by

DR. MANOJ KUMAR SHARMAPh.D., F.L.S. (London)

Janta Vedic College,Baraut, Baghpat (U.P.)

(An ISO 9001:2008 Certified Company)

Vayu Education of India2/25, Ansari Road, Darya Ganj, New Delhi-110 002

CYTOGENETICS ANDBIOTECHNOLOGY

Cytogenetics and Biotechnology

Copyright © VAYU EDUCATION OF INDIA

ISBN: 978-93-83758-02-9

First Edition: 2014

Price: 320/-

All rights reserved. No part of this publication may be reproduced, stored in a retrievalsystem, or transmitted, in any form or by any means, electronic, mechanical,photocopying, recording or otherwise, without the prior permission of the Author.

Published by:

AN ISO 9001:2008 CERTIFIED COMPANY

VAYU EDUCATION OF INDIA2/25, Ansari Road, Darya Ganj, New Delhi-110 002Ph.: 91-11-43526600, 41564445Fax: 91-11-41564440E-mail: vayueducation@rediffmail.com, vayueducation1@gmail.comWebsite: www.veiindia.com

The present book has been written according to the latest syllabussuggested by U.G.C. It is written in simple and easy language. Thisbook provides knowledge about cell biology, genetics and biotechn-ology. As we know that cell biology, genetics and biotechnology has awide scope and taught as foundation course to U.G. This book providesbasic knowledge to all undergraduates students like B.Sc. (Micro-biology), B.Sc. (Industrial micro biology), B.Sc. (Biotechnology),B.Sc. (Biochemistry) and B.Sc. (Biology).

First of all I thank to almighty for giving me strength for thisnoble cause. My respectful thank to Dr. Y. S. Tomar Principal,Dr. Sudhir Kumar, Dr. Baljeet Singh, Dr. S. P. Singh, Dr. A. K. Sharma,Dr. Rajeshwari Sharma, Dr. Bharatveer, Dr. K.P. Singh and mycolleagues Dr. Sanjay, Mr. Manoj, Dr. Amarpal and Dr. Umendra.

Last but not least, I would like to pay thanks to my parents andother family members-Ranjna, Varusha and Harshit Bhardwaj and Ialso thank to my publisher Dr. R.K.Jain and all those who are directlyor indirectly engaged in the publication of this book.

Dr. Manoj Kumar Sharma

P R E FP R E FP R E FP R E FP R E FAAAAAC EC EC EC EC E

CONTENTCONTENTCONTENTCONTENTCONTENT

Preface v

Chapter–1: Cell 1Chapter–2: Cell division 68Chapter–3: Chromosome 91Chapter–4: Chromosomal Mutations-Structural Changes 114Chapter–5: Chromosomal Mutations-Numerical Changes 125Chapter–6: Nucleic acid 145Chapter–7: Genetic Code 190Chapter–8: Protein 216Chapter–9: Gene and its Regulation 228Chapter–10: Genetics 265Chapter–11: Gene Intrection 297Chapter–12: Mutation 314Chapter–13: Genetic Linkage 340Chapter–14: Extra Nuclear Inheritance 357Chapter–15: Biotechnology 368Chapter–16: Recombinant DNA Technology 373Chapter–17: Cloning Vector 385Chapter–18: Biotechnology and its Applications 446Chapter–19: Growth Medium 472Chapter–20: Tissue Culture 484Index 509

The cell was discovered by Robert Hooke in 1665. The celltheory, first developed in 1839 by Matthias Jakob Schleidenand Theodor Schwann, states that all organisms are composedof one or more cells, that all cells come from pre-existing cells, thatvital functions of an organism occur within cells, and that all cellscontain the hereditary information necessary for regulating cellfunctions and for transmitting information to the next generation ofcells. The cell is the basic structural, functional and biological unit ofall known living organisms. Cells are the smallest unit of life that isclassified as a living thing, and are often called the “building blocksof life”.

