Artificial chromosomes like BACs, YACs and MACs are very efficient vectors for eukaryotic gene transfers.



The nomenclature of plasmid cloning vectors:-

The name 'pBR322' conferms with the standards rule for vectors nomenclature.

'p' indicate that this is indeed a plasmid.

'BR' identifies the laboratory in which the vector was originally constructed (BR strand for Boliver and Rodriguez the researchers who developed pBR322.

322 distinguishes this plasmid from other developed in same laboratory (there are also plasmid called pBR325, pBR327, pBR328, etc.).



Tumour-inducing (Ti) Plasmid:-

1. One of the most interesting plasmids is the tumour-inducing (Ti) plasmid of Agrobacterium tumefaciens.

2. Agrobacterium tumefaciens is the causative agent of a common plant disease termed crown gall disease.

3. Agrobacterium tumefaciens is an natural genetic engineered; it infects a wound, and injects a short stretch of DNA called T-DNA into some of the cells around the wound.

4. It is possible to remove the tumour-producing genes that are transferred to the plant and substitute useful genes such as the B. thuringiensis toxin genes.

5. The toxin is naturally produced by the bacterium B. thuringiensis as it forms endospores.

6. The toxin binds to specific receptors in the insect gut, causing gut to dissolve.

7. These useful genes are then transferred and integrated into the plant.

8. When the genes for B. thuringiensis toxin are transferred to a plant, the plant becomes toxic to many insect pests, thus, conferring insect resistance.



Transduction:-

1. Transfer of live genetic material is involved in transduction and sexduction.

2. Transduction is the transfer of bacterial genetic material from one bacterium to another using a phage as the vector.

3. Sexduction is the process in which a fragment of genetic material from one bacterium is carried with the sex-factor F to a second bacterium.





Polymerase Chain Reaction (PCR):-

1. Polymerase Chain Reaction (PCR) was developed by Kary Mullis in 1983 while he was a staff chemist at the Cetus Corporation in California.

2. In 1993, PCR won Kary Mullis the Nobel Prize for chemistry.

3. PCR is nicknamed as 'People Choice Reaction'.

4. PCR is a method for amplifying a specific piece of DNA molecule without the requirement for time-consuming cloning procedures.

5. A direct procedure to copy the gene sequence of interest is called the PCR.

6. PCR starts with double stranded DNA molecule from which millions of identical copies of a select region can be produced.

7. Four ingredients of the PCR are:

(i) the double stranded DNA to be amplified called the target DNA.

(ii) a heat stable DNA polymerase, which work at optimum temperature of 72oC (usually Taq DNA polymerase obtained from the thermophile Thermus aquaticus a bacterium lives in hot water at a temperature 75oC. )

(iii) each of the four nucleotides (dATP, GTP, dCTP, dTTP); and

(iv) small single stranded strands of DNA of about 20 nucleotides called primers.

(v) a primer is short nucleotide sequence that can be extended by DNA polymerase in PCR.







































































8. An automatic thermal cycler is used in PCR which has a microprocessor controlled temperature cycling.

9. PCR technique has now been automated and is carried out by a specially designed machine.

10. Currently a PCR machine can carry out 25 cycles and amplify DNA 105 times as little as 57 minutes.

11. PCR has revolutionized many aspects of science and medicine because it permits the investigation of minute samples of DNA, PCR is used in the test of HIV.



Nucleic Acid Blotting:-

1. Nucleic acid blotting techniques are employed for the characterization of cloned sequences.

2. Four nucleic acid blotting techniques are Southern blot, Northern blot, Western blot and Southwestern blot.

3. One of the most useful methods developed by Edward Southern for identifying a specific gene is the Southern blot.

4. Southern blotting technique is used for separating DNA fragments and identification of cloned genes.

5. Northern blotting technique can be used to determine whether a cloned gene is transcriptionally active in a given cell or tissue type, this technique uses RNA.

6. Western blotting technique involves hybridization of protein bound to a filter.

7. Southwestern blotting technique is a variant of the Southern blot used to find protein molecules that stick to DNA molecules.

8. Gel electrophoresis and autoradiography are employed in nucleic acid blotting.



Medical Diagnosis of Diseases:-

1. Diagnosis of diseases can be done with DNA probes, short segment of single stranded DNA attached to a radioactive or fluorescent marker.

2. Small oligonucleotides capable of recognizing complementary sequence are known as molecular probes.

3. A molecular probe might be used to find a nucleotide sequence.

4. In Southern blotting, DNA is visualized by DNA probes.

5. DNA probes are used for identification of infectious agents like Salmonella (food poisoning), Staphylococcus (pus forming), hepatitis virus, HIV, etc.



Applications of Recombinant DNA Products



Recombinant Product

Potential Use

Blood-clotting factor VIII

Treatment of haemophilia

Calcitonin

Treatment of osteomalacia

Erythropoietin

Treatment of anaemia

Growth hormone

Growth promotion

Insulin

Treatment of diabetes

Interferons

As antiviral, antitumour and anti-inflammatory agents

Interleuking

Treatment of immune disorders and tumours

Relaxin

Aid to childbirth

Serum albumin

Plasma supplement

Stomatostatin

Treatment of acromegaly

Streptokinase

Anticoagulant

Tissue plasminogen activator

Anticoagulant



Gene Therapy:-

1. The use of bioengineered cells or other biotechnology techniques to treat human genetic disorders is known as gene therapy.

2. Gene therapy is the transfer of normal genes into body cells to correct a genetic defect.

3. Gene therapy can be used to treat genetic diseases like sickle-cell anaemia and Severe Combined Immuno-Deficiency (SCID).

4. SCID is caused by a defect in the gene for the enzyme adenosine deaminase (ADA).

5. SCID patients have no functioning T lymphocytes.

6. SCID patients are treated with the injections of their white blood cells that have been engineered to carry the normal ADA alleles.





2. CLONING



1. Clone is a population of cells or individuals which are genetically identical.

2. The term 'clone' is derived from the Greek word 'Klan' meaning 'twig'.

3. Cloning is the process of producing many identical organisms or cones.

4. Cloning is traditionally done by horticulturists who develop a new plant by just planting a twig from parental plant.

5. There are two types of cloning, gene cloning at molecular level and cloning of organisms.

6. Cloning is meant for preservation of the genotype of the organism.

7. With the death of an organism, a particular genotype is lost.



Microbial Cloning:-

1. Each of the genetically altered or modified microbial cell can be duplicated every time it divides.

2. In just a few days there will be millions of cloned cells, each one canying a copy of the original donated gene.

3. Several improved genetically altered strains of microorganisms can be cloned in large numbers for various applications.



Genetically Engineered Microbes and their Applications



Microbe

Application

Bacillus thuringiensis

Production of Bt toxin

Escherichia coli

Production of human insulin, growth hormone, interferon, etc.

Pseudomonas fluorescence

Prevention of frost damage to plants

Pseudomonas putida

Scavenging of oil spills by digesting hydrocarbons of crude oil.

Rhizobium meliloti

Nitrogen fixation by "nif" gene in cereal crops.





Landmarks in Recombinant DNA Technology



1958 DNA polymerase purified.

1970 A complete gene synthesized in vitro.

Discovery of the enzymes restriction endonuclease and reverse transcriptase.

1972 First recombinant DNA molecules.

1973 Use of plasmid vectors for gene cloning.

1975 Southern blot technique for detecting specific DNA sequences.

1976 Genetech founded, a biotechnology company.

1977 Methods for rapid DNA sequencing.

Discovery of "split genes" and somatostatin synthesized using recombinant DNA.

Genetech produce first human protein (somato statin) in a micro organil.

1978 Human genome library constructed.

1979 Insulin synthesized using recombinant DNA.

First human viral antigen (hepatitis B) cloned.

1982 Commercial production of E. coli genetically engineered human insulin (Eli Lilly & co.). Isolation, cloning and characterization of a human cancer gene (Oncogene ).

1983 Engineered Ti plasmid used to transform plants.

Development of the Polymerase Chain Reaction (PCR) technique.

1988 The first successful production of a genetically engineered staple crop (soyabeans).

Development of the gene gun.

1991 Development of transgenic pigs and goats capable of manufacturing proteins such as human haemoglobin.

First test of gene therapy on human cancer patients.

1994 The Flavr Savr tomato introduced, the first genetically engineered whole food approved for sale.

Fully human monoclonal antibodies produced in genetically engineered mice

1995 Haemophilus influenzae genome sequenced.

1996 Methanococcus jannaschii and Saccharomyces cerevisiae genomes sequenced.

1997 Human clinical trials of antisense drugs and DNA vaccines begun E. coli genome sequenced.

1998 First cloned mammal (the sheep Dolly)

The complete sequence of the nematode Caenorhabditis elegans genome.

2000 Complete sequence of the euchromatic portion of the fruitfly Drosophila melanogaster genome

Complete sequence of the weed Arabidopsis thaliana (Mouse Ear Cress) genome.

2001 International Human Genome Sequencing Consortium and Celera Genomics published the first drafts of the sequence of the human genome.

2002 International Rice Genome Sequencing Project and Syngenta published first draft of the gnomic sequence of two rice subspecies.



