Molecular Biology.ppt

1
Introduction To Molecular Biology
By
Salwa Hassan Teama (M.D)

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Molecular Biology
 Molecular biology; the study of biology at the
molecular level.
 Molecular biology; the study of gene structure and
functions at the molecular level to understand the
molecular basis of hereditary, genetic variation, and the
expression patterns of genes.
 The Molecular biology field overlaps with other
areas, particularly genetics and biochemistry.

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The Genome
 The genome of an organism is the totality of genetic
information and is encoded in the DNA (or, for some
viruses, RNA).
commons.wikimedia.org
/
wiki/Image:Genome.jpg

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Genome Database
 The database is organized in six major organism groups:
 Eukaryotes, Bacteria, Archaea, Viruses, Viroids and Plasmids.

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All living things are grouped into three domain:
 Eukaryotes;
 Prokaryotes and
 Archaea.
Three Domain of Life

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The Cell
The cell is the smallest
living unit, the basic
structural and functional
unit of all living things.
Some organisms, such as
most bacteria, are
unicellular (consist of a
single cell). Other
organisms, such as
humans, are multicellular.

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The Cell
 Cells are stacked
together to make up
structures, tissues and
organs. Most cells have
got the same information
and resources and the
same basic material.
Cells can take many
shapes depending on
their function.
Function of cells
 Secretion (Produce
enzymes).
 Store sugars or fat.
 Brain cells for memory
and intelligence.
 Muscle cells to contract.
 Skin cell to perform a
protective coating.
 Defense, such as white
blood cells.

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Eukaryotic Cell
 Eukaryotes are generally more advanced than
prokaryotes. There are many unicellular organisms which
are eukaryotic, but all cells in multicellular organisms are
eukaryotic.
 Eukaryotic cells are found in animals; plants; fungi and
protists cell.

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Eukaryotic Cell
 Cell with a true nucleus, where
the genetic material is
surrounded by a membrane;
 Eukaryotic genome is more
complex than that of
prokaryotes and distributed
among multiple chromosomes;
 Eukaryotic DNA is linear;
 Eukaryotic DNA is
complexed with proteins called
"histones;
 Numerous membrane-bound
organelles;
 Complex internal structure;
 Cell division by mitosis.

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Prokaryotic Cell
 Unicellular organisms, found in
all environments. These
include bacteria and archaea.
 Without a nucleus; no nuclear
membrane (genetic material
dispersed throughout
cytoplasm ;
 No membrane-bound
organelles;
 Cell contains only one circular
DNA molecule contained in the
cytoplasm;
 DNA is naked (no histone);
 Simple internal structure; and
 Cell division by simple binary
fission.

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Archaea
 Archaea are
prokaryotes; organisms
without nucleus but some
aspect of their molecular
biology are more similar
to those of eukaryotes.

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Eukaryotic Cell Cycle
Eukaryotic Cell Cycle :
defined as the sequence of
events that occurs during the
lifetime of a cell and is
traditionally divided into four
phases:
 G1 = Growth and preparation
of the chromosomes for
replication
 S = Synthesis of DNA
 G2 = Preparation for mitosis
 M = Mitosis

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Central Dogma of Molecular Biology
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html
The flow of genetic information as follows:

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Deoxyribonucleic Acid (DNA)
Deoxyribonucleic Acid
(DNA), the genetic
material of all cellular
organisms and most
viruses, the gigantic
molecule which is used to
encode genetic
information for all life on
Earth.

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http://genome.gsc.riken.go.jp/hgmis/graphics/slides/01-0085jpg.html
U.S. Department of Energy Human Genome Program, http://www.ornl.gov/hgmis.