Cells consist of protoplasm enclosed within a membrane,which contains many biomolecules such as proteins and nucleicacids. Organisms can be classified as unicellular (consisting of asingle cell; including most bacteria) or multicellular (includingplants and animals). While the number of cells in plants and animalsvaries from species to species, humans contain about 100 trillion (1014)cells.

PROK ARYOTIC CELLThe prokaryotes are a group of organisms whose cells lack amembrane-bound nucleus (karyon). The organisms whose cells do havea nucleus are called eukaryotes. Most prokaryotes are unicellularorganisms, although a few such as myxo-bacteria have multicellularstages in their life cycles or create large colonies like cyanobacteria.The word prokaryote comes from the Greek "pro" before and "karyon"nut or "kernel". Prokaryotes do not have a nucleus, mitochondria, orany other membrane-bound organelles. In other words, all theirintracellular water-soluble components (proteins, DNA andmetabolites) are located together in the same area enclosed by thecell membrane, rather than separated in different cellularcompartments.

C e l lCHAPTER

2 Cytogenetics and Biotechnology

Prokaryot ic Cel l Structure1. Flagellum2. Glycocalyx3. Cell wall (except Mycoplasma)4. Cell membrane5. Cytoplasm6. Ribosome7. Nucleoid8. Inclusions

A Prokaryotic Cel l has three Architectural Regions1. On the outside, flagella and pili project from the cell's surface.

These are structures (not present in all prokaryotes) made ofproteins that facilitate movement and communication betweencells.

2. Enclosing the cell is the cell envelope generally consisting of acell wall covering a plasma membrane though some bacteriaalso have a further covering layer called a capsule. The envelopegives rigidity to the cell and separates the interior of the cellfrom its environment, serving as a protective filter. Thoughmost prokaryotes have a cell wall, there are exceptions suchas Mycoplasma (bacteria) and Thermoplasma (archaea). Thecell wall consists of peptidoglycan in bacteria, and acts as anadditional barrier against exterior forces. It also prevents thecell from expanding and bursting (cytolysis) from osmoticpressure due to a hypotonic environment. Some eukaryoticcells (plant cells and fungal cells) also have a cell wall.

3. Inside the cell is the cytoplasmic region that contains thegenome (DNA), ribosomes and various sorts of inclusions. Theprokaryotic chromosome is usually a circular molecule (anexception is that of the bacterium Borrelia burgdorferi, whichcauses Lyme disease). Though not forming a nucleus, the DNAis condensed in a nucleoid. Prokaryotes can carry extra-chromosomal DNA elements called plasmids, which are usuallycircular. Plasmids encode additional genes, such as antibioticresistance genes.

Morphology of Prokaryot ic Cell sProkaryotic cells have various shapes;

The four basic shapes are:• Cocci- spherical• Bacilli - rod-shaped

Cell 3

• Spirochaete - spiral-shaped• Vibrio - comma-shaped

Rep r od uc t i o nBacteria and archaea reproduce through asexual reproduction, usuallyby binary fission. Genetic exchange and recombination still occur, butthis a form of horizontal gene transfer and is not a replicative process,simply involving the transference of DNA between two cells, as inbacterial conjugation.

DNA TransferDNA transfer between prokaryotic cells occurs both in bacteria andarchaea, although it has been mainly been studied in bacteria. Inbacteria, gene transfer occurs by three processes. These are

(1) Transduction, bacterial virus (bacteriophage)-mediated(2) Conjugation, plasmid-mediated(3) Transformation

Tr a n s d uc t i o nTransduction of bacterial genes by bacteriophage appears to reflectan occasional error during intracellular assembly of virus particles,rather than an adaptation of the host bacteria. The transfer of bacterialDNA is under the control of the bacteriophage's genes rather thanbacterial genes.

