Ebook Molecular biology - Different facets (1st): Part 1

MOLECULAR BIOLOGY
Different Facets



MOLECULAR BIOLOGY
Different Facets

Anjali Priyadarshini, PhD
Prerna Pandey, PhD


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Priyadarshini, Anjali, author
Molecular biology : different facets / Anjali Priyadarshini, PhD, Prerna Pandey, PhD.
Includes bibliographical references and index.
Issued in print and electronic formats.
ISBN 978-1-77188-641-3 (hardcover).--ISBN 978-1-315-09927-9 (PDF)
1. Molecular biology. I. Pandey, Prerna, author II. Title.
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Names: Priyadarshini, Anjali, author. | Pandey, Prerna, author.
Title: Molecular biology : different facets / Anjali Priyadarshini, Prerna Pandey.
Description: Toronto ; New Jersey : Apple Academic Press, 2018. | Includes bibliographical references

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ABOUT THE AUTHORS
Anjali Priyadarshini, PhD
Anjali Priyadarshini, PhD, is an Assistant Professor at Delhi University,
India. Dr. Priyadarshini is a Council of Scientific and Industrial Research
(CSIR) Government of India awardee. Her field of research and interest
includes biotechnology and nanotechnology. Dr. Priyadarshini has published
papers in peer-reviewed journals in the biomedical field.
Prerna Pandey, PhD
Prerna Pandey, PhD, is a biotechnologist with several years of wet lab
research experience. She has worked at the International Center for Genetic
Engineering and Biotechnology, New Delhi, India. Her PhD research
involved isolation and molecular characterization of Geminiviruses,
genome sequencing, gene annotation, and gene silencing using the RNA
interference technology. She has also worked at Transasia Biomedicals and
Advance Enzyme Technologies as a scientist. Dr. Pandey has published
papers in peer-reviewed journals in the field and has submitted a number
of annotated Geminiviral genome sequences to GenBank, including two
novel ones. She has also completed editing and proofreading courses from

the Society for Editors and Proofreaders (SfEP) and now works as a freelance scientific writer and editor.
Author Details:

Dr. Anjali Priyadarshini, MSc, PhD

Recipient of CSIR: JRF, SRF.
Address: 74 B, Ayodhya Enclave, Sector 13 Rohini, New Delhi 85, India.
Affiliation: Assistant Professor, SRM University, Sonipat, Haryana, India.
E-mail: ;

Dr. Prerna Pandey, MSc, PhD

Address: B 1403, Jasper, Hiranandani Estate, Thane 400607, Maharashtra, India.
Affiliation: Freelance scientific writer and editor.
Phone: +919167932133.
E-mail: ;



CONTENTS



List of Abbreviations..................................................................................... ix

Preface........................................................................................................ xiii
Introduction...................................................................................................xv
1.Cell................................................................................................................. 1
2.

Genes and Genetic Code............................................................................ 29

3.

Molecular Biology of Microorganisms..................................................... 99

4.

Plant Molecular Biology.......................................................................... 157

5.

Genetic Manipulation by Recombinant DNA Technology................... 219

6.

Molecular Diagnostics............................................................................. 275

Index.................................................................................................................. 311



LIST OF ABBREVIATIONS

AAV
adeno-associated virus
ABA
abscisic acid
ACC1-aminocyclopropane-1-carboxylate
aCGH

array comparative genomic hybridization
AD
activation domain
AdVadenovirus
AFLP
amplified fragment length polymorphism
AOH
absence of heterozygosity
ARMS
amplification refractory mutation system
ARS
autonomous replicating sequences
BAC
bacterial artificial chromosome
BD
DNA-binding domain
cAMP
cyclic adenosine mono phosphate
CAP
catabolite activator protein
CasCRISPR-associated
Cas9
CRISPR-associated proteins
CBF
C-repeat binding factors
CBLs
calcineurin B-like proteins
CENcentromeres
CIAP
calf-intestinal alkaline phosphatase