Cell Cloning:-

1. Cell cloning is based on the property of totipotency (in plants) or pluriopotency (in animals).

2. Totipotency is the potential ability of a plant cell to grow into a complete plant.

3. Pluriopotency is the potential ability of a cell into develop any type of the cell in the animal body for instance nerve cells or kidney cells or heart cells.

4. Virtually all plants are totipotent but in animals only fertilized egg and stem cells in the embryonic blastocyst are totipotent.

5. Moreover in contrast to animals, there is no real distinction in plants between somatic cells and germ line cells.



Plant Cloning:-

1. Many orchids producing beautiful flowers are cloned plants.

2. Scientists have genetically engineered agronomically important crop plants.

3. Clones of plants are easy to get by vegetative propagation or tissue culture.

4. Rapid production can be achieved using actively dividing meristematic cells.

5. These occur at the root and shoot apices that are growth areas of the plant.

6. For the agriculture, disease, drought, insect pest-resistant as well as herbicide-tolerant crops have been successfully produced by gene manipulation.

7. Genetically Modified Food (GMF) like vitamin A-rich rice (Golden rice) and lysine-rich pulse seeds are now becoming components of human staple food.





Animal Cloning:-

1. Animal cloning is more difficult than plant cloning.

2. This is because animal cells lose their totipotency on reaching the gastrula stage of development.

3. Simian cloning (cloning of monkeys) was camed out by Don Wolf (USA) in 1996 from an eight-cell embryo.

4. ANDI was the first clone monkey by embryo splitting derived its name from a backward abbreviation for 'Inserted DNA'.

5. ANDI was the world's first genetically altered primate produced by inserting a jellyfish gene into the embryo of a rhesus monkey.

6. Ian Wilmut of Roslin Institute, Edinburgh, U.K. has produced a clone of adult lamb named "Dolly" (Feb. 1997).

7. The scientists extracted genetic material from the udder cells of one sheep and implanted it into another sheep's egg after removing its genetic material.

8. The fused cell developed into an embryo which was planted into the uterus of another sheep which acted as surrogate mother.

9. Recent genetic analysis of Dolly's DNA has shown that she is a "chimera", not a perfect clone.

10. Dolly has two genetic mothers as confirmed by the analysis of her mitochondria by Eric Schon and Ian Wilmut in 1999.

11. Dolly has nuclear genes from the ewe who supplied the udder cell an mitochondrial genes from the egg cytoplasm of the second ewe.

12. The Scottish scientists who cloned Dolly have (July 1997) produced Molly and Polly, two lambs clone with a human gene for blood clotting Factor IX.

13. The milk of "Molly" and "Polly" contains Factor IX that can be extracted for us in treating human haemophilia.

14. Steven Stice and James Robl (USA) have developed a technique for genetically customized calves that will be able to produce medicines for humans in their milk.

15. The first cloned calves George and Charlie were born in January 1998.



Human Cloning:-

1. Brigitte Boisslier, a 46-year old French chemist announced the creation of the world's first cloned human baby nicknamed "Eve" (December 2002).

2. Brigitte is the president of the Clonaid human cloning society which believes that man kind was created by extra-terrestrials.

3. Standard DNA profiling of both the baby and mother are not available to confirm the claim made by Brigitte Boisslier.

4. If the baby is a clone, its DNA will match both the nuclear DNA and the mitochondrial DNA of the woman who was said to be cloned.



3. TRANSGENICS



1. A gene that has been introduced into a cell or organism is called a transgene (for transferred gene) to distinguish it from endogenous genes.

2. The organism carrying the introduced foreign gene is said to be transgenic.

3. Thransgenics or transgenic organisms are also called Genetically Modified Organisms (GMOs).

4. Transgenics were first created and expressed in microorganisms, now techniques are available to make transgenic animals and higher plants.



Transgenic Animals:-

1. Two methods are used to produce transgenic animals:

(i) microinjection of DNA into fertilized eggs and

(ii) infection of preimplantation embryos with retroviral vectors.

2. Most of the transgenic animals studied to date were produced by microinjection of DNA into fertilized eggs.

3. Prior to microinjection, the eggs are surgically removed from female parent and fertilized in vitro.

4. The DNA is then microinjected into the male pronucleus of the fertilized egg through a very fine-tipped glass needle.

5. Usually, several hundred to several thousand copies of the gene of interest are injected into each egg.

6. The integration on injected DNA molecules appears to occur at random sites in the genome.

7. The first transgenic animal produced was the 'supermouse' by the incorporation of the gene for human growth hormone by Richard Palmiter and Ralph Brinster in 1981.

8. Transgenic animals have been genetically engineered with human genes in order to make products that can be used in human medicine.

9. Transgenic cattle (cow, sheep and goat) have been produced with the intention of increased milk yield, as well as, therapeutic human proteins.

10. The transgenic cattle secrete these useful proteins in their milk from where it can be 'harvested'.

11. Transgenic pigs have been given human genes so that their organs carry human antigens.

12. Transgenic pigs organs like heart, kidney, pancreas can be transplanted into humans. Surprisingly, these are not rejected.

13. Transgenic pigs show undesirable side effects of the higher growth hormone levels.

14. Most the female transgenic pigs are sterile, in addition both sexes are lethargic with weak muscles and are highly susceptible to arthritis and ulcers.



Transgenic Plants:-

1. Plants have been genetically manipulated by plant breeders for decades.

2. At present plant breeders can directly modify the DNA of plants; they can quickly add genes from other species to plant genomes by recombinant DNA techniques.

3. Transgenic plants can be produced by several different procedures.

4. One widely used procedure, called microprojectile bombardment, involves shooting DNA-coated tungsten or gold particles into plant cells.

5. Another procedure, called electroporation, uses a short burst of electricity to get DNA into the cell.

6. The most widely used method of generating transgenic plants, at least in dicots, is probably Agrobacterium tumefaciens mediated transformation.

7. The gene coding for the insecticidal protein form Bacillus thuringiensis can be transferred to the cotton plant and this transgenic cotton plant known as genetically modified cotton Bt is resistant to bollworm.

8. The genetic engineering permits the production of transgenic GMO tomato called Flavr Savr.

9. Flavr Savr tomato has much longer and more flavourful shelf-life than conventional tomatoes, because of delayed ripening.

10. Delayed ripening is possible by reducing the amount of cell wall degrading enzyme 'polygalacturonase' responsible for fruit softening.



4. DNA FINGERPRINTING



1. DNA fingerprinting also known genetic fingerprinting was developed by a British geneticist Prof. Alec Jefferys in 1984 at the University of Leivester.

2. A more recent and more sensitive version is known as DNA profiling.

3. DNA fingerprinting was first used in Britain to verify the parentage of an immigrant who had left the country and then wanted to return.

4. Each of us is genetically unique (with the exception of identical twins) and genetic variation could be used to identify individuals, much as conventional fingerprints does.

5. The chromosomes of very human cell contain scattered through their DNA short, highly repeated 15 nucleotide segments called "mini-satellites" or Variable-Number Tandem Repeat (VNTR).

6. The location and number of repeats of any particular minisatellites are so highly variable that no two people are alike.

7. The probability of two unrelated individuals having same pattern of location and repeat number of minisatellites is one in ten billion (in a world population of just over 5.4 billion); no two persons are alike.

8. Restriction Fragment Length Polymorphism (RFLP) distributed throughout human genomes are useful for gene mapping and DNA fingerprints.

9. The basis of DNA fingerprinting is the occurrence of restriction fragment length polymorphism.

10. DNA fingerprint method is very useful for DNA tests for identity and relationships, forensic studies and polymorphism.



Technique for DNA Fingerprinting:-

11. Only a small amount of tissues like blood or semen or skin cells or the hair root follicle is needed of DNA fingerprinting.

12. Typically DNA content of about 100,000 cells or about 1 microgram is sufficient.

13. The procedure of DNA fingerprinting involves the following major steps:

(i) DNA is isolated from the cells in a high-speed refrigerated centrifuge.

(ii) If the sample of DNA is very small, DNA can be amplified by Polymerase Chain Reaction (PCR).

(iii) DNA is then cut up into fragments of different length using restriction enzymes.

(iv) The fragments are separated according to size using gel electrophoresis through an agarose gel. The smaller fragments move faster down the gel than the larger ones.

(v) Double stranded DNA is then split into single stranded DNA using alkaline chemicals.

(vi) These separated DNA sequences are transferred to a nylon or nitrocellulose sheet placed over the gel. This is called 'Southern Blotting' (after Edward Southern, who first developed this method in 1975).

(vii) The nylon sheet is then immersed in a bath and probes or markers that are radioactive synthetic DNA segments of known sequences are added. The probes target a specific nucleotide sequence which is complementary to VNTR sequence and hybridizes them.

(viii) Finally, X-ray film is exposed to the nylon sheet containing radioactive probes. Dark bands develop at the probe sites which resemble the bar codes used by grocery store scanners to identify items.



Application of DNA Fingerprinting:-

This technique is now used to:

(i) Identify criminals in forensic laboratories.

(ii) Settle paternity disputes.

(iii) Verify whether a hopeful immigrant is, as he or she claims, really a close relative of already an established resident.

(iv) Identify racial groups to rewrite biological evolution.