Co n j ug a t i o nConjugation in the well-studied E. coli system is controlled by plasmidgenes, and is an adaptation for distributing copies of a plasmid from

4 Cytogenetics and Biotechnology

one bacterial host to another. Infrequently during this process, aplasmid may integrate into the host bacterial chromosome, andsubsequently transfer part of the host bacterial DNA to anotherbacterium. Plasmid mediated transfer of host bacterial DNA(conjugation) also appears to be an accidental process rather than abacterial adaptation. Usually, the DNA is spread throughout the entirecell, where it is readily accessible to be transcribed into messengerRNA (mRNA) that is immediately translated by ribosomes into protein.Sometimes, when biologists prepare prokaryotic cells for viewingunder a microscope, the DNA will condense in one part of the cellproducing a darkened area called a nucleoid. The prokaryoticchromosome is intimately associated with special proteins involvedin maintaining the chromosomal structure and regulating geneexpression. In addition to a single large piece of chromosomal DNA,many prokaryotic cells also contain small pieces of DNA calledplasmids. These circular rings of DNA are replicated independentlyof the chromosome and can be transferred from one prokaryotic cellto another through pili, which are small projections of the cellmembrane that can form physical channels with the pili of adjacentcells. The transfer of plasmids between one cell and another is oftenreferred to as "bacterial sex".

The genes for antibiotic resistance, or the gradual ineffectivenessof antibiotics in populations, are often carried on plasmids. If theseplasmids get transferred from resistant cells to nonresistant cells,bacterial infection in populations can become much harder to control.For example, it was recently learned that the superbug MRSA, ormultidrug-resistant Staphylococcus aureus, received some of its drug-resistance genes on plasmids. Natural bacterial transformationinvolves the transfer of DNA from one bacterium to another throughthe intervening medium.

Tra n s fo r ma t io nUnlike transduction and conjugation, transformation is clearly abacterial adaptation for DNA transfer, because it depends on numerousbacterial gene products that specifically interact to perform thiscomplex process. For a bacterium to bind, take up and recombine donorDNA into its own chromosome, it must first enter a specialphysiological state called competence. About 40 genes are required inBacillus subtilis for the development of competence. The length of DNAtransferred during B. subtilis transformation can be as much as a thirdto the whole chromosome. Transformation is a common mode of DNA

Cell 5

transfer, and 67 prokaryotic species are thus far known to be naturallycompetent for transformation. The development of competence innature is usually associated with stressful environmental conditions,and appears to be an adaptation for promoting repair of DNA damagein recipient cells.

Among archaea, Halobacterium volcanii forms cytoplasmic bridgesbetween cells that appear to be used for transfer of DNA from onecell to another. Another archaeon, Sulfolobus solfataricus, transfersDNA between cells by direct contact. Frols et al. found that exposureof S. solfataricus to DNA damaging agents induces cellularaggregation, and suggested that cellular aggregation may enhanceDNA transfer among cells to provide increased repair of damagedDNA via homologous recombination.

Evolut ion of ProkaryotesThe current model of the evolution of the first living organisms is thatthese were some form of prokaryotes, which may have evolved out ofprotobionts. In general, the eukaryotes are thought to have evolvedlater in the history of life. However, some authors have questionedthis conclusion, arguing that the current set of prokaryotic speciesmay have evolved from more complex eukaryotic ancestors through aprocess of simplification. Others have argued that the three domainsof life arose simultaneously, from a set of varied cells that formed asingle gene pool.

Phylogenetic tree showing the diversity of prokaryotes, comparedto eukaryotes.

6 Cytogenetics and Biotechnology

There is no consensus among biologists concerning the positionof the eukaryotes in the overall scheme of cell evolution. Currentopinions on the origin and position of eukaryotes span a broadspectrum including the views that eukaryotes arose first in evolutionand that prokaryotes descend from them, that eukaryotes arosecontemporaneously with eubacteria and archeabacteria and hencerepresent a primary line of descent of equal age and rank as theprokaryotes, that eukaryotes arose through a symbiotic evententailing an endosymbiotic origin of the nucleus, that eukaryotes arosewithout endosymbiosis, and that eukaryotes arose through a symbioticevent entailing a simultaneous endosymbiotic origin of the flagellumand the nucleus, in addition to many other models, which have beenreviewed and summarized elsewhere.