CIPKs
CBL-interacting protein kinases
CKIs
Cdk inhibitors
co-IPco-immunoprecipitation
CRISPR
clustered regularly interspaced short palindromic repeats
ddNTPsdideoxynucleotides
DGGE
denaturing gradient gel electrophoresis
DHPLC
denaturing high-performance liquid chromatography
DNA
deoxyribonucleic acid
dNTPdeoxyribonucleotides
DREB
dehydration-responsive element-binding proteins
dsRNA
double-stranded RNA
EMS
ethyl methanesulfonate
ER
endoplasmic reticulum


x

List of Abbreviations

EST

expressed sequence tag
ETethylene
F-SSCP
fluorescent SSCP
FISH
fluorescent in situ hybridization
GE
genetically engineered
GEAC
Genetic Engineering Approval Committee
GGE
gradient gel electrophoresis
GINA
Genetic Information Non-discrimination Act
GM
genetically modified
gRNA
guide RNA
GST
glutathione S-transferase
GWAS
genome-wide association studies
HAD
heteroduplex analyses
HBV
hepatitis B virus
HCN
hydrogen cyanide
HDR
homology-directed repair

Hfr
high frequency recombination
HPV
human papilloma virus
HR
homologous recombination
IAA
indole-3-acetic acid
IFimmunofluorescence
IHF
integration host factor
IPimmunoprecipitation
IRES
internal ribosome sites
IRGSP
International Rice Genome Sequencing Project
IS
insertion sequence
ISO
allele-specific oligonucleotide
IVF
in vitro fertilization
IVSP
in vitro synthesized protein assay
JA
jasmonic acid
KSI
ketosteroid isomerase
LOH
loss of heterozygosity

LVslentivirus
MAPH
multiplex amplifiable probe hybridization
MAPKs
mitogen-activated protein kinases
MBP
maltose-binding protein
MCS
multiple cloning site
miRNAsmicroRNA


List of Abbreviationsxi

MLPA
multiplex ligation-dependent probe amplification
messenger RNA
mRNA
mRNP
mRNA protein
NGS
next-generation sequencing
NHEJ
nonhomologous end joining
NMPs
nucleoside monophosphates
NPR
nodule promoting rhizobacteria
NTPs
nucleoside triphosphates

OLA
oligonucleotide ligation assay
PAM
protospacer adjacent motif
PCR
polymerase chain reaction
PEG
polyethylene glycol
PGD
pre-implantation genetic diagnosis
PGPR
plant growth promoting rhizobacteria
PHPR
plant health promoting rhizobacteria
PM
personalized medicine
PMF
plant molecular farming
PPipyrophosphate
PPV
produced Plum pox virus
PR
plant resistance
pRNA
primer RNA
PTGS
posttranscriptional gene silencing
PTM
posttranslational modification
PTT

protein truncation test
QTL
quantitative trait locus
RBS
ribosome binding site
RCM
rolling circle mechanism
RFLP
restriction fragment length polymorphisms
RISC
RNA-induced silencing complex
RNA
ribonucleic acid
RNAP
RNA polymerase
RNPribonucleoprotein
ROS
reactive oxygen species
rRNA
ribosomal RNA
SA
salicylic acid
sgRNA
single-guide RNA
shRNAs
short hairpin RNAs
SINEs
short interspersed nuclear elements

xii

List of Abbreviations

siRNAs
small interfering RNAs
single nucleotide polymorphisms
SNPs
SSB
single-stranded binding protein
SSCP
single-strand conformation polymorphism
SSO
single-strand origin
SSRs
simple sequence repeats
STPs
signal transduction pathways
SUMO
small ubiquitin related modifier
TBP
TATA-binding protein
TEV
tobacco etch virus
TFs
transcription factors
TGGE
temperature gradient gel electrophoresis
TM
theta mechanism

TMV
tobacco mosaic virus
tPA
tissue plasminogen activator
tracrRNA
trans-activating crRNA
Trptryptophan
TrxA
thioredoxin A
TTGE
temporal temperature gradient gel electrophoresis
UPD
uniparental isodisomy
UVultraviolet
VEGF
vascular endothelial growth factor
VIGS
virus-induced gene silencing
WCR
Western corn rootworm
YAC
yeast artificial chromosome
YCp
yeast centromeric plasmid


