solutions manual for biotechnology fundamentals … · q3. explain mendelian’s genetics. answer:...

SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN

SOLUTIONS


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Q4 True

Q5 Human insulin

Q6 1991

Q7 False

Q8 Restriction endonucleases

Q9 True

Q10 Golden rice

Section C: Critical Thinking

Answers are not provided in this section as the students are encouraged to write their

own viewpoints.

CHAPTER 2 GENES & GENOMICS

Section A: Descriptive Type

Q1. What is the cell theory?

Answer: The idea of the cell theory is coined by a Frenchman, H.J. Dutrochet, and the

credit for formulating the cell theory is given to a German botanist, M.I. Schleiden and a

German zoologist, T. Schwann, who clearly outlines the basic features of the theory in

1839. Moreover, in the year 1858, R. Virchow extended the cell theory and suggested

that all living cells arise from pre-existing living cells. To prove Virchow hypothesis,

Louis Pasteur performed experiments suggested that living things are composed of cells

and all living cells are arise from pre-existing cells. There is an exception that does not fit

into cell theory, such as viruses which may be defined as an infectious subcellular and

ultramicroscopic organism. The viruses are simple as they lack the internal organization

which is the main characteristics of a living cell and due to this unique characteristics,

viruses, mycoplasma, viroids, and prions do not easily fit in the definition of a cell

theory. In addition, there are other organisms in protozoa and algae, which also don’t fit

in the definition of cell theory.

Q2. Describe the characteristics of a prokaryotic cell.

Answer: The prokaryote cell is simpler than a eukaryote cell, lacking a nucleus and most

of the other organelles of eukaryotes. There are two kinds of prokaryotes: bacteria and

archaea; they share a similar overall structure. A prokaryotic cell has three architectural

regions: (a) on the outside, flagella and pili project from the cell's surface. These are



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structures (not present in all prokaryotes) made of proteins that facilitate movement and

communication between cells; (b) enclosing the cell is the cell envelope generally

consisting of a cell wall covering a plasma membrane though some bacteria also have a

further covering layer called a capsule. The envelope gives rigidity to the cell and

separates the interior of the cell from its environment, serving as a protective filter.

Though most prokaryotes have a cell wall, there are exceptions, such as Mycoplasma

(bacteria) and Thermoplasma (archaea)). The cell wall consists of peptidoglycan in

bacteria, and acts as an additional barrier against exterior forces. It also prevents the cell

from expanding and finally bursting (cytolysis) from osmotic pressure against a

hypotonic environment. Some eukaryote cells (plant cells and fungi cells) also have a cell

wall; (c) inside the cell is the cytoplasmic region that contain the cell genome (DNA) and

ribosomes and various sorts of inclusions. A prokaryotic chromosome is usually a

circular in a shape with an exception is that of the bacterium Borrelia burgdorferi, which

causes Lyme disease, DNA is linear in shape. One of the distinct features of prokaryote is

the absence of nucleus in the cytoplasm; DNA is usually condensed in a nucleoid.

Prokaryotes can carry extra-chromosomal DNA elements called plasmids, which are

usually circular. Plasmids enable additional functions, such as antibiotic resistance. The

presence of plasmid DNA in bacteria not only made bacteria a distinct from animal or

plant but also put the bacteria in the unique position as an important genetic engineering

tool.

Q3. Explain Mendelian’s genetics.

Answer: For thousands of years, farmers and herders have been selectively breeding

their plants and animals to produce more useful hybrids. It was somewhat of a hit or miss

process since the actual mechanisms governing inheritance were unknown. Knowledge of

these genetic mechanisms finally came as a result of careful laboratory breeding

experiments carried out over the last century and a half. By the 1890's, the invention of

better microscopes allowed biologists to discover the basic facts of cell division and

sexual reproduction. The focus of genetics research, then shifted to understanding what

really happens in the transmission of hereditary traits from parents to children. A number

of hypotheses were suggested to explain heredity, but Gregor Mendel, a little known