The division to prokaryotes and eukaryotes reflects two distinctlevels of cellular organization rather than biological classification ofspecies. Prokaryotes include two major classification domains: thebacteria and the archaea. Archaea were recognized as a domain oflife in 1990. These organisms were originally thought to live only ininhospitable conditions such as extremes of temperature, pH, andradiation but have since been found in all types of habitats.

Relat ionship to EukaryotesThe division between prokaryotes and eukaryotes is usuallyconsidered the most important distinction among organisms. Thedistinction is that eukaryotic cells have a "true" nucleus containingtheir DNA, whereas prokaryotic cells do not have a nucleus. Onecriticism of this classification points out that the word "prokaryote"is based on what these organisms are not (they are not eukaryotic),rather than what they are (either archaea or bacteria). Anotherdifference is that ribosomes in prokaryotes are smaller than ineukaryotes. However, two organelles found in many eukaryotic cells,mitochondria and chloroplasts, contain ribosomes similar in size andmakeup to those found in prokaryotes. This is one of many pieces ofevidence that mitochondria and chloroplasts are themselves descendedfrom free-living bacteria. This theory holds that early eukaryotic cellstook in primitive prokaryotic cells by phagocytosis and adaptedthemselves to incorporate their structures, leading to the mitochondriawe see today.

The genome in a prokaryote is held within a DNA/protein complexin the cytosol called the nucleoid, which lacks a nuclear envelope.The complex contains a single, cyclic, double-stranded molecule of

Cell 7

stable chromosomal DNA, in contrast to the multiple linear, compact,highly organized chromosomes found in eukaryotic cells. In addition,many important genes of prokaryotes are stored in separate circularDNA structures called plasmids.

Prokaryotes lack mitochondria and chloroplasts. Instead,processes such as oxidative phosphorylation and photosynthesis takeplace across the prokaryotic cell membrane. However, prokaryotesdo possess some internal structures, such as prokaryoticcytoskeletons, and the bacterial order Plancto-mycetes have amembrane around their nucleoid and contain other membrane-boundcellular structures. Both eukaryotes and prokaryotes contain largeRNA / protein structures called ribosomes, which produce protein.

Prokaryotic cells are usually much smaller than eukaryotic cells.Therefore, prokaryotes have a larger surface-area-to-volume ratio,giving them a higher metabolic rate, a higher growth rate, and as aconsequence, a shorter generation time than eukaryotes.

In 1977, Carl Woese proposed dividing prokaryotes into theBacteria and Archaea (originally Eubacteria and Archae-bacteria)because of the major differences in the structure and genetics betweenthe two groups of organisms. This arrangement of Eukaryota (alsocalled "Eukarya"), Bacteria, and Archaea is called the three-domainsystem, replacing the traditional two-empire system.

S i z eThe sizes of prokaryotes relative to other organisms and biomolecules

Most prokaryotes are between 1 µm and 10 µm, but they can varyin size from 0.2 µm to 750 µm (Thiomargarita namibiensis).

E n v i r o n me n tProkaryotes live in nearly all environments on Earth. Some archaeaand bacteria thrive in harsh conditions, such as high temperatures

8 Cytogenetics and Biotechnology

(thermophiles) or high salinity (halophiles). Organisms such as theseare referred to as extremophiles. Many archaea grow as plankton inthe oceans. Symbiotic prokaryotes live in or on the bodies of otherorganisms, including humans.

Eukaryot ic Cell sComponents of a typical cell:

1. Nucleus2. Nucleolus3. Ribosome (little dots)4. Vesicle5. Rough endoplasmic reticulum6. Golgi apparatus (or "Golgi body")7. Cytoskeleton8. Smooth endoplasmic reticulum9. Mitochondrion

10. Vacuole11. Cytosol (fluid that contains organelles)12. Lysosome13. CentrosomePlants, animals, fungi, slime moulds, protozoa, and algae are all

eukaryotic. These cells are about fifteen times wider than a typicalprokaryote and can be as much as a thousand times greater in volume.The main distinguishing feature of eukaryotes as compared toprokaryotes is compartmentalization: the presence of membrane-bound compartments in which specific metabolic activities take place.Most important among these is a cell nucleus, a membrane-delineatedcompartment that houses the eukaryotic cell's DNA. This nucleus givesthe eukaryote its name, which means "true nucleus." Other differencesinclude:

1. The plasma membrane resembles that of prokaryotes infunction, with minor differences in the setup. Cell walls mayor may not be present.