PREFACE

The book Molecular Biology: Different Facets includes a comprehensive
description of the basic tenets of molecular biology, from mechanisms to

their elaborate role in gene regulation. The initial sections describe the
history of genetics and molecular biology. The book highlights the significance of the molecular approaches for all biological processes in both
simple and complex cells. The text also incorporates the most recent references and has been written for students as well as for teachers of molecular
biology, molecular genetics, or biochemistry. The authors have described
experimental approaches wherever necessary to explore the evidence that
led to the development of important concepts and hypotheses that led to
significant advances in molecular biology. The book is divided into six
chapters; the initial topics cover basic information that help in understanding the advanced topics covered later in the chapters.



INTRODUCTION

The field of molecular biology has taken several giant steps that have a
broad range of applications. Various aspects of life came to be known
by the scientific pursuits done in molecular biology. Molecular biology
has been found to have very significant use in disease diagnostics and
therapeutics. These outcomes have been initiated from the discovery of
the structure of DNA. This discovery led us to deciphering of the genetic
code and its outcome. A very important tenet that forms the core of life
processes is the central dogma of life, which was established when the
genetic code was decoded. The various aspects of the expression of this
genetic code opened up a wide arena of its role played in various biochemical processes. Once the genetic basis of multiple diseases was established,
the path was paved for its diagnostic and therapeutic use. Not only its role
is there in disease but also aids in regulation with the help of regulatory
RNA. All this goes in it tandem with the aid of various other branches of
science such as microbiology, genetics, biochemistry, to name a few. So
much is known and still much is left to be known. This is a vast field of
micromolecules that affects our well-being so much.
The molecular mechanisms underlying the various processes taking

place in a cell such as replication, transcription, processing of RNA,
and translation offer various new avenues for research with potential in
understanding the complex system of life. The study of the cell cycle
and various intricacies in its control could serve as checkpoints in understanding diseases and potential drug targets.
Several processes in bacteria like conjugation, transformation, and
transduction ushered in an era of excitement and research. The understanding of “simple” bacterial molecular biology changed the paradigm
of viewing these microscopic cells with all their intricacies. These molecular mechanisms opened up new avenues as their potential in mapping
genes and related genetic studies and their use in recombinant DNA technology, which has and is revolutionizing science. The same statement may
be extended to another kingdom of fungi that have their own molecular
pathways.


xviIntroduction

The molecular aspects of the so-called threshold organisms, such
as viruses, show immense complexities and elaborately sophisticated
pathways.
This book covers aspects of molecular biology in brief, as each topic
is an ocean to be delved into. Nevertheless, though each of the topics is
presented as “tips of the icebergs,” they have been presented and elucidated in concise terms with relevant research. The book serves to ignite
the minds of students and academicians to pursue research or just serve as
reading material.


CHAPTER 1

CELL

CONTENTS
Abstract...................................................................................................... 2

1.1 Introduction to Cell........................................................................... 2
1.2 Organization and Structure of Cells.................................................. 5
1.3 Shared Properties of Biological Systems........................................ 15
1.4Mitosis............................................................................................ 16
1.5 Genetic Regulation of Cell Cycle................................................... 21
1.6Meiosis............................................................................................ 24
1.7Summary......................................................................................... 27
1.8 Review Questions........................................................................... 27
Keywords................................................................................................. 27
References................................................................................................ 28