Central European monk, was the only one who got it more or less right. His ideas



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published in 1866 but largely went unrecognized until 1900, which was long after his

death. While Mendel's research was with plants, the basic underlying principles of

heredity that he discovered also apply to people and other animals because the

mechanisms of heredity are essentially the same for all complex life forms. Through the

selective cross-breeding of common pea plants (Pisum sativum) over many generations,

Mendel discovered that certain traits show up in offspring without any blending of

parental characteristics. For instance, the pea flowers are either purple or white--

intermediate colors do not appear in the offspring of cross-pollinated pea plants. Mendel

observed seven traits that were easily recognized and apparently only occurred in one of

two forms:

Flower color is purple or white;

Flower position is terminal

Stem length is long or short

Seed shape is round or wrinkled

Seed color is yellow or green

Pod shape is inflated or constricted

Pod color is yellow or green

This observation that these traits do not show up in offspring plants with intermediate

forms were critically important because the leading theory in biology at the time was that

inherited traits blend from generation to generation. Most of the leading scientists in the

19th century accepted this "blending theory." Charles Darwin proposed another equally

wrong theory known as "pangenesis". This held that hereditary "particles" in our bodies

are affected by the things we do during our lifetime. These modified particles were

thought to migrate via blood to the reproductive cells and subsequently could be inherited

by the next generation. This was essentially a variation of Lamarck's incorrect idea of the

"inheritance of acquired characteristics." Mendel picked common garden pea plants for

the focus of his research because they can be grown easily in large numbers and their

reproduction can be manipulated. Pea plants have both male and female reproductive

organs. As a result, they can either self-pollinate themselves or cross-pollinate with

another plant. In his experiments, Mendel was able to select cross-pollinate purebred

plants with particular traits and observe the outcome over many generations. This was the



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basis for his conclusions about the nature of genetic inheritance. He came to three

important conclusions from these experimental results (i)- That the inheritance of each

trait is determined by "units" or "factors" that are passed on to descendants unchanged

(these units are now called genes), (ii)- That an individual inherits one such unit from

each parent for each trait, and (iii) That a trait may not show up in an individual but can

still be passed on to the next generation. It is important to realize that, in this experiment,

the starting parent plants were homozygous for pea seed color. That is to say, they each

had two identical forms (or alleles) of the gene for this trait--2 yellows or 2 greens. The

plants in the F1 generation were all heterozygous. In other words, they each had inherited

two different alleles--one from each parent plant. It becomes clearer when we look at the

actual genetic makeup, or genotype, of the pea plants instead of only the phenotype, or

observable physical characteristics. Note that each of the F1 generation plants inherited a

Y allele from one parent and a G allele from the other. When the F1 plants breed, each

has an equal chance of passing on either Y or G alleles to each offspring. With all of the

seven pea plant traits that Mendel examined, one form appeared dominant over the

other. Which is to say, it masked the presence of the other allele? For example, when the

genotype for pea seed color is YG (heterozygous), the phenotype is yellow. However, the

dominant yellow allele does not alter the recessive green one in any way. Both alleles can

be passed on to the next generation unchanged.

Q4. Explain supercoiling in a DNA molecule.

Answer: One of the interesting characteristics of DNA is its ability to form a coil like

structure and this process of making coiling is called as DNA supercoiling. With DNA in

its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4

base pairs, but if the DNA is twisted the strands become more tightly. If the DNA is

twisted in the direction of the helix, this is positive supercoiling, and the bases are held

more tightly together. If they are twisted in the opposite direction, this is negative

supercoiling, and the bases come apart more easily. In nature, most DNA has slight

negative supercoiling that is introduced by enzymes called topoisomerases. These

enzymes are also needed to relieve the twisting stresses introduced into DNA strands

during processes such as transcription and DNA replication.

Q5. Describe the role of DNA polymerase in replication.