5. GENOMICS



1. The term genome has been introduced by Winkler in 1920.

2. Genome refers to one complete copy of the genetic information (DNA) or one complete set of chromosomes (monoploid or haploid) of an organism.

3. The term genomics is relatively new, coined by Thomas Roderick in 1986.

4. Genomics is the sub-discipline of genetics devoted to the mapping, sequencing and functional analysis of genomes.

5. Genomics is subdivided into structural genomics and functional genomics.

6. Structural genomics is the study of genome structure, deals with the complete nucleotide sequences of the organisms.

7. Functional genomics is the study of genome function which includes transcriptome and proteome.

8. Transcriptome is a complete set of RNAs transcribed from a genome.

9. Proteome is a complete set of proteins encoded by a genome and aims at the determination of the structure and functions of all the proteins in living organisms.

10. During the last two decades, the complete DNA sequences of following have been determined:

(i) The genomes of 599 viruses and viroids.

(ii) 205 naturally occurring small intracellular self-replicating DNA molecules called plasmids.

(iii) 185 chromosomes of organelles such as chloroplasts and mitochondria.

(iv) Genomes of 32 eubacteria, 7 archaea, a fungus, a plant and 2 animals.

(v) In addition, two drafts of the sequences of the human genome have been published.



11. The nucleotide sequence of the genomes of two subspecies of rice were published in April 2002.

12. Human body contains 100 million cells of over 260 different kinds.

13. There is a complete set of instruction in each of the cell needed to make a whole new human being.

14. There are 23 different chromosomes containing packed DNA in a haploid set of human genome.

15. Additional DNA is in cell's mitochondria which is inherited from one's mother.



The Human Genome Project:-

1. The Human Genome Project, sometimes called "biology's moon shot", was launched on October 1, 1990 for sequencing the entire human genome of 2.75 billion (2.75  109 or 2750000 bp or 2750000 kilobase pairs or 2750 megabase pairs) nucleotide pairs.

2. The goals of this project are:

(i) To map all the human genes

(ii) To construct a detailed physical map of the entire human genome, and

(iii) To determine the nucleotide sequence of all 24 human chromosomes by the year 2005.

3. An international Human Genome Organization (HUGO) was organized to coordinate the efforts of human geneticists around the world.

4. James Watson was the first director of human genome project; after leadership during the first two years of the project, he was replaced by Francis Collins in 1993.

5. Two important scientists associated with human genome are: Francis Collins, director the Human Genome Project and J. Craig Venter, founding president of Celera Genomics.

6. The complete sequencing of the first human chromosome, small chromosome 22, was published in December 1999.

7. The complete sequence of human chromosome 21 followed in May 2000.

8. The first-draft sequences of entire human genome has appeared in the February 15, 2001 issue of Nature and the February 16, 2001 issue of Science.

9. According to present estimate there are about three billion nucleotide base pairs in human genome.

10. The number of genes in human genome is approximately 30,000 to 40, 000.

11. The number of genes in mice genome is almost same as human and nine-tenths of human genes are identical to that of mice.

12. Human being possess more than twice as many as genes in Drosophila.

13. Human beings are 99.9 per cent identical with each other at the DNA level.

14. Every alive human being is exactly the same and even bacteria are our cousins in code.

15. Different human genes vary widely in the length often over thousands of base pairs.

16. While -globin and insulin gene are less than 10 kilobase pairs, the gene responsible for Duchenne Muscular Dystrophy on 'X' chromosome is made up of 2400 kilobase pairs which is probably the longest gene known.

17. Though lily plant produces fewer proteins than human being, it has 18 times more DNA.

18. Only less than 2 per cent of the genome is known to include the exons, the protein coding sequences.

19. Approximately 1 million copies of short 5 to 8 base pair repeated sequence clustered around the centromeres and near the ends of the chromosomes represent 'the junk DNA'.

20. Associated with the Human Genome Project the parallel efforts to obtain gene maps and complete sequence of the genomes of a number of other model organisms, including E. coli. Yeast, Drosophila melanogaster, mouse, etc.

21. Sequencing of the somplete yeast, Saccharomyces cerevisiae, genome was completed in 1996; yeast was the first eukaryotic organism to have its entire genome sequenced.

22. Yeast genome contains an estimated 6,000 genes having 12 million base pairs.

23. In early 1997, the complete 4.7 million base pairs genome with 4000 genes of E. coli was reported.

24. E. coli is the most studied and best understood cellular organism on our planet.

25. Drosophila genome contains 180 million base pairs and 13,000 genes, fewer than the 18,000 genes reported in the nematode Caenorhabditis elegans, however the latter has only 97 million base pairs.

26. Drosophilla Melanogaster (fruit fly) is fondly regarded as Queen of Genetics or Cindrella of Genetics.

Genome of Model Organisms



Organism

No. of Base pairs

No. of genes

Bacteriophage

10 thousand

–

Escherichia coli

4.7 million

4,000

Saccharomyces cerevisiae

12 million

6,000

Caenorhabditis elegans

97 million

18,000

Drosophila melanogaste

180 million

13,000

Human

3 billion

30,000

Lily

106 billion

–



Prospects and Implications of Human Genome:-

1. The genome project is being compared to the discovery of antibiotics.

2. We will soon have details of more than 1200 genes that are responsible for common cardiovascular ailments, endocrine diseases, neurological disorders like Alzheimer's disease, deadly cancers and many more.

3. Efforts are in progress to determine genes that will revert cancerous cells to normal.

4. The human genome sequencing not only holds promise for a healthier living, it also holds the prospects of cast database of knowledge about designer drugs, genetically modified diets and finally our genetic identity.



6. GENE LIBRARIES AND GENE BANKS



1. A gene library is a collection of gene clones that contains all the DNA present in some source.

2. If the original source of the DNA was original DNA from a living organism, then the library seeks to include clones of all that DNA; it is called a genomic gene library.

3. Gene libraries can also be created using RNA.

4. If a gene library is created by enzymatic copying of RNA by reverse transcriptase (RNA-dependent DNA polymerase), it would be called c-DNA library.

5. A gene bank is repository of clones of known DNA fragments, genes, gene maps, seeds, spores, frozen sperms or eggs or embryos.

6. These are stored for possible use in genetic engineering and breeding experiments where species have become extinct.

7. Gene banks can be used increasingly as the rate of extinction increases, depleting the Earth's biodiversity and genetic variety.

8. If the function of the gene is unknown, its nucleotide sequence can be compared with thousands of gene sequences stored in three large computer gene banks, one is Germany, a second in Japan and a third in the United States.

9. Arobidopsis thalina (cruciferae family) is used as 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. The genomes has 130  106 bp and estimated 26000 genes.

10. C. elegans is a free living nematode. It is the first multicellular animal whose genome was completely sequenced

11. Many human disease networks are highly conserved in fruitfly. Thus this insect diseases, including neurological disorders.

12. Lesch Nyhan Syndorme is X linked disorder characterized by Mental retardation & bizarre behavior of affected children, including self mutilation. The syndrome is caused by mutations in the gene for hypoxanthine guanine phosphoribosyl transferees (HPRT).

FUNDAMENTALS OF MICROBIOLOGY-For Various Competitive Exams.

1. MICROBIOLOGY: INTRODUCTION

i.Micro-biology is study of microorganisms like viruses, Bacteria, Protozoa, 1-celled algae and 1-celled fungi etc. Antonie van Leeuwenhoek of Holland, Louis Pasteur of France, Robert Koch of Germany and Joseph Lister of England were earlier, workers in Microbiology. Leeuwenhoek is recognized as 'pioneer' worker in microbiology, he discovered bacteria for the first time as 'animalcules' or the 'wretched beastes'. Pasteur was fist to study role of microorganisms in fermentation process involved in wine preparation. Koch studied anthrax disease, caused by bacteria Bacillus anthracis. On the basis of his study Koch proposed Law of Pathogenicity, which was later on recognized as Koch's Postulates. Koch's Postulates are not applicable for viruses & organisms causing TB and leprosy. Lister introduced antiseptic surgery and also prepared culture of bacteria first time.

ii.The two major research institutes of microbiology in world are: Pasteur Institute of Microbiology in Paris, France and Lister Institute of Microbiology, England. Workers at Lister Institute discovered 'naked' bacteria i.e. bacteria without cell of mucopolysaccharide complex. They were called 'L-form' of bacteria, named after Lister Institute. Mycoplasma like organisms, PPLO, is called L-form of Bacteria.

iii.Triple Antigen Programme includes, immunization against Diptheria, Pertusis (Whooping cough) and Tetanus. It is also called D.P.T. Oral drops of polio are delivered separately. BCG vaccines are first vaccines given to new borns after 3-4 weeks of birth. Vaccines of: Polio was discovered by Salke; Small pox by Jenner, BCG by Calmett-Guire; Anthrax by Koch and Rabies by Pasteur. Vaccines of anthrax were prepared first. Pasteur, for developing vaccines of rabies was rewarded handsomely by Czar of Russia.