2

Molecular Biology: Different Facets

ABSTRACT
The volume of research that has gone into study and advancement of
cell biology requires it to be dealt with independently as a subject. Cell
biology deals with the cellular organization of various life forms including
prokaryotes, eukaryotes as well as the cellular forms such as viruses.
The variations within a group lead to its classification which is very
much dependent on the surrounding environment as well. Continuity of
the living forms requires division of cell and formation of daughter cells
within a generation or to form cells involved in formation of progeny for
next generation. This chapter is an attempt to give a panoramic view of the
entire process and the subject.
1.1  INTRODUCTION TO CELL
Human mind has been very curious to know when, why, and how of any
event, regarding all the biological processes. Our understanding of all the

biological process gained a momentum since the invention of simple and
complex microscope. First and foremost, breakthrough came with the
observations of the cells and the microorganisms by Robert Hooke and
Anton von Leeuwenhoek. This led to more detailed study of such microscopic structures by more intriguing minds, thus forming foundation for
various branches of science such as genetics, biochemistry, molecular
biology, cell biology, etc. The subsequent parts of this chapter deals with
the various aspects related to aforesaid branches of science. The main
focus of this chapter is on the comparative study of animal, plant, and
microbial cell apart from dealing with acellular microorganism which is
virus.
Molecular biology as the name suggests is the branch of science which
deals with the minuscules. All the life processes can now be viewed and
analyzed at the microscopic level. This has been made possible by cumulative efforts of number of scientist and researchers. I begin my work by
thanking all the known, very well-known, and the unknown whose pursuit
to find answers to many questions has led to our understanding of molecular biology.
Now that the world has seen the smallest of the smallest material
ranging in the diameter of nanoscale and less, there was a time when


Cell3

discovery of the basic unit of life, that is, a cell was hailed among
the biggest discovery of the smallest. This discovery by Robert Hook
laid the foundation of the study of the small as the next big thing. His
discovery was the culmination of his simple quest to know the basis
of cork functioning to hold air in a bottle. He observed a honeycomb
like pattern in cork under microscope, which was nothing but empty
cell walls of dead plant tissue as we know now. Robert Hook was able
to tantalize our understanding along with another great observer and
discoverer Anton van Leeuwenhoek, about the yet to be identified and

analyzed microscopic world. Robert Hook was a microscopist and Anton
van Leeuwenhoek, a Dutch merchant having a very curious bent of mind
and disposition. As Robert Hook is credited with the discovery of cell
(Fig. 1.1), Anton van Leeuwenhoek (Fig. 1.2) has the distinction of
discovering the animalcules or the microscopic organisms, for example,
the bacteria.

FIGURE 1.1  Robert Hooke’s microscope and dead cork cells as seen by him. The cells
had hexagonal shape and gave an appearance of bee hive.


4

Molecular Biology: Different Facets

FIGURE 1.2  Father of microbiology Anton van Leeuwenhoek.

The next query to be addressed was the ubiquitous nature of cell. If
at all, cell could be hailed as the basic unit of life. This was satisfactorily
realized by Matthias Schleiden, a German botanist in 1838 and Theodor
Schwann, a German zoologist in 1839. Matthias Schleiden in his work
concluded that plants were made of cells; similar claims were made by
Theodor Schwann regarding cellular organization of animal and proposal
of the first two tenets of The Cell Theory was made:
1) All organisms are composed of one or more cells.
2) The cell is the structural unit of life.
Their belief that cells could arise from noncellular material or the spontaneous generation theory was put to rest by Rudolf Virchow in 1855. He
was a German pathologist who added to the tenets of cell theory. Later, the
third tenet was added.
3) Cells can arise only by division from a preexisting cell.

As the third tenet suggests, the formation of new cells by division of
preexisting cell has and is being demonstrated in laboratories across the
world having basic cell culture facility. Cells can be extracted from plant
as well as animals to be “grown” in laboratory. The first human cell to be


Cell5

cultured was obtained from malignant tumor of cervix. It was christened
HeLa cells after the donor Henrietta Lacks.
Regarding the first two tenets of cell theory, there are certain properties
which are adherent to all the cells irrespective of their origin. They are as
follows:
• Response to stimuli: response of a cell against external stimuli with
the help of various receptors present on the surface.
• Reproduction: division of cell leading to formation of new daughter
cells.
• Acquisition and utilization of energy: photosynthetic and respiratory activities.
• Possession of information in the form of genetic code: following the
central dogma of life where the genetic information is transcribed
and translated to perform various cellular function.
• Site of various chemical reaction for various life processes: anabolism + catabolism = metabolism.
• Capability to do various mechanical work: transport of various
material within a cell or to different locations in the body of multicellular organism.
• Self-regulatory mechanisms: ability to correct malformed genetic
information and self-destruction of cells which are beyond repair
(apoptosis).
The work of these pioneer researchers in basic science laid the foundation of modern day field of molecular biology, which has helped a great
deal in various facets of life including healthcare, diagnostics, therapeutics, and prosthetics to name a few.
1.2  ORGANIZATION AND STRUCTURE OF CELLS