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Answer: DNA polymerases are a family of enzymes that carry out all forms of DNA

replication. A DNA polymerase can only extend an existing DNA strand paired with a

template strand; it cannot begin the synthesis of a new strand. To begin synthesis of a

new strand, a short fragment of DNA or RNA, called a primer must be created, and

paired with the DNA template. Once a primer pairs with DNA to be replicated, DNA

polymerase synthesizes a new strand of DNA by extending the 3’ end of an existing

nucleotide chain, adding new nucleotides matched to the template strand one at a time via

the creation of phosphodiester bonds. The energy for this process of DNA polymerization

comes from two of the three total phosphates attached to each unincorporated base. Free

bases with their attached phosphate groups are called nucleoside triphosphates. When a

nucleotide is being added to a growing DNA strand, two of the phosphates are removed

and the energy produced creates a phosphodiester bond that attaches the remaining

phosphate to the growing chain. The energetic of this process also help explain the

directionality of synthesis if DNA were synthesized in the 3’ to 5’ direction, the energy

for the process would come from the 5' end of the growing strand rather than from free

nucleotides. DNA polymerases are generally extremely accurate, making less than one

error for every 107 nucleotides added. Even so, some DNA polymerases also have

proofreading ability; they can remove nucleotides from the end of a strand in order to

correct mismatched bases. If the 5’ nucleotide needs to be removed during proofreading,

the triphosphate end is lost. Hence, the energy source that usually provides energy to add

a new nucleotide is also lost.

Q 6 What are topoisomerases and helicases?

Answer: Topoisomerases are enzymes with both nuclease and ligase activity. These

proteins change the amount of supercoiling in DNA and some of these enzyme work by

cutting the DNA helix and allowing one section to rotate, thereby reducing its level of

supercoiling; the enzyme then seals the DNA break. Other types of these enzymes are

capable of cutting one DNA helix and then passing a second strand of DNA through this

break, before rejoining the helix. Topoisomerases are required for many processes

involving DNA, such as DNA replication and transcription. Helicases are proteins that

are a type of molecular motor. They use the chemical energy in nucleoside triphosphates,

predominantly ATP, to break hydrogen bonds between bases and unwind the DNA



12

double helix into single strands. These enzymes are essential for most processes where

enzymes need to access the DNA bases.

Q7. How does DNA methylation occur?

Answer: DNA methylation has been proven by research to be manifested in a number of

biological processes such as regulation of imprinted genes, X chromosome inactivation,

and tumor suppressor gene silencing in cancerous cells. It also acts as a protection

mechanism adopted by the pathogen DNA mainly bacterial against the endonuclease

activity that destroys any foreign DNA. The expression of genes is influenced by how the

DNA is packaged in chromosomes, in a structure called chromatin. Base modifications

can be involved in packaging, with regions that have low or no gene expression usually

containing high levels of methylation of cytosine bases. For example, cytosine

methylation produces 5-methylcytosine, which is important for X-chromosome

inactivation. The average level of methylation varies between organisms - the worm

Caenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels,

with up to 1% of their DNA containing 5-methylcytosine. Despite the importance of 5-

methylcytosine, it can deaminate to leave a thymine base, methylated cytosine are

therefore particularly prone to mutations. Other base modifications include adenine

methylation in bacteria and the glycosylation of uracil to produce the "J-base" in

kinetoplastids.

Q 8 What is a PCR?

Answer: Polymerase chain reaction (PCR) is a technique to amplify a single or few

copies of a piece of DNA across several orders of magnitude, generating thousands of

millions of copies of a particular DNA sequence. PCR is developed in 1983 by Kary

Mullis, and now it is most common and indispensable technique used in medical and

biological research labs for a variety of applications. These include DNA cloning for

sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of

hereditary diseases; the identification of genetic fingerprints, and the detection and

diagnosis of infectious diseases. The method relies on thermal cycling, consisting of

cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic

replication of the DNA. Primers which are basically short DNA fragments containing

sequences complementary to the target region along with a DNA polymerase are key



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components to enable selective and repeated amplification. As PCR progresses, the DNA

generated is itself used as a template for replication, setting in motion a chain reaction in

which the DNA template is exponentially amplified. PCR can be extensively modified to

perform a wide array of genetic manipulations. Almost all PCR applications employ a

heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated

from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a

new DNA strand from DNA building blocks, the nucleotides, by using single-stranded

DNA as a template and DNA oligonucleotides which is also called as DNA primers,

which are required for initiation of DNA synthesis. The vast majority of PCR methods

use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined

series of temperature steps. These thermal cycling steps are necessary first to physically

separate the two strands in a DNA double helix at a high temperature in a process called