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 Thread like structure.
 Located in the cell
nucleus.
 The storage place for all
genetic information.
 The number of
chromosomes varies from
one species to another.
The Chromosome

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In normal human cell
DNA contained in the
nucleus, arranged in 23
pairs of chromosomes ;
22 pairs of chromosomes
(autosomes) ; the 23
chromosome pair
determines the sex of
individual and is
composed of either two
(x) chromosomes
(female) or an (x) and (y)
chromosome (male).
The Chromosome

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 The basic units of
inheritance; it is a
segment within a very
long strand of DNA with
specific instruction for the
production of one specific
protein. Genes located
on chromosome on it's
place or locus.
The Gene

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 DNA and RNA are long
chain polymers of small
compound called
nucleotides. Each
nucleotide is composed
of a base; sugar (ribose
in RNA or deoxyribose in
DNA) and a phosphate
group. The phosphate
joins the sugars in a DNA
or RNA chain through
their 5` and 3` hydroxyl
group by phosphodiester
bonds.
General Structure of Nucleic Acid

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 The structure of DNA was described by British
Scientists Watson and Crick as long double helix
shaped with its sugar phosphate backbone on the
outside and its bases on inside; the two strand of
helix run in opposite direction and are anti-parallel to
each other. The DNA double helix is stabilized by
hydrogen bonds between the bases.
 This structure explains how genes engage in
replication, carrying information and acquiring
mutation.
 The G+C content of a natural DNA can vary from 22-
73% and this can have a strong effect on the physical
properties of DNA, particularly its melting
temperature.

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 There are four different types of nucleotides found in
DNA, differing only in the nitrogenous base: A is for
adenine; G is for guanine; C is for cytosine and T is for
thymine.
 These bases are classified based on their chemical
structures into two groups: adenine and guanine are
double ringed structure termed purine , thymine and
cytosine are single ring structures termed pyrimidine.
 The bases pair in a specific way: Adenine A with
thymine T (two hydrogen bonds) and guanine G with
cytosine C (three hydrogen bonds).
 Within the structure of DNA, the number of thymine is
always equal to the number of adenine and the
number of cytosine is always equal to guanine.
 In contrast to DNA; RNA is a single stranded, the
pyrimidine base uracil (U) replaces thymine and ribose
sugar replaces deoxyribose.

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Genomic DNA organization
Eukaryotic genes: DNA
molecules complexed
with other proteins
especially basic proteins
called histones, to form a
substance known as
chromatin. A human cell
contains about 2 meters
of DNA. DNA in body
could stretch to the sun
and back almost 100
times. So it is tightly
packed.

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Eukaryotic Chromatin
 Eukaryotic chromatin
is folded in several ways.
The first order of folding
involves structures called
nucleosomes, which have
a core of histones,
around which the DNA
winds ( four pairs of
histones H2A, H2B,H3
and H4 in a wedge
shaped disc, around it
wrapped a stretch of 147
bp of DNA).

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DNA Replication
 DNA Replication: The
DNA (all gene) duplication; the
transfer the genetic information
from a parent to a daughter cell ;
the DNA base sequence are
precisely copied.
 Replication proceeds in a
semiconservative manner, each
strand of the DNA helix serves as
a template for the synthesis of
complementary DNA strands. This
lead to the formation of two
complete copies of the DNA
molecule, each consisting of one
strand derived from the parent
DNA molecule and one newly
synthesized complementary
strand.

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Mitochondrial DNA
 Mitochondria is a membrane-
enclosed organelle found in most
eukaryotic cells.These organelles
range from 1–10 micrometers
(μm) in size.
 Mitochondria generate most of
the cell's supply of adenosine
triphosphate (ATP).
 Mitochondria are involved in a
range of other processes, such as
signaling, cellular differentiation,
cell death, as well as the control of
the cell cycle and cell growth.
 Mitochondria have been
implicated in several human
diseases, including mental
disorders,cardiac dysfunction,[and
may play a role in the aging
process.
 Mitochondria has its own DNA.

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Mitochondrial DNA
 Mitochondrial DNA contains 37 genes, all of which
are essential for normal mitochondrial function. Thirteen
of these genes provide instructions for making enzymes
involved in oxidative phosphorylation.
 Oxidative phosphorylation is a process that uses oxygen
and simple sugars to create adenosine triphosphate
(ATP), the cell's main energy source.
 The remaining genes provide instructions for making
molecules called transfer RNAs (tRNAs) and ribosomal
RNAs (rRNAs).
 Mitochondrial genes are among the estimated 20,000 to
25,000 total genes in the human genome.

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Function of The DNA
 Deoxyribonucleic Acid (DNA), the gigantic
molecule which is used to encode genetic information
for all life on Earth.
 The chemical basis of hereditary and genetic variation
are related to DNA.
 DNA directs the synthesis of RNA which in turn
directs protein synthesis.