All living things fall broadly into one of the two categories:
• prokaryotes
• eukaryotes
pro = means “prior to,” eu = means “true,” and karyote = means
“nucleus.”


6

Molecular Biology: Different Facets

The distinction is based on whether or not a cell has a nucleus.
Prokaryotic cells do not have nuclei, while eukaryotic cells do. Also,
eukaryotic cells have organelles (Fig. 1.3). Although there are many
differences between a prokaryotic cell and a eukaryotic cell, there are
many similarities too, which point to the fact that they have a common
ancestor.

FIGURE 1.3  Schematic diagrams of (a) animal, (b) plant, and (c) bacterial cell. An
animal cell lacks cell wall and plastids which is the site for protein synthesis. The plant cell
is characterized by presence of large vacuoles. Both plant and animal cell are eukaryotic
with a well-defined nucleus. The bacterial cell, which is prokaryotic, lacks a well-defined
nucleus, with presence of cell wall made up of peptidoglycan as well as flagella for
locomotion.


Cell7

1.2.1  EARLY EVOLUTION OF CELLS
Contemporary evidence favors the view that all living organisms should

be grouped into three lineages (or classes)1,2,3:
• archaebacteria
• eubacteria (or just/true bacteria)
• eukaryotes
It is believed (estimated) that evolution of all three lineages occurred
approximately 3.5 billion years ago from a common ancestral form
called progenote (Table 1.1). Archaea and eubacteria are both prokaryotic because they are single-cell organisms without nucleus. Physiological
morphological and ecological diversity has been used to a great extent for
classification of prokaryotes. However, it is now accepted that eukaryotic cells are composed of various prokaryotic contributions, for example,
mitochondria and chloroplast are considered to be of prokaryotic origin
living in a symbiotic association with the eukaryotic cell making the
dichotomy artificial.
In contrast to eukaryotes, prokaryotes do not have structural diversity
or higher cellular organization such as tissue and organ (General Microbiology, 5th edition, Stanier).
1.2.2 ARCHAEBACTERIA
• The word archaea is derived from the word “ancient.”
• It is most recently discovered lineage.
• Archaebacteria are similar in shape to bacteria, but genetically they
are as distinct from bacteria as they are from eukaryotes.
(Whole genome sequencing of the archaeon Methanococcus jannaschii
showed 44% similarity to the known genes in eubacteria and 56% of genes
that were new to science)
Based on their physiology, archaea can be classified into three
subcategories:
a) Methanogens—prokaryotes that produce methane (CH4)
b) Halophiles—prokaryotes that live at very high concentrations of
salt (NaCl)


Autotroph or

heterotroph

Unicellular

Unicellular

Number of Cells

Mode of Nutrition Autotroph or
heterotroph

Cell wall without Cell wall of cellulose Cell wall with
peptidoglycan
in some, some have chitin
chloroplast

Cell wall with
peptidoglycan

Autotroph or
heterotroph

Most unicellular,
some colonial and
some multicellular
Heterotroph

Eukaryote

Animal

Autotroph

Heterotroph

Multicellular

Cell wall of cellulose No cell wall of
and chloroplast
chloroplast

Eukaryote

Most unicellular, Multicellular
some unicellular

Eukaryote

Cell structure

Eukaryote

Prokaryote

Plant

Eukaryote

Prokaryote

Fungi

Cell type

Archaebacteria

Archaebacteria Protist

Bacteria

Eubacteria

Kingdom

Domain

TABLE 1.1  Classification of Living Things.
8
Molecular Biology: Different Facets