DNA melting. At a lower temperature, each strand is then used as the template in DNA

synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity

of PCR results from the use of primers that are complementary to the DNA region

targeted for amplification under specific thermal cycling conditions. Most PCR methods

typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some

techniques allow for amplification of fragments up to 40 kb in size. The PCR is

commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–

0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes

to achieve the temperatures required at each step of the reaction (see below). Many

modern thermal cyclers make use of the Peltier effect which permits both heating and

cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-

walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal

equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of

the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of

the reaction mixture or a ball of wax inside the tube.

Q9 How does forensic DNA profiling done with a PCR tool?

Answer: Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a

crime scene to identify a matching DNA of an individual, such as a perpetrator. This

process is called genetic fingerprinting, or more accurately, DNA profiling. In DNA



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profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats

and minisatellites, are compared between people. This method is usually an extremely

reliable technique for identifying a matching DNA. However, identification can be

complicated if the scene is contaminated with DNA from several people. DNA profiling

was developed in 1984 by British geneticist Sir Alec Jeffreys, and first used in forensic

science to convict Colin Pitchfork in the 1988 Enderby murders case. People convicted of

certain types of crimes may be required to provide a sample of DNA for a database. This

has helped investigators solve old cases where only a DNA sample was obtained from the

scene. DNA profiling can also be used to identify victims of mass casualty incidents. On

the other hand, many convicted people have been released from prison on the basis of

DNA techniques, which were not available when a crime had originally been committed.

Section B: Multiple Choice

Q1 100 trillion

Q2 Leeuwenhoek

Q3 No

Q4 Plasmids

Q5 Mitochondria

Q6 True

Q7 Both contain their own genome

Q8 Fungi

Q9 True

Q10 True

Q11 Genes

Q12 Sugar

Q13 Positive

Q14 Protein synthesis

Q15 Ribosome

Q16 False

Q17 Polymerase

Q18 DNA mutation

Q19 Kary Mullis



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Q20 DNA Section C: Critical Thinking

Answers are not provided in this section as the students are encouraged to write their

own viewpoints.

CHAPTER 3 PROTEINS & PROTEOMICS

Section A: Descriptive Type

Q1: Explain the function of proteins as enzymes.

Answer: The best-known role of proteins in the cell is as enzymes, which catalyze

chemical reactions. Enzymes are usually highly specific and accelerate only one or a few

chemical reactions. Enzymes carry out most of the reactions involved in metabolism, as

well as manipulating DNA in processes such as DNA replication, DNA repair, and

transcription. Some enzymes act on other proteins to add or remove chemical groups in a

process known as post-translational modification. About 4,000 reactions are known to be

catalyzed by enzymes. The rate acceleration conferred by enzymatic catalysis is often

enormous as much as 1017-fold increase in rate over the uncatalyzed reaction in the case

of orotate decarboxylase. The molecules bound and acted upon by enzymes are called

substrates. Although enzymes can consist of hundreds of amino acids, it is usually only a

small fraction of the residues that come in contact with the substrate, and an even smaller

fraction 3 to 4 residues on average that are directly involved in catalysis. The region of

the enzyme that binds the substrate and contains the catalytic residues is known as the

active site.

Q2. Explain the role of proteins in cell signaling.

Answer: Many proteins are involved in the process of cell signaling and signal

transduction. Some proteins, such as insulin, are extracellular proteins that transmit a

signal from the cell in which they were synthesized to other cells in distant tissues. Others

are membrane proteins that act as receptors whose main function is to bind a signaling

molecule and induce a biochemical response in the cell. Many receptors have a binding

site exposed on the cell surface and an effectors domain within the cell, which may have

enzymatic activity or may undergo a conformational change detected by other proteins

within the cell. Antibodies are protein components of adaptive immune system whose

main function is to bind antigens or foreign substances in the body, and target them for



Courtesy of CRC Press/Taylor & Francis Group

Bacterial cell

Plasma membrane

CapsuleCell wall

Circular DNAPlasmid

Ribosome

Pili

Cytoplasm

Flagella

Human cell

CytoplasmMitochondriaNuclear pores

Rough endoplasmicreticulum Cell wall

Centrioles

Microfilaments

Plasma membrane

Golgi bodies

Microtubules

Smoothendoplasmic

reticulum

Nucleus

Nucleolus

Chromatin

Peroxisome

Chromatin

Lysosome

Ribosome

Figure 2.1 Prokaryotic cell and eukaryotic cell: Diagrammatic illustration.