2. MICROBIOLOGY: STUDY OF VIRUSES

2a) Viruses: Introduction

i.Study of viruses is called virology and Beijerinck is recognized as 'father of virology'. He reported enormous power of multiplication of viruses inside host cell. He also studied mechanism of nitrogen fixation by microorganisms like bacteria.

ii.The term 'virus' has been derived from Greek word 'virion' which means 'poison'. Viruses are defined as hyperparasites and 'filterable entities' not having any cellular organization and defined as hyperparasites and 'filterable entities' not having any cellular organization and because of this cell theory is not applicable on them. They are thus exceptions to cell theory. They form crystals when taken out of the host cell. This shows non-living nature of viruses, which was first revealed by Stanley while working on 'Tobacco Mosaic Virus'. Nucleoprotein structure of viruses was first studied by Bawden & Pierre. In nucleoprotein structure of viruses, core of nucleic acid is surrounded by coat of protein. Viruses are unique as they contain only one type of nucleic acid. Mutation in viruses was first reported by Delbruck. Mutation and enormous power of reproduction confirm living nature of viruses. Viruses are thus the only entities, which have characters of non-living and living entities both. They are thus 'connecting links' between non-living entities and lining entities.



2b) Viruses: Historical Studies

Studies in viruses started with independent works of Pasteur and Adolf Mayer. Pasteur prepared vaccines of rabies virus and used the term 'virus' for first time. Mayer reported mosaic like spots on leaves of tobacco plant infected with virus. He described the virus as Tobacco Mosaic Virus [TMV]. Ivanowski passed the cell sap of infected tobacco leaves through bacteria proof bacteria proof filter and sprayed the filtrate on healthy leaves. Mosaic spots appeared again. Ivanowski through his experiment described viruses as filterable entities. Famous conclusion of Beijerinck about viruses i.e. 'contagium vivum fluidum' means sap retains the infection of viruses (living fluid infectant). Takami of Holland studied transmission of viruses through various agencies. He observed that insects are important agencies in transmission of viruses. Insects that transmit viruses are called 'vertors'. Viruses-vector relationship is highly specific.

Culture of viruses was prepared for the first time by Ender. It was poliomyelitis virus. Hershey and Chase observed that nucleic acid is the infective part of virus. They selected Bacteriophages for their experiment. Bacteriophages are DNA containing viruses which attack bacteria. They were discovered by Twort; further studies were made by DeHerelle. Experiment of Hershey & Chase is another example which confirms status of DNA as genetic material. Safferman & Morris discovered 'Cyanophages' i.e., viruses which attack blue green algae like Lyngbya, Phormidium & Plectonema. They are also called LPP-1 on the basis of hosts. Mycophages were discovered by Sinoten. They contain ds RNA.

Issac & Lindeman discovered a protein called Interferon secreted by host cells against infection of viruses. They selected cold virus for their experiment. Gibbs described viruses in coded form which is called 'Cryptogram of viruses' Cryptogram of viruses is based on 4 pairs of characters:



First pair : Type of nucleic acid in virus i.e. R or D

Number of thread in nucleic acid 1 or 2

Second pair : Molecular weight of viruses in millions i.e. 2

Percentage of nucleic acid of the viruses 5

Third pair : Shape of the virus in total i.e. E (elongeted) or S (spiral) or

X (unknown)

Shape of the protein coat of the virus E or S or X

(E' means elongated structure or rod shaped structure with blunt ends)

Fourth pair : Host/vector i.e. S (seed plant), I(invertebrate), V(vertebrate),

B(bacterium), Fu(fungus), Di(diptra), Af(aphid), Zero or 0 (novector),

X(unknown vector) Vector of the virus X



Thus, for example R/1; 2/5; E/E; S/X is cryptogram of TMV.

For Polia Virus (Poliovirus Primus) cryptogram is R/1; 5/30; S/S; V/O.

For Influenza Virus (mixovirus influenzae) R/1; 2-3/10; S/E; V/O.

For T4 phage D/2; 130/40; X/X; B/O.



2c) Viruses: Some Terms

i.A complete virus having a core of nucleic acid and coat of protein is called virion. Protein coat of the virus is termed as capsid; units of capsid are capsomeres. A virus drops its infective part i.e., nucleic acid alone inside host cell; empty protein coat remains outside. This empty protein coat, which remains outside the host called ghost protein, disappears later on. Raw material for new protein coat of the progeny viruses is contributed by the host.

 A few proteins in some viruses are enzymatic in functions e.g. lysozyme in bacteriophages, reverse transcriptase(RNA dependent DNA polymerase) in retrovituses, neuraminidase in influenza virus etc.

ii.Viriod: Subviral agent having infectious nucleic acid alone is called viroid. Viroids are RNA & particles lacks protein coat. Spindle disease in photo tuber is caused by viroid, which is PSTV. Viroids were discovered by Diener & Rayner.

iii.Prion or Slow Virus: Protein macromolecules causing infections are called prions. They lack ucleic acids. Scrappes & Kuru diseases (Severe brain infection) is sheep are caused by prions. They were first reported by Priusner who was awarded Nobel Prize for reporting Mad Cow disease caused by PrP (prion related protein). Bovine spongiform encephalopathy or Creulzfeldt Jacob disease (a type of insanity in young people), Kuru disease (the laughing death), Alzheimer's disease are caused by prions. In 1976 D.C.Gujdusek was awarded Nobel Prize for his work on prion based diseases.

iv.Viruses transmitted by insects are called Arboviruses. Viruses causing Dengue, encephalitis etc are arboviruses.

v.Viruses which contain ss-DNA are called 'Gemini viruses'. X174 phages are geminiviruses. They were reported by Sinsheimer.

vi.Viruses which enter inside host cell as intact structures, are called Adeno viruses. Well defined protein coat is absent in Adenoviruses.

vii.Virusoids or Satellilte RNAs (Rundle et. Al 1981). These are small circular RNAs similar to viroids, but are located inside the protein coat of a true virus. Virusoids required the assistance of virus for their replication.



2d) Viruses: Classification and Nomenclature









































Earlier it was believed that plant viruses i.e. Phytophagineae contain RNA whereas animal viruses i.e. Zoophagineae contain DNA. This is, however, no more correct now. The only thing which is true about viruses is presence of only one type of nucleic acid. Cold virus i.e. Rhinovirus and poliovirus i.e. Enterovirus is together placed under Picorna virus.



2e) Viruses: Disease on Plants – Symptoms

i.Mosaic like spots may develop on leaves.

ii.Leaves may show rolling or curling.

iii.Leaves may show loss of chlorophyll (Chlorosis) or death of tissues (necrosis).

iv.Plants may show abnormal or stunted growth.

v.Plants may show uncontrolled cell division i.e. hyperplasia(cell number increase) and unwanted swelling i.e. Hypertrophy in host.

vi.Plants may show loss of entire inflorescence or flowers may become green i.e. virescence in flowers.



2f) Viruses: Some Common Viral Diseases

1. Plant Viral Diseases. Tobacco Mosaic, Potato Mosaic Pumpkin Mosaic, Apple Mosaic, Tulip Mosaic (Broken Tulip), Wheat Mosaic Streak, Bhindi Yellow Vein Mosaic, Potato Leaf Roll, Tobacco Leaf Curl, Papaya Leaf Curl. Banana Bunchy Top, Tomato Bunchy Top, Rice Tungro etc.



2. Animal Viral Diseases. Foot and Mouthm, Enquine encephalitis, Rinderpest, Kysanur Forest Disease (KFD) of monkeys (also humans), New Castle or Ranikhet of Fow].



3. Human Viral Diseases. Poliomyelitis, Rabies, Mumps, Measles, German Measles, Chickenpox, Smallpox (eradicated), Dengue, Encephalitis, Common cold, Flu, some gastrointestinal diseases, Yellow Fever, Herpes, Hepatitis, SARS, AIDS.



Some Important Modern Viral Diseases



1. Cancer or Carcinoma:

Despite the fact that virus causing cancer is yet to be identified, still cancer is considered like a viral disease because of typical viral symptoms like Hyperplasia & hypertrophy, are reported in cancer. Certain genes called proto-oncogenes (c-onc) are present in human cells, which are involved in normal cell function. Mutation of proto-oncogenes may give rise to oncogenes, which stimulate cell division leading to an uncontrolled growth of the cells causing cancer. All retoveiruses and some DNA containing viruses (such as poxvirus, adenovirus, Herpes virus, hepatitis-B virus etc.) possess oncogene (v.onc) in their genome. These viruses are potential cancer producing pathogens. Study of cancer is called Oncology. Temin and Baltimore's studies on Rous sarcoma virus [RSV] have provided new dimensions in the knowledge of cancer. RSV is RNA containing virus and RNA of RSV forms DNA inside host cell through reverse transcription or 'Teminism'. Newly formed DNA develops malignant tumours in host. Harmful tumor is called malignant tumours whereas tumours, which are not harmful, are termed as benign tumours. Are lethal in advanced stage.







2. Acquired Immunodeficiency Syndrome (AIDS):

AIDS is a viral disease in which our acquired immunity defense system is gradually shattered, and the patient dies of multiple infectious diseases. The first indication of AIDS disease came to light in USA in 1981 amongst a group of young homosexuals who were addicts of herion and other narcotics. Luc Montagnier (1983) and Robert Gallo (1984) discovered that a new type of retrovirus is the causative pathogen of AIDS. In 1986, The International Committee of virus Nomenclature (ICVN) named the AIDS virus as Human Immunodeficiency Virus (HIV).