2. The eukaryotic DNA is organized in one or more linearmolecules, called chromosomes, which are associated withhistone proteins. All chromosomal DNA is stored in the cellnucleus, separated from the cytoplasm by a membrane. Someeukaryotic organelles such as mitochondria also contain someDNA.

3. Eukaryotes can move using motile cilia or flagella. Eukaryoticflagella are less complex than those of prokaryotes.

Cell 9

CELL WALL OF PROKARYOTIC CELLAround the outside of the cell membrane is the bacterial cell wall.Bacterial cell walls are made of peptidoglycan (also called murein),which is made from polysaccharide chains cross-linked by unusualpeptides containing D-amino acids. Bacterial cell walls are differentfrom the cell walls of plants and fungi which are made of cellulose andchitin, respectively. The cell wall of bacteria is also distinct from thatof Archaea, which do not contain peptidoglycan. The antibioticpenicillin is able to kill bacteria by preventing the cross-linking ofpeptidoglycan and this causes the cell wall to weaken and lyse. Thelysozyme enzyme can also damage bacterial cell walls.

There are broadly speaking two different types of cell wall inbacteria, called Gram-positive and Gram-negative. The namesoriginate from the reaction of cells to the Gram stain, a test long-employed for the classification of bacterial species.

Gram-positive bacteria possess a thick cell wall containing manylayers of peptidoglycan and teichoic acids. In contrast, Gram-negativebacteria have a relatively thin cell wall consisting of a few layers ofpeptidoglycan surrounded by a second lipid membrane containing

10 Cytogenetics and Biotechnology

lipopolysaccharides and lipoproteins. Most bacteria have the Gram-negative cell wall and only the Firmicutes and Actino-bacteria(previously known as the low G+C and high G+C Gram-positivebacteria, respectively) have the alternative Gram-positivearrangement.

Showing the arrangement of N-Acetyl glucosamine

Showing arrangement of N-Acetyl muramic acid

Cel l Wal l of Eukar yot ic Cel lThe cell wall is the tough, flexible but sometimes fairly rigid layerthat surrounds some types of cells. It is located outside the cellmembrane and provides these cells with structural support andprotection, in addition to acting as a filtering mechanism. A majorfunction of the cell wall is to act as a pressure vessel, preventing over-expansion when water enters the cell. Cell walls are found in plants,

Cell 11

bacteria, fungi, algae, and some archaea. Animals and protozoa do nothave cell walls.

Plant cell wall structure

The material in the cell wall varies between species, and can alsodiffer depending on cell type and developmental stage. In bacteria,peptidoglycan forms the cell wall. Archaean cell walls have variouscompositions, and may be formed of glycoprotein S-layers, pseudo-peptidoglycan, or polysaccharides. Fungi possess cell walls made ofthe glucosamine polymer chitin, and algae typically possess walls madeof glycoproteins and polysaccharides.

The walls of plant cells must have sufficient tensile strength towithstand internal osmotic pressures of several times atmosphericpressure that result from the difference in solute concentrationbetween the cell interior and external water. Plant cell walls varyfrom 1/10 to several µm thick.

L a ye r sUp to three strata or layers may be found in plant cell walls:

• The middle lamella, a layer rich in pectins. This outermostlayer forms the interface between adjacent plant cells and gluesthem together.

Cytogenetics and Biotechnology By Dr.Manoj Kumar Sharma

Publisher : Vayu Education ISBN : 9789383758029 Author : Dr. Manoj KumarSharma

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