002x001.eps




Protein channel

Hydrophobic region

Inside of cell(cytoplasm)

Outside of cell

Phospholipid bilayer

Hydrophilic region

Hydrophilic region

Figure 2.2 Cell membrane: Diagrammatic illustration.

002x002.eps




N

(a)

(b)

N

N N

O NH

H

H

H

H T

C

H

G

H

N

N

NA N

O

O

O

N

N

N

Adenine

GuanineCytosine

Thymine

N

N

N

Figure 2.3 (a) A GC base pair with three hydrogen bonds. (b) An AT base pair with two hydrogen bonds. Non-covalent hydrogen bonds between the pairs are shown as dashed lines.

002x003.eps




S

S

S

P

P

P

S

S

S

P

P

P

Hydrogen bonds

T

G

C

A

C

G

3΄

5΄

5΄

Phosphodiesterbonds

3΄

Purine

Pyrimidine

Figure 2.4 DNA base pairing.

002x004.eps




Double helix of DNA

S

S

S

A

G

T

P

P

P

S

S

S

T

C

A

P

P

PBase pairs

Hydrogen bondsNucleotide

Sugar phosphatebackbone

Figure 2.5 The structure of DNA: pieces of DNA are pairs of molecules, which entwine like vines to form a double helix. DNA strands are composed of four nucleo-tide subunits. These are adenine (A), thymine (T), cytosine (C), and guanine (G). Each base forms hydrogen bonds readily to only one other—A to T and C to G. The entire nucleotide sequence of each strand is complementary to that of the other.

002x005.eps




Relaxed

Positive

SupercoilUncoilloop

Negative

Figure 2.6 DNA supercoiling.

002x006.eps




S

S

S

U

A

C

P

P

P

S

S

S

A

U

G

P

P

PBase pairs

Hydrogen bonds


Figure 2.7 RNA structure.

002x007.eps




ACCUGCU

U U

U

CCGGU

TGG

AG

C

UA C

A

GGGCGUG

GGCCD

C C

GG

G G G

G

D A G CC

DC

GG

G

GGG

ICGI

CCC

CU

AA

UU

C3́

T CG loopD loop

Anticodon

5́

CAmino acid

Anticodon loop

Figure 2.8 Transfer RNA (tRNA) structure.

002x008.eps




mRNA Small rRNA subunit

Psite

Asite

Large rRNA subunit

P A

Polypeptide chainNew amino acid added

5́ 3́

Codons

tRNAs

Figure 2.9 Ribosomal RNA (rRNA).

002x009.eps




Parent chromosomes

Chromosomes replicate

Like chromosomes pair upand swap sections of DNA

Chromosomepairs divide

Daughter nucleidivide again

Figure 2.10 Stages of meiosis.

002x010.eps




46 chromosomesInterphase

Doubling of chromosomesProphase

Nucleus dissolvedand microtubules attach to

centromeresPrometaphase

Chromosomes align atmiddle of cellMetaphase

Separation ofchromosomesAnaphase

Microtubules disappearand start to divide Telophase

Daughter cells with46 chromosomesCytokinesis

Figure 2.11 Stages of mitosis.

002x011.eps




1.

Parental DNA

Single-strand binding protein stabilize the

unwound parental DNA

The leading strand is synthesizedcontinuously in the 5 3direction by DNA polymerase

3.

4.

5.

Replication fork

Primase

DNA primer

DNA ligase

DNA polymerase

Okazaki fragmentbeing made

3́

5́ 5́

3́

3́

5́

The lagging strand issynthesized discontinuously

After RNA primer is replaced by DNA,DNA ligase joins the Okazaki fragment to the growing strand

Helicases unwind the parental double helix

2.