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The Genetic Code
 The purine and pyrmidine bases of the DNA molecule
are the letters or alphabet of the genetic code. All
information contained in DNA represented by four letters:
A,T,C,G.
 Three nucleotides of DNA (1st, 2nd and 3rd) form triplet
codons. A group of codons constitute the genetic code,
that can be translated into amino acid of proteins.
 RNA Codon tRNA Amino Acids

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The Genetic Code
 The sequence of codons
in the mRNA defines the
primary structure of the
final protein. Since there
are 64 possible codons,
most amino acids have
more than one possible
codon. Out of the 64
possible 3-base codons,
61 specify amino acids;
the other three are stop
signals (UAG, UAA, or
UGA).

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The RNA
 Three major classes of RNA: messenger (mRNA),
transfer (tRNA) and ribosomal (rRNA). Minor classes
of RNA include small nuclear RNA ; small nucleolar
RNA;………..

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The RNA
- The concentration of
purine and pyrimidine
bases do not necessarily
equal one another in RNA
because RNA is single
stranded. However, the
single strand of RNA is
capable of folding back
on itself like a hairpin and
acquiring double strand
structure.

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Messenger RNA
 mRNA molecules represent
transcripts of structural genes
that encode all the information
necessary for the synthesis of
a single type polypeptide of
protein.
 mRNA; intermediate carrier
of genetic information; deliver
genetic information to the
cytoplasm where protein
synthesis take place.
 The mRNA also contains
regions that are not translated:
in eukaryotes this includes the
5' untranslated region, 3'
untranslated region, 5' capand
poly-A tail.

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Transfer RNA(tRNA)
 All tRNAs share a
common secondary
structure represented by
a coverleaf. They have
four-paired stems
defining three stem loops
(the D loop, anticodon
loop, and T loop) and the
acceptor stem to which
amino acids are added in
the charging step.
 RNA molecules that carry
amino acids to the
growing polypeptide.

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Ribosomal RNA (rRNA)
Ribosomal RNA (rRNA) is the central component of
the ribosome, 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.

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Ribosomes
 Ribosomes ; Factory for
protein synthesis; are
composed of ribosomal
RNA and ribosomal
proteins (known as a
Ribonucleoproteinor
RNP). They translate
messenger RNA (mRNA)
to build polypeptide
chains using amino acids
delivered by transfer RNA
(tRNA).

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Ribosomes
 Eukaryotic ribosomes are larger. They consist of two
subunits; a 60S subunit holds (three rRNAs 5S, 5.8S,
28S and about 40 proteins) and a 40S subunit contains
(an18S rRNA and about 30 proteins) , which come
together to form an 80S particle compared with
prokaryotic 70S ribosome

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 Most mRNA are
translated by more than
one ribosome at a time;
the result, a structure in
which many ribosomes
translate an mRNA in
tandem, is called a
polysomes.
Polysomes

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The Protein
 Proteins are the basic building materials of a cell, made by cell
itself; the final product of most genes.
 Proteins are chain like polymers of a few or many thousands of
amino acids. Amino acids are represented by codons, which are 3-
nucleotide RNA sequences. Amino acids joined together by peptide
bonds (polypeptide). Proteins can be composed of one or more
polypeptide chains.
 Proteins have many functions: provide structure that help cells
integrity and shape (e.g. collagen in bone); serve as enzymes and
hormones; bind and carry substance and control of activities of
genes….

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Four levels of a protein's structure:
 Primary structure: Formed by joining the amino acid
sequence into a polypeptide.
 Secondary structure: Different conformation that can
be taken by the polypeptide: alpha helix and strands of
beta sheet.
 Tertiary structure : Result from folding the secondary
structure components of the polypeptide into three-
dimensional configuration.
 Quaternary structure : complex of several protein
molecules or polypeptide chains, usually called protein
subunits, which function as part of the larger assembly or
protein complex.

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Gene Expression
 Gene expression process by which a gene product (an
RNA or polypeptide ) is made.
 In transcription steps, RNA polymerase make a copy
of information in the gene (complementary RNA)
(mRNA) complementary to one strands of DNA.
 In translation step, ribosomes read a messenger
RNA and make protein according to its instruction. Thus
any change in gene sequence may lead to change in the
protein product.