Although AIDS is hardly a two and half decades old disease, it has spread virtually all over the world, becoming pandemic. According to the estimates of world Health Organization (WHO), about 52 million people in the world harbour HIV and thousands of new cases are detected every day. In India HIV infection was first detected in 1986 in some sex workers of Tamil Nadu. Since then, about 4 million cases have been detected and infection is fast spreading.



Structure of HIV:

HIV is a spherical, enveloped virus of about 90-120 nm diameter. Its genome consists of a single stranded RNA filament segmented into two identical filaments and associated with a 'reverse transcriptase enzyme'. Due to the presence of two identical filaments of RNA, HIV is considered to be 'diploid'. The envelope is comprised of a lipid bilayer derived from host cell membrane, and projecting, knob like glycoprotein spikes with pedicels formed of virus coded glycol-proteins (gp).



Pathogenesis and Symptoms:

The sentinels of our defense system are a type of white blood cells called lymphocytes. These cells are of different types to play different roles in acquired immunity. One type of lymphocytes called T4(TH) lymphocytes, play the crucial role in activation of other components of the immune system and production of antibodies. HIV attacks T4 lymphocytes and destroy them. As a result the T4 lymphocyte count in blood gradually diminishes (< 200). From the blood plasma the virus also permeates into other body fluids like semen, vaginal fluids saliva, tears, urine, sweat, breast milk etc. Gradual destruction of T4 lymphocytes through several years ultimately leads to a full blown AIDS disease, during which a patient suffers from persistent fever and diarrhea, loss of appetite and weight, weakness and fatique, body pain, dry cough, white lesions in mouth and alimentary canal and warts upon anus and genitals. Eventually, the patient loses immunity and dies of multiple infectious diseases.



Transmission:

HIV is present in blood and other body fluids of infected persons. Hence it is transmitted from one to another person through activities involving exchange or transfer of body fluids. Thus, HIV is primarily transmitted through sexual activities. Unprotected sex, sharing of syringes and needles by intravenous drug addicts, use of unsterilized syringes by doctors and health workers etc. are responsible for transmission of HIV. Transfusion of infected blood and blood products, transplantations from infected to healthy one through breast milk feeding after birth are the other potential mode of transmission of HIV.



Diagnosis:

Some genes of HIV genome code for synthesis of some antigens which are released into the blood by infected T4 lymphocytes. The immune system of body produces antibodies against these antigens, which can be detected by serological test called Elisa (Enzyme Linked Immunosorbent Assay), western blotting and PCR polymerase reaction test are the confirmatory test.

Symbol used to describe AIDS disease is ' ' (red ribbon). Noted Hollywood star Rock Hudson & Lawn Tennis Player Arthur Ashe of US were notable AIDS-causalities.

NARI (National AIDS Research Institute Puna).

NACO (National AIDS Control Organisation).

ARV (Anti Retro Viral Drug).

Prevention and Treatment:

General precautions such as safer sex and extensive use of condoms, strict screening of blood and blood products used for transfusions and tissue and organ transplantations, and extensive health education can prevent AIDS. December, 1 is observed as World's AIDS Day to arouse general awareness amongst people about HIV infection.

At present, there is no effective drug to save the immune system of AIDS patient from collapse. However, there are some drugs which are known to delay the progress of AIDS. These include Zidovudine (Azidothymidine-AZT), XQ-9302, Amphotericine-B and its methyl ester etc.



3. Pox:

Following types of pox diseases are reported in man:

Small Pox : Caused by DNA containing Pox viridae, Varoila.

Chicken Pox : Caused by DNA containing Herpes Virus, Vacicella.

German Pox : Caused by RNA containing Para influenza virus, Rubella.

Measles or Khasra : Caused by RNA containing Para influenza virus or Paramyxoviridae with minus strand RNA. AS already mentioned, vaccines of small pox were prepared by Jenner. Earlier small pox was very lethal disease in epidemic form in India. Officially, it has been eradicated from India with the help of WHO.



Natural Defenses against Viral Infections:



1. Interferons (Isaacs and Lindenmann, 1957). Interferons are antiviral proteins (a class of glycoproteins) which diffuse out from infected cells, enter into the cells in the neighbourhood and impart a defense mechanism to these cells against viral infections by inhibiting the synthesis of viral proteins. These are therefore used for therapeutic and preventive purposes in viral infections, interferons are always secreted by host cells against viral infection.



2. Body Immunity. Viruses are good antigens to induce active production of specific antibodies (by lymphocytes).



3. Immunoprophylaxis – Vaccines: Vaccines contain inactivated (attenuated) virions, which cannot cause the concerned diseases, but still act as antigens to induce antibody production by the immune system of the body.



4. Chemotherapy – Antiviral Drugs: The chemotherapeutic agents antibiotics which are used in the treatment and control of bacterial diseases are ineffective for viral diseases. However, some antiviral drugs have been prepared. These include (i) Thiosemicarbozone (for treatment and prevention of smallpox), (ii) Ribavirin or Virozole (for influenza),
(iii) Deoxyuridines, Acylovir (for herpes), (iv) Foscarnet (for hepatitis-B) etc.



2g) Viruses: Study of Bacteriophages

i.Discovery of bacteriophages is associated with Twort when he reported viral disease in bacteria. The term bacteriophages was coined by De Herelle. They are viruses, which attack bacteria. A typical bacteriophage looks like a tadpole/sperm comprising a hexagonal head of protein and a tail also a protein they show binal symmetry. Head is 1000A long and 650A in width; length of the tail is 1000A. Thus, head and tail are equal in length. Infective part of the bacteriophage in most of the cases is ds-linear DNA and it is present in hexagonal head. Joint of head and tails is neck, which is covered by collar of protein. Tail ends in end plate that develops 3 pairs of tail fibers of protein. Bacteriophages that attack E.coli are called coliphage or T-phages. They were numbered as T1, T2, T3, T4, T5, T6, T7 etc by Delburck, T2, T4, T6 etc are T-even phages T1, T3, T5, etc or T-odd phages.

ii.A bacteriophage comes into contact with the bacterial (host) cell through tail fibres. A passage is created in the host cell once the mucocomplex cell wall of bacteria is broken by lysozyme of bacteriophage. Once the passage is created, ds-linear DNA of bacteriophage is dropped inside the host cell. Empty protein coat i.e. Ghost protein remains out the host cell and disappears later on. Infective part or genetic material in X174 phages is ss circular DNA and it is RNA in MS-2 & R17 phages.

iii.Phage DNA dropped inside host cell may be virulent or temperate. Bacterial cell is destroyed if phage DNA is virulent, bacterial cell is not destroyed is phage DNA is temperate.

iv.Destruction of bacterial cell by virulent phage DNA is called lysis. The sequence of events leading to lysis of bacterial cell are:

Virulent phage DNA dropped inside bacterial cell.

Multiplication of phage DNA inside host cell i.e. Formation of prophage. Formation of complete phages. (End of latent period or period of eclipse of phages).

Destruction or lysis of bacterial cell (Total time: 15-20 minutes).*

v. A bacterial cell is not destroyed if phage DNA is temperate. Temperate phage DNA is attached with host DNA; this is called lysogeny. A lysogenic bacterial cell behaves normally till temperate phage DNA is attached. Lysogeny in bacteria was first reported by Lwoff. Lwoff also coined the term -phage for those phages in which tail terminates into single tail fibre.

Lysogeny may be of 3 types. They are -phage lysogeny; P1-phage lysogeny and mu-phage lysogeny. In -phage lysogeny, temperate phage DNA is attached with bacterial DNA at specific place i.e. at 'gal' & 'bio' genes of bacterial DNA. In P1-phage lysogeny, temperate phage DNA is attached with any place other than gal's genes. In mu-phage lysogeny, temperate phage DNA is never attached with bacterial DNA, remain in bacterial cytoplasm as plasmid.

A lysogenic bacterial cell shows different behaviour when exposed to different dosages of UV rays:

































3. Microbiology: Study of Bacteria

3a) Bacteria: Introduction

i.Study of Bacteria is called 'Bacteriology'. Leeuwenhoek is recognized as pioneer worker in Bacteriology. Pasteur, Koch & Lister are other earlier workers in Bacteriology.

ii.The term bacteria were coined by Ehrenberg for the smaller 'animalcules' or the 'wretched beasts' of Leeuwenhoek. Bacteria are found everywhere and in all sorts of climates however they do not survive in acidic medium of Pickle solution or highly concentrated sucrose solution due to plasmolysis.

iii.Bacteria activity is reduced greatly at low temperature; this explains why foodstuff remains safe for longer period in cold devices like fridge and refrigerators etc. Bacteria behave in different manner in different range of temperature.























3b) Bacteria : Forms

i.On the basis of shape, bacteria may be cocci (Singular coccus), Bacilli (Bacillus), Vibrio, Spirillum etc. Cocci are spherical bacteria, smallest in size. They may be single (monoococucus), in pairs (Diplococcus), in chains (streptococcus), in bunches (staphylococcus) or cuboid (sar cinnae). Bacteria causing pneumonia are Diplococcus Bacilli are rod shaped bacteria they may be single or in chains (Streptobacilli). Majority of parasitic bacteria are rod shaped bacteria. Streptobacilli surrounded by mucilaginous sheath form Zoogloea. Vibro are comma shaped bacteria. Bacteria causing cholera are vibrio bacteria. Spirillum and spirochaete are zigzag spring like bacteria, Spirillum volutans is recognized as largest bacteria. The terms like coccus, bacillus, vibrio etc were coined by Mueller.

ii.On the basis of cilia, a bacterial cell may be:

Atrichous : Bacterial cell without cilia.

Monotrichour : Bacteria cell with single cilia.

Lopotrichous : Bacteria cell with tuft of cilia at one end.

Cephalotrichous : Bacterial cell with tuft of cilia at both the ends.

Amphitrichous : Bacterial cell with one cilia at both the ends.

Peritrichous : Bacterial cell with cilia throughout the body.



Bacteria causing cholera disease is comma shaped & Lophotrichous.

iii.On the basis of nutrition, bacteria may heterotrophy or autotroph. Heterotroph bacteria are further divided as saprophytic bacteria & parasitic bacteria. Similarly, autotroph bacteria are further divided as chemosynthetic and photosynthetic bacteria.

Bacteria, which obtain their food from dead organic matter, are saprophytic bacteria. They perform significant role of decomposers in ecosystem. Bacteria, which obtain their food from other living entity, are called parasitic bacteria. The living entity, which provides food to parasite, is termed as host. Host-parasite relation is specific. Parasite is called pathogen when it spreads in host.

Bacteria, which obtain their food through the inorganic reactions in which they participate, are called chemosynthetic bacteria. They are named after the inorganic reactions, us that they are involved. Thus, N-Bacteria S-Bacteria & Fe-Bacteria etc are examples of chemosynthetic bacteria. Chemosynthetic bacteria are basically autotroph but do not require pigment chlorophyll for energy requirement. Chemosynthetic bacteria will survive alone if chlorophyll pigment disappears from the world; rest will perish.

Bacteria, which obtain chemical energy through glucose formation, are called photosynthetic bacteria. They are autotrophs and depend on chlorophyll like pigments i.e., Bacteriochlorophyll & chlorobium chlorophyll (Bacterio viridies) for glucose formation. Green-S-Bacteria, Purple-S-Bacteria & Rhodospirillium etc. are examples of photosynthetic bacteria.



Comparison between normal and bacterial photosynthesis

Normal Photosynthesis

Bacterial Photosynthesis

1. Raw materials are CO2 & H2O

1. Raw materials are CO2 & H2S

2. H for reduction of CO2 into glucose comes from H2O

2. H- for reduction of CO2 into glucose comes from H2S.

3. Principal pigment is chlorophyll a

3. Principal pigment Bacteriochlorophyll a.

4. Intermediate H-acceptor is NADP

4. Intermediate H-acceptor is NAD.

5. PS I & PS II both are present in light phase.

5. PS I only is present in light phase; PS II is absent.

6. O2 is released as by product

6. S is released as by product

7. Biochemical reaction

6CO2 + 12H2O = C6H12O6+6H2O+6O2

7. Biochemical reaction.

6CO2 + 12H2O = C6H12O6+6H2O+12S

Generalised equation for both of the above

6CO2 + 12H2A = C6H12O6 + 6H2O + 12A

A may be oxygen or sulfur.



Photosynthesis in bacteria was first studied by Van Neil of Germany who was first to support the idea of Robino Hill that O2 released in normal photosynthesis comes from H2O and not from CO2.



3c) Bacteria: Disadvantages

Bacteria are our 'foes' as they are associated with several diseases of plants and animals including man due to parasitic mode of nutrition of some bacteria. Bacterial attack is less severe on plants than it is on animals.

Important bacterial diseases on plants are:

(a)Canker of Citrus (citrus canker) Xanthomonas citri

(b)Angular leaf spot of cotton X. malvacearum

(c)Fire blight of apples Erwinnia amylovora

(d)Crown gall of crops Agrobacterium tumefaciens

(e)Tondu of wheat Corynebacterium tritici

Important bacterial diseases on man are:

(f)Cyphillis Tryponema pallidum

(g)Plague (Black Death) Pasteurella pestis (Y. pestis)

(h)Chlorea Vibrio cholerae

(i)Anthrax Bacillus anthracis

(j)Pneumonia Diplococcus pneumonae

(k)Tuberculosis (TB) Mycobacterium tuberculoses

(l)Typhoid (Enteric fever) Salmonella typhi

(m)Bacterial dysentery Bacillus dysentrae (Shigella)

(n)Amoeboid dysentery Entamoeba histolytica (Protozoa)

(o)Tetanus (Lock jaw) Closteridium tetani

(p)Ptomaine (deadly food poisoning) Closteridium botulinum

(q)Leprosy Bacillus lepri (Mycobacterium lepri)

(r)Diptheria Corynebacterium diptherae

(s)Erysepalas (deadly blood Streptococcus

Poisoning; Death of RBC's)

(t)Whooping cough Bordetella partusis

(u)Gonorrhoea Neisseria gonorrhoae

Bacterial diseases like cholera, typhoid, dysentery etc are water borne diseases: tuberculosis, plague etc are air-borne diseases whereas disease like tetanus spreads through wounds. Syphilis is transmitted through sexual contact. Transmission of leprosy through contact is a matter of dispute. India based Mother Teresa was awarded Nobel Prize for her service to leprosy patients in West Bengal and other parts of India.



3d) Bacteria: Advantages:

Bacteria are our friends as they are associated with several advantages in various forms. Some of them are:

i.Bacteria & Ecosystem: Saprophytic bacteria along with saprophytic fungi perform useful role of decomposers in ecosystem. Decomposition of organic matter in absence of O2 is called putrefaction and breakdown of organic matter in presence of O2 is called decay. Highly un-decomposed organic matter is litter; partly decomposed organic matter is humus and highly decomposed organic matter is called humus.

ii.Bacteria & Industry: Lactobacillus laciti and Streptococcus lacti convert milk into curd. Acetobacillus aciti changes sugar solution into vinegar. Flavour in tea leaves by Micococcus condensa and in tobacco leaves by Micococus megatherium develops due to fermentation activity of bacteria. Retting involves separation of plant fibers after decomposition of non-cellulosic materials by various species of Closteridium like C.butyricum and C.felsineum.

iii.Bacteria and N-cycle system: Nitrogen is first essential element in terms of requirement for proper growth of plants. It is required in protein synthesis, chlorophyll synthesis and also in synthesis of nucleic acids. Plants cannot utilize molecular nitrogen directly. The plants mainly in the form of nitrates absorb it. Nitrogen changes into nitrate via ammonia formation. Some bacteria control N-cycle, which comprises Nitrogen fixation, Nitrification and Denitrification. Nitrogen fixation involves conversion of N2 into ammonia. Bacteria which participate in N-fixation are Closteridium, Azotobacter and Rhizobium etc. Closteridium and Azotobacter and asymbiotic biological N fixing bacteria whereas Rhizobium is symbiotic biological N-fixing bacteria are present in root nodules of Leguminous or papilionaceous plants. Klebsiella is leaf nodule N-fixing bacteria. Frankia is non-leguminous N-fixing bacteria 'nif' genes control biological N-fixation. Mechanism of biological N-fixation by bacteria was first studied by Heijerinck whereas Winogradasky studied biological N2 fixation by some blue green algae.

Conversion of NH3 into nitrite is termed as initial stage of Nitrification. A bacterium which participates in initial stage of nitrification is nitrosomonas Conversions of nitrite into nitrate is called final stage of nitrification. Nitrobacter is the bacteria which participates in final stage of nitrification.

Release of Nitrogen from Macromolecules of Nitrogen is called denitrification. Bacillus denitrificans is the bacteria involved in denitrification. The summary of Nitrogen cycle is given in the following figure:







































Summary of various N-fixing bacteria:

Azotobacter, Thiobacillus, Mycobacterium etc. : Free living & aerobic.

Closteridium Klebsiella : Free living & anaerobic.

Rhizobium, Aopzprirllium Frankia : Symbiotic & anaerobic

Citrobacter : Symbiotic and anaerobic

Bacteria & Antibiotics: Though first antibiotic penicillin was obtained from ascomycetes fungus Penicillium notatum by Alexander Flemming in 1929, yet soil-borne bacteria Streptomyces of actinomycetes (ray fungi) are rich source of various types of antibioitics. Antibiotics are chemical inhibition. The term 'antibiotic' was coined by Salman Waksman. Antibiotics are known as chemotherapeutic agents. Some of the antibiotics extracted from bacteria are bacitracin, subtilin, polymyxin, gramicidin etc.



Salient features of some antibiotics chemotherapeutic agents



Name of the antibiotics

Name of the discover

Source of antibiotics

Action of antibiotics

Penicillin

Alexander Flemming

P. notatum

P. chrysogenum

Breaks muco complex cell wall of bacteria.

Streptomycin

Waksman

Streptomyces griesus

Inhibits synthesis of 30S fragment of ribosome in bacteria.

Erythromycin

Mc Guire

S. erythreus

Inhibits synthesis of 50S fragment of ribosome in bacteria

Chloromycin

Burkholder

S. venezualae

Inhibits initiation of protein synthesis in bacteria.

Aureomycin

Duggar

S. auerofaciens

Inhibits formation of tRNA amino acid complex.

Terramycin

Pfizer Ltd.

S. rimosus

Inhibits formation of tRNA amino acid complex.



Penicillin is not broad spectrum antibiotic because it is more active on gram positive bacteria, less effective on gram negative bacteria. Streptomycin is first brad spectrum antibiotic which is equally active on gram positive and gram negative bacteria both.

Bacteria & Miscellaneous uses

(a)Aerobacter aerogenes decomposes DDT whereas Flavobacterium and Corynebacterium decompose 2, 4-D etc. DDT and 2, 4-D are non-biodegradable chemicals, which may be lethal even.

(b)Bacillus pudita, pseudomonas and some other oil-eating bacteria help in cleaning up oil spills. They thus help in preventing water pollution of seas/oceans caused due to oil refineries.

(c)Anand Mohan Chakroborty has developed genetically engineered bacteria named Superbug that feeds on oil spills.

(d)Bacterium Methylophylus methylotropous is source of single cell protein (SCP) the product was called 'Pruteen'. Fungus Fusarium is also used for the purpose i.e. preparation of mycoprotein on larger scale.

(e)Batch culture & continuous culture are two main methods of fermentation on larger scale. The former is closed system whereas latter is open system.







3e) Bacteria: Classification:

Bacteria and blue green algae are prokaryotes as they lack true nucleus in absence of nuclear membranes. They were placed together under Schizophyta by Cohn in 1853. However, Whittaker in his famous five kingdom classification placed prokaryotes under Monera, a term coined by Stanier. Bergey's Manual in Determinative Microbiology (1974) is widely recognized in medical science:































3f) Bacteria: Structure of a Cell:

i.Bacteria is placed in plant kingdom because contains rigid & non-living cell wall which is outer most in position. Bacterial cell wall is made up of muco-complex or peptidoglycan. Muco-complex is a complex formed between few simple sugars and proteins: The sugars are N-Acetyl glycosamine (NAG) and N-Acetyl Muramic Acid (NAM) or murein. Protein of mucocomplex are formed by few amino acid i.e. Glycine, Lysine, glutamic acid and diaminopimelic acid (DAP): Peptidoglycan linkage of bacterial cell wall is broken by lysozyme enzyme of bacteriophages or by antibiotic penicillin.

Staining of bacterial cell wall was first introduced by Christian Gram. It involves staining of bacterial cell with crystal-violet (Gram Stan). With this bacterial cells appear deep violet coloured. Crystal violet staining is followed by treatment with organic solvent. If deep violet colour is retained by bacterial cell after treating with organic solvent then bacteria are called Gram positive. If deep violet colour is lost after treating with organic solvent then bacteria are termed as Gram negative.

Difference between Gram Positive and Gram Negative Bacteria

Mucocomplex content is high in gram positive bacteria

Mucocomplex content is low in gram negative bacteria

Teichoic acid is present

Teichoic acid is absent

Penicillin is more effective

penicillin is less effective

Lipid content is low, this explains why deep violet colour is retained after treatment with organic solvent

Lipid content is high, this explains why deep violet colour is lost after treatment with organic solvent lipid is dissolved in organic solvent

Example: Bacillus subtilis (Haybacillus)

Example: E. coli

Mucocomplex cell wall is surrounded by slime layer, which changes into capsule layer during unfavourable condition. Capsulated stage in bacteria is resistant to antibiotics to larger extent.

ii.True nucleus is absent in bacteria because nuclear membrances are absent in prokaryotes, Incipient nucleus in prokaryotes is called 'NUCLEOID' and it is represented by a double stranded circular DNA only. Nucleolus is also absent. ds circular DNA without proteins in bacteria may also be considered as bacterial chromosome.

iii.Membranou cell-organelles remain absent in prokaryotes including bacteria, however in some bacteria, inward folding of cell membrane may form structures like mesomomes & chromatophores: Mesosomes, first reported by Frtiz-James, develop in aerobic bacteria for complete breakdown of macromolecules in presence of O2. Definite role of mesosomes is also reported during replication of DNA. Chromatophores or photosynthetic lamellae develop for photosynthesis. Ribosomes for protein synthesis are 70S in size and they remain embedded directly in cytoplasm in absence of ER. Since ribosomes remain embedded directly in cytoplasm this explains why density of prokaryotic cytoplasm is more than density if eukaryotic. The two fragments of 70S ribosomes are 50S & 30S.

Carbohydrates are stored in the form of glycogen whereas storage of protein is in the form of volutin granules. Storage of inorganic substances is in the form of granulose.

iv.A bacterial cell may or may not have flagella. Bacterial flagellum is different from eukaryotic flagellum because bacterial flagellum is made of flagellin proteins and 9+2 arrangement is also not present. Flagella like structures but much smaller in length are called fimbrae or Pilli. Fimbrae or pili may be specialized to form sex pili, which play significant role during conjugation in bacteria.

v.Extra inheritable material, present in cytoplasm bacterial cell is called 'plasmid' discovered by Lederberg. Some plasmids are capable of combining with main genetic material i.e. DNA of bacterial cells. They are called Episomes. Therefore, it may be concluded that all episomes are plasmids but all plasmids are not episomes. R Factor in bacteria is purely a plasmid; it provides resistance against antibiotic. The other examples of common plasmids in bacteria are pBR322 and Col-factor, which produces colicin. F factor (fertility-factor) in bacteria is an example of episome. It plays significant role in conjugation. Bacterial cell, which contains F factor, is cytoplasm called F+ cell or donor cell and bacterial cell, which lack F factor, is called F– cell or recipient cell. Bacterial cell is termed as High frequency Recombinant (HFR) when F factor combines with DNA of bacteria.

3g) Bacteria: Reproduction:

i.Asexual reproduction through binary fission is very common method of reproduction in Bacteria. It takes place during favourable conditions. One parent bacterial cell splits to form two daughter bacterial cells in binary fission.

A bacterial cell divides every minute; time required to form 128 cells of bacteria will be? Number of divisions required to form 128 bacterial of cells will be:

1 Cell ---- Istdiv-----2 Cells ----IInddiv----4 Cells ----IIIrddiv---8 Cells---IVthdiv---16 Cells---Vthdiv---32 Cells----VIthdiv-----64 Cells---VIIthdiv---128 cells. Thus, numbe of divisions required to form 128 bacterial cells=7. Therefore, time required to form 128 bacterial cells: 7 divisions X 1 minute = 7 minutes.

A bacterial cell divides every minute it takes one hour to fill a cup; time required to fill half & quarter of the cup will be?











From the above, it is clear that: (X + 1+ 1) + 60. Hence, X = 58 minute.

Therefore time required to fill ¼ cup will be: 58 minutes and time required to form ½ cup will be 58 + 1 = 59 minutes.

ii.Conidia like structure develops during asexual reproduction in fungi like bacteria i.e., in Actinomycetales.

iii.Bacteria forms endospore during unfavourable conditions it is the hardest structure of bacteria. Bacterial cell under goes capsulated stage and entire inner contents shrink to form endospore. One bacterial cell forms one endospore during unfavourable conditions.

iv.Sexual reproduction or syngamy involves fusion of two haploid units or gametes. The two haploid units are formed after meiosis or reduction division. In bacteria meiosis or reduction division is not possible because bacterial cell contains single chromosome. This sexual reproduction or syngamy is not reported in bacteria in absence of meiosis. Sexual reproduction plays significant role in evolution because it is main method of genetic recombination in living entities. Though well-defined sexual reproduction is missing in bacteria, yet bacteria shows some important methods of genetic recombination.

3h) Bacteria: Methods of Genetic Recombination:

Three main methods of genetic recombination are reported in bacteria. They are conjugation, transformation and transduction. In addition to these three, sexduction is also a method of genetic recombination in bacteria.

i.Conjugation, a method of genetic recombination in bacteria was first reported by Lederberg & Tatum in bacterium E. coli. It involves transfer of 'F factor' from donor bacterial cell F+ to recipient bacterial cell F– through direct contact. The two types of bacterial cells i.e. F+ and F– come in contact with each other through sex pili and a passage called conjugation tube develops for transfer of F factor. F factor is doubled before transfer and F– cell becomes F+ after transfer of F factor. Thus genetic recombination takes places in recipient cell:





















ii.Transformation another method of genetic recombination in bacteria was first reported by Griffith in 1926-27 in bacterium Diplococcus pneumonae. The event is in vitro (in Lab) only and not in vivo (not in nature). It involves transfer of a part of DNA of donor bacterial cell to recipient bacterial cell through medium solution. Part of DNA of donor bacterial cell, which is transferred, is called transformosome. It is 1/1000 of original DNA content of donor bacterial cell.























Griffith prepared two strains of Diplococcus pneumonae i.e. non-capsulated, non-virulent type (R-Type) and capsulated, virulent type (S-Type). The two types were infected in mice and following observations were observed:

R-Type : all mice healthy

S-type : all mice died of pneumonia

S-type Heat killed : all mice healthy

R-type + S-type (heat killed) : all mice died of pneumonia

The observation that all mice died when R-type + S-type (heat killed) was injected, prompted Griffith to conclude that there was transfer of 'something' from heat killed S-type to R-type, which made R-type virulent. Griffith could not identify this something all that time. Avery, Macleod & McCarty (Avery et al) later on repeated the experiment of Griffith in 1948 and identified that this something was part of DNA of S-type (heat killed), which was transferred to R-type. These parts of DNA called transformosome made R-type virulent. Avery et al. with this finding established the status of DNA as every genetic material. Work of Hershey & Chase involving DNA as infective part of Bacteriophage is another experiment, which establishes status of DNA as genetic material.

iii.Transduction yet another method of genetic recombination, was discovered by Zinder & Laederberg in bacterium Salmonella typhi. It involves transfer of a part of DNA (from donor bacterial ell to recipient bacterial cell through bacteriophages. The part of DNA transferred is about 1/1000 of original DNA content. The event is rare but it is reported in nature. New phages may carry a part of DNA of bacterial cell. Bacterial cell B' (recipient cell) receives a part of DNA of Bacterial cell A' (donor cell) through bacteriophages. In generalised transduction, recipient cell receives plasmid of donor cell.



























iv.SEXDUCTION was reported by Addelburg & Burns in E. coli. They observed that during conversion of HFR into F+ dissociation of F factor is not always accurate. F factor some times carries a part of main DNA of Bacteria. This F factor with a part of bacterial DNA is called F prime of F' factor. When this cell conjugates and transfers the gene attached with F prime, recipient cell becomes 'partially diploid' for the particular gene. This phenomenon has been described as sexduction in bactera.



Methods of genetic recombination in bacteria are tools of genetic engineering that is a nascent branch of biotechnology genetic engineering aims at developing superior character of desired results by manipulating at gene level. Bacterial plasmids like pBR322, Ti factor (Tumor inducing factor) etc are of immense value in genetic engineering. Several diseases like Diabetes etc may be cured with the help of genetic engineering. High yielding & disease resistant varieties of crops may also be developed with the help of genetic engineering. Thus genetic engineering in near future may prove boon to mankind: but at the same time it may prove to be a curse as well. The biggest threat of genetic engineering is of biological warfare. Pathogens of several dreaded diseases of past may be regenerated again which may cause huge destruction to mankind.



4. Microbiology: Miscellaneous

i.Mycoplasma were discovered by Nocard & Roux as Mollicutes. They are 'L-form' of bacteria, which are filterable like viruses. They are called 'L-form' because they lack well defined mucocomplex cell wall of typical bacteria. Due to this they are polymorphic entities i.e. they may exist in several forms; hence the popular name of 'joker' in biological kingdom. Due to absence of cell wall, mycoplasma looks like a boiled fried egg. Mycro Plasma are gram negative and are not affected by antibiotics penicillin (inhibitor of cell wall formation) but are inhibited by tetracyclin Mycoplasma has a definite cellular organization; a cell of mycoplasma is the smallest cell. Genetic material is ds DNA that is highly coiled and attached with a disc at one end. Cytoplasm contains RNA & 70S ribosomes. Mycoplasma require sterols for the growth. Some biologists place mycoplasma between viruses & bacteria. Dieseases like 'Little leaf of brinjal' and 'witches broom' of sugarcane etc are caused by Mycoplasma and related mollicutes placed under Pleuro Pneumonia like organism (PPLO). Some microbiologists have named mycoplasma like organism as Spiroplasma, which cause diseases on plants.

ii.Rickettsias are tiny obligate, gram negative pleomorphic but walled parasites present in blood sucking insects like ticks, lices, mites etc. They may be rod-shaped or spherical. They have mucopeptide cell wall and genetic material is ds circular DNA. Cytoplasm contains RNA and 70S ribosomes. They are sensitive to antibiotics and multiply by fission. Diseases like Typhus, Q-fever, Trench fever rocky-mountain spotted fever etc are caused by Rickettsias. Ricketts first observed them but Proper description was made by Rocha-levis later on.

iii.Spirochaetes are protozoa like aflagellate bacteria. They have an axial filament called crista, which is spirally coiled. They show gliding movement and multiply by fission. Diseases like Syphilis, Jaundice etc are caused by them.

iv.Chlamydiae: They are Gram(-ve) intracellular parasites of about 0.25 m. size, often grouped along rickettsiae. However, their reproductive cycle differs from rickettsiae in the formation of initial or reticulate bodies (RB) and elementary bodies inside host phagosome. They have cell wall similar to typical bacterial wall. They lack ATP formation and obtain the same from outside chlamydiae are thus energy parasites. The common diseases caused by chlamydiae include Conjuntivitis, nongonococcal urethritis and lymphogram losa venecatum, pneumonia and bronchopneumonia, psittacosis.



SARS (Severe Acute Respiratory Syndrome), this viral disease was first reported in Guangdong Province in Southern China in Nov. 2002. The cause of SARS was identified as a member of the Corona Viridae (single stranded RNA viruses).

SARS causing virus transmitted by droplets from coughs and sneezes.

It produces severe form of pneumonia, fever of 100.4 oF (some time with chills), headache, bodyaches and malaise.

High Security Animal Disease laboratory Bhopal, is the main center where SARS virus study carried out.





Vaccination:

Vaccination is a preventative strategy that aims to stimulate the host immune system, by exposing it to the infectious agent in question in an inactivated or incomplete form. There are four types of virus vaccines:

1. Attenuated (='weakened') vaccines contains 'live' viruses, but ones whose pathogenicity has been greatly reduced. E.g. Polio Virus vaccine developed by Albert Sabin in the 1960s. The cowpox virus used by Edward Jenner in his pioneering vaccination work in the late 18th century was a naturally occurring attenuated version of the smallpox virus.

2. Inactivated vaccines contain viruses which have been exposed to a denaturing agent such as formalin. This has the effect of rendering them non-infectious, while at the same time retaining their ability to stimulate an immune response. Vaccines directed against influenza are of this type.

3. Subunit vaccines depend on the stimulation of an immune response by just a part of the virus. Since the complete virus is not introduced, there is no change of infection, so vaccines of this type have the attraction of being very safe. Subunit vaccines are often made using recombinant DNA technology; the first example to be approved for human use was the hepatitis B vaccine, which consists of part of the protein coat of the virus produced in specially engineered yeast cells.

4. DNA vaccines are also the product of modern molecular biology techniques. DNA coding for virus antigens is directly injected into the host, where it is expressed and triggers a response by the immune system. Vaccines of this type have not so far been approved for use in humans.





Satellite Viruses:-

i.Satellite viruses are also known as 'incomplete viruses'.

ii.The replication of this virus is dependent of coinfection of a second 'helper' virus.

iii.Satellite virus and helper virus are separate entities; they encode their own capsids and infect the cell independently.

स्टेम सेल से श्वास नली का सफल प्रतिरोपण

श्वास नली का सफल प्रतिरोपण




चिकित्सा जगत में तीसरी बार श्वास नली का प्रत्यारोपण किया गया है

ब्रिटेन में 10 साल के लड़के की श्वास नली का उसके ख़ुद के स्टेम सेल से सफल प्रतिरोपण किया गया है.



चिकित्सा जगत में इस ऑपरेशन को बड़ी उपलब्धि माना जा रहा है. क़रीब नौ घंटे तक ग्रेट ऑरमंड स्ट्रीट हॉस्पिटल में चले इस ऑपरेशन में श्वास नली के लिए स्टेम सेल इस लड़के के शरीर से ही लिया गया.



हालाँकि श्वास नली का दुनिया में पहला प्रतिरोपण 2008 में स्पेन में किया गया था, लेकिन इस ऑपरेशन में श्वास नली के बहुत छोटे हिस्से का प्रतिरोपण कर दिया गया था.



डॉक्टरों के कहना है कि लड़के के स्वास्थ्य में तेज़ी से सुधार हो रहा है और सामान्य रूप से सांस ले रहा है.



उपलब्धि



इस लड़के को जन्म से ही सांस लेने में मुश्किल होती थी और इसकी वजह थी उसकी श्वास नली का बेहद संकरा होना. डॉक्टरों के अनुसार जन्म के समय लड़के की श्वास नली सिर्फ़ एक मिलीमीटर चौड़ी थी.



पहली बार किसी बच्चे का उपचार स्टेम सेल से किया गया है और ये अब तक की सबले लंबी प्रत्यारोपित श्वास नली है

मार्टिन ब्रिशेल

डॉक्टरों ने इस लड़के की श्वास नली का रास्ता चौड़ा करने के लिए पिछले साल नवंबर में भी ऑपरेशन किया था, लेकिन ये सफल नहीं हो सका और मरीज़ को कई दिक्कतों का सामना करना पड़ा.



इसके बाद डॉक्टरों ने लड़के के बोन मैरो (मेरु रज्जा) से स्टेम सेल लेकर धातु की श्वास नली के अंदर और बाहर प्रत्यारोपित करने का निर्णय लिया. डॉक्टरों को भरोसा है कि अगले महीने तक स्टेम सेल स्पेशलाइज़्ड सेल में बदल जाएगी.



दो साल पहले दो बच्चों की माँ 30 वर्षीय क्लाउडिया कास्टिलो स्टेम सेल से बनी श्वास नली को प्रत्यारोपित करने वाली पहली शख्स थीं. तपेदिक के चलते उनकी श्वास नली ख़राब हो गई थी.