Figure 2.12 DNA replication process.

002x012.eps




A T TC5́ 3́

G T A A3́ 5́

Eco RI

DNA fragments removed

C5́ 3́

G T A A3́ 5́

C

G T A A

DNA fragments join at sticky ends

RecombinantDNA

Figure 2.13 Role of restriction enzyme in making recombinant DNAATTCCC5.

002x013.eps




O O

N

NH2 NH2

N

N

N

CH3

5΄-CpG-3΄3΄-GpC-5΄

DNAmethyl transferases

Cancer DNA

Normal DNA

Methylated CpG site

Unmethylated CpG site

Exon A Exon B

Exon A Exon B

Figure 2.14 DNA methylation.

002x014.eps




Mutation point

Mutated DNA sequence Normal DNA sequence

T

T

GG

A

A

C

GGC

C

TG

GG

CC

C

Figure 2.15 DNA mutation.

002x015.eps




DNA sample

Forward and reverse primers

Sample preparation

PCR machine

Multi-�cation of DNAhappened in the PCR

Sample loading on gel

Gel electrophoresisGel documentation system

Final PCR product

Figure 2.16 Steps in the PCR.

002x016.eps




G

GG

G

C5 ΄

3΄

3΄

3΄

3΄

3΄

3΄ 3΄

3΄ 3΄

3΄

5΄

5΄

5΄

5΄ 5΄

5΄ 5΄

5΄

5΄

5΄

5΄

3΄3΄

C C

CG G

G

T

T T TA

A AA

Multiple copies of DNA

C CC GT T TACG GT A AA C C C GT T TA

CGT A AA

C C C

CG G

G

T

T T TA

A AA

Primers binds to template DNA strands at 55°C

Separation of DNA strands due to heat at 95°C

Multiplication of DNA strands using Taq DNApolymerase at 72°C

Multiple copies of DNA

G

Figure 2.17 Amplification of DNA fragment.

002x017.eps



Major fields of biotechnology

Title in here1

Chapter # 1




Title in here2

• Cell is the building block of human body and it is critical to understand its structure and function so that better treatment modalities can be achieved.

• There is similarity and difference of intracellular and extracellular aspects of cells of prokaryotic and eukaryotic origin.

Biotechnology Fundamentals by Firdos Alam Khan

Chapter # 2

CELL BIOLOGY




Title in here3

• The cell is the structural and functional unit of all known living organisms.

• It is the smallest unit of a living organism, and is often called the building

block of life.

• Humans have approximately 100 trillion or 1014 cells, an example of a

multicellular organism. On the other hand, a single-celled bacterium is called

unicellular.

• A typical cell size is 10 micrometer while a typical cell mass is 1 nanogram.

All animals and plants are made of cells and such concept was originally

coined by Aristotle (384-322 BC).


Chapter # 2

CELL AS A BUILDING BLOCK OF LIFE




4

• All animals and plants are made of cells and such concept was originally

coined by Aristotle (384-322 BC).

• In 1665, Robert Hooke observed for the first time the structure of a cell under

a very primitive microscope.

• In 1674, Antonie van Leeuwenhoek discovered cells with structural

organization within the cell.

• In 1824, H.J. Dutrochet, a French scientist, gave the idea of the cell theory


Chapter # 2

CELL AS A BUILDING BLOCK OF LIFE




Title in here5

• There are two different types of cells, the eukaryotic and prokaryotic cells.

• The prokaryote cell is simpler than a eukaryote cell, lacking a nucleus and

most of the other organelles of eukaryotes.

• There are two kinds of prokaryotes, bacteria and archaea, These prokaryotes

share a similar overall structure

• Eukaryotic cells are about 10 times the size of a typical prokaryote

• The major difference between prokaryotes and eukaryotes is that eukaryotic

cells contain membrane-bound compartments in which specific metabolic

activities take place


Chapter # 2

CLASSIFICATION OF CELL



Title in here6Biotechnology Fundamentals by Firdos Alam Khan

Chapter # 2

ORGANIZATION OF A CELL

External Organization

• Cell membrane• Cell capsule• Flagella

Internal Organization

Cytoplasmic Parts

• Mitochondria• Chloroplast• Ribosome• Endoplasmic reticulum• Golgi apparatus• Lysosomes and

Peroxisomes • Centrosomes• Vacuoles • Cytoskeleton

Nuclear Parts

• DNA• RNA• mRNA• tRNA• rRNA• snRNA• Nucleoli• Chromatins




Chapter # 2

MACRO-MOLECULES OF BODY

• Living cells are primarily made up of water, a number of other molecules are also abundant within a cell such as macromolecules.