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Types of control in Eukaryotes
 Transcriptional, prevent
transcription, prevent mRNA
from being synthesized.
 Posttranscriptional,
control mRNA after it has been
produced.
 Translational, prevent
translation; involve protein
factors needed for translation.
 Posttranslational, after
the protein has been produced.

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Mutation
 Mutation include both gross alteration of chromosome
and more subtle alteration to specific gene sequence.
 Gross chromosomal aberrations include: large deletions;
addition and translocation (reciprocal and nonreciprocal).
 Mutation in a gene's DNA sequence can alter the amino
acid sequence of the protein encoded by the gene. Point
mutations are the result of the substitution of a single base.
Frame-shift mutations occur when the reading frame of the
gene is shifted by addition or deletion of one or more bases.

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Mutation
Mutations can have harmful,
beneficial, neutral, or uncertain
effects on health and may be
inherited as autosomal
dominant, autosomal
recessive, or X-linked traits.
Mutations that cause serious
disability early in life are
usually rare because of their
adverse effect on life
expectancy and reproduction.

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Common Tools in Molecular Biology
 Nucleic acid fractionation
 Polymerase chain reaction
 Probes, Hybridization
 Vector, Molecular cloning
 Nucleic acid enzymes
 Microarray
 DNA sequencing
 Electrophoretic separation of nucleic acid
 Detection of genes:
 *DNA: Southern blotting; inSitu hybridization; FISH
Technique
 *RNA: Northern blotting
 *Protein: Western blotting, immunohistochemistry

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Human Genome Project
Goals
 Identify all the approximately 20,000-25,000 genes in
human DNA,
 Determine the sequences of the 3 billion chemical base
pairs that make up human DNA, store this information in
databases,
 Improve tools for data analysis,
transfer related technologies to the private sector, and
 Address the ethical, legal, and social issues (ELSI) that may
arise from the project.

50
Molecular Biology : Uses
 Various methods in molecular biology diagnose the
different human diseases; diagnosis of an infectious
agent, in malignancy, the presence of the genetic
disease and in transplantation, paternity and forensic
analysis.
The Most Recent Applied Technologies
 Genetic engineering
 DNA finger-printing in the social and forensic science.
 Pre and postnatal diagnosis of inherited diseases.
 Gene therapy.
 Drug Design.

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Molecular biology is
facilitating research in
many field including
biochemistry,
microbiology,
immunology and
genetics,…………………
…
Molecular biology
allows the laboratory to
be predictive in nature,
it gives information that
the patients may be at
risk for disease (future).

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Glossary
 Alleles are forms of the same gene with small differences in their sequence of DNA bases.
 Exon (Coding DNA): A gene sequence contains protein coding information.
 Introns (intervening sequence) (A noncoding DNA sequence ): Intervening stretches of DNA that separate exons.
 Primary transcript: The initial production of gene transcription in the nucleus; an RNA containing copies of all exons and introns.
 RNA gene or non-coding RNA gene: RNA molecule that is not translated into a protein. Noncoding RNA genes produce
transcripts that exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA (tRNA) and
ribosomal RNA (rRNA), small RNAs such as snoRNAs, microRNAs, siRNAsand piRNAs and lastly long ncRNAs.
 Enhancers and silencers: are DNA elements that stimulate or depress the transcription of associated genes; they rely on tissue specific
binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer depending on what is bound to it.
 Activators: Additional gene-specific transcription factors that can bind to enhancer and help in transcription activation.
 Open reading frame (ORF) : A reading frame that is uninterrupted by translation stop codon (reading frame that contains a start codonand
the subsequent translated region, but no stop codon).
 Directionality: in molecular biology, refers to the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention
of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5' end and a 3' end ( "five prime end" and "three prime end"). The
relative positions of structures along a strand of nucleic acid, including genes, transcription factors, and polymerases are usually noted as being
either upstream (towards the 5' end) or downstream (towards the 3' end).
 3' flanking region: Present adjacent to 3' end of the gene; often contain sequences which affect the formation of the 3` end of the message
and may contain enhancers or protein