• Macromolecules provide structural support, store fuel, store and retrieve genetic information, and speed up biochemical reactions.

• There are four major types of macromolecules that play important functions in the life a cell:

• PROTEINS, • CARBOHYDRATES, • NUCLEIC ACID, AND • LIPIDS



Title in here8

In most living organisms (except for viruses), genetic information is stored in DNA, which resides in the nucleus of living cells. It gets its name from the sugar

molecule contained in its backbone (deoxyribose). However, it gets its significance from its unique structure. Four different nucleotide bases occur in DNA: adenine

(A), cytosine (C), guanine (G), and thymine (T).


Chapter # 2

DNA STRUCTURE

S

S

S

P

P

P

S

S

S

P

P

P

Hydrogen bonds

T

G

C

A

C

G

3’

5’

5’

3’

Phosphodiesterbonds

purine

pyrimidine



Title in here9

Ribonucleic acid (RNA) is a biologically important type of molecule that consists of a long chain of nucleotide units. RNA and DNA are both nucleic acids, but differ in three main ways. First, unlike DNA which is double-stranded, RNA is a single-stranded molecule in most of its biological roles and has a much shorter chain of nucleotides. Second, while DNA contains deoxyribose, RNA contains ribose, (there is no hydroxyl group attached to the pentose ring in the 2' position in DNA).


Chapter # 2

RNA STRUCTURE

S

S

S

U

A

C

P

P

P

S

S

S

A

U

G

P

P

PBase pairs

Hydrogen bonds




Title in here10

Transfer RNA (tRNA) is a small RNA molecule (usually about 74-95 nucleotides) that transfers a specific active amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation


Chapter # 2

TRANSFER RNA STRUCTURE3’

5’

Amino acid

D loop

Anticodon loopAnticodon loop

Anticodon

T CG loop



Title in here11

Ribosomal RNA (rRNA) is the central component of the ribosome, the protein manufacturing machinery of all living cells. The function of the rRNA is to provide a mechanism for decoding mRNA into amino acids and to interact with the tRNAs during translation by providing peptidyl

transferase activity. The tRNA then brings the necessary amino acids corresponding to the appropriate mRNA codon


Chapter # 2

RIBOSOMAL RNA STRUCTURE

mRNA

Small rRNA subunit

P site

Asite

large rRNA subunitP A

polypeptide chainNew amino acid added

5’ 3’

codons

tRNAs




Chapter # 2

CELL DIVISION

• All living cells undergo cell division in order to multiply and grow and there are two ways the cell can be divided such as meiosis and mitosis

• When cells are divided to yield two daughter cells, the genetic material must be divided equally so that each daughter cell contains identical DNA copies.



13

Meiosis is a process of reduction division in which the number of chromosomes per cell is reduced to half. In animals, meiosis always results in the formation of gametes, while in other organisms it can give rise to spores.


Chapter # 2

MEIOSIS

Parent chromosomes

Chromosomes Replicate

Like chromosomes pair up& swap sections of DNA

Chromosomes pairs divide

Daughter nuclei divide again



14

Mitosis is the process in which a eukaryotic cell separates the chromosomesin its cell nucleus into two identical sets in two daughter nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two daughter cells containing roughly equal shares of these cellular components


Chapter # 2

MITOSISInterphase

Prophase

Prometaphase

Metaphase

Anaphase

Telophase

Cytokinesis

46 chromosomes

Doubling of chromosomes

Nucleus dissolved and microtubules attach to centromeres

Chromosomes align at Middle of cell

Separation of chromosomes

Microtubules disappearand start to divide

Daughter cells with 46 chromosomes



Title in here15

In a cell, DNA replication begins at specific locations in the genome, called "origins". Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork


Chapter # 2

DNA REPLICATION PROCESS

1 DNA Unwinding

Parental DNA

2 protein stabilize

the unwound parental DNA

3 DNA synthesis by

DNA polymerase

4 The lagging stand is synthesized

discontinuously

5 Okazaki fragment to

the growing strand

Replication fork

Primase

DNA primer

DNA ligase

DNA polymerase

Okazaki Fragment being made

5’

3’

3’

5’

5’

3’



16

A restriction enzyme is an enzyme that cuts DNA at specific recognition nucleotidesequences (with Type II restriction enzymes cutting double-stranded DNA) known as restriction sites


Chapter # 2

ROLE OF RESTRICTION ENZYME

A T T C CC5 ‘ 3 ‘

G T A A G G

3 ‘ 5 ‘

CC5 ‘ 3 ‘

G T A A G G

3 ‘ 5 ‘

C

G

C5 ‘

G T A A G

3 ‘

Eco RI

DNA fragments removed

DNA fragments join at sticky ends

RecombinantDNA



Title in here17

DNA methylation is one such post-synthesis modification. DNA methylation has been proven by research to be manifested in a number of biological processes such as regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in cancerous cells


Chapter # 2

DNA METHYLATION

O

N

NH2

NO

N

NH2

N

CH3

5’-CpG-3’3’-GpC-5’

DNAMethyl transferases

Cancer DNA

Normal DNA

Methylated CpG site

Unmethylated CpG site

Exon A Exon B

Exon A Exon B



Title in here18

DNA mutation is a change in the sequence of DNA. It can be caused by copying errors in the genetic material during cell division, by exposure to ultraviolet/ionizing radiation, chemical mutagens, or viruses, or by cellular processes such as hyper-mutation. It can also be induced by the organism itself.


Chapter # 2

DNA MUTATION PROCESS

X-rayexposure

DNA Before

DNAAfter

StructuralChanges




Chapter # 2

DNA AMPLIFICATION BY POLYMERASE CHAIN REACTION (PCR)

Figure 2.18DNA Mutation

DNA sample

Reverse Primer

Forward Primer

PCR machine Sample loading on gel

Gel electrophoresisGel documentation system

Final PCR Product

Sample preparation




Chapter # 2

DNA MUTATION PROCESS

A T T C C G TC5 ‘ 3 ‘

G T A A G G C A3 ‘ 5 ‘

A T T C C G TC5 ‘ 3 ‘

G T A A G G C A3 ‘ 5 ‘

Separation of DNA strands due to heat at 95 degree C

Primers binds to Template DNA strands at 55 degree C

5 ‘ 3 ‘

3 ‘ 5 ‘

5’3’5’ 3’

A T T C C G TC5 ‘ 3 ‘G T A A G G C A

3 ‘ 5 ‘

A T T C C G TC5 ‘ 3 ‘G T A A G G C A

3 ‘ 5 ‘

Multiple copies of DNAMultiple copies of DNA

Multiplication of DNA strands using Taq DNA Polymerase at 72 degree C



21

Q1. What is the cell theory?

Q2. Describe the characteristics of a prokaryotic cell.

Q3. Explain Mendelian’s genetics.

Q4. Explain supercoiling in DNA molecule.

Q5. Describe the role of DNA polymerase in replication.

Q6. What are topoisomerases and helicases?

Q7. How does DNA methylation occur?

Q8. What is a polymerase chain reaction?

Q9. How does forensic DNA profiling done with PCR tool?


Chapter # 2

FIND A SOLUTION FOR A PROBLEM



22

Q1 In order to identify the real culprit among a group of crime suspects, what

technique can be used to establish the identity of the culprit? Explain with

suitable examples.

Q2 Is it possible to study the genetic information of an individual by working

with mRNA only? Explain.

Q3 What would be the status of gene expression in case mRNA is not available?

Q4 What will happen if nuclear DNA is circular in shape and mitochondrial

DNA is linear in shape?


Chapter # 2

CRITICAL THINKING



solutions manual for biotechnology fundamentals … · q3. explain mendelian’s genetics. answer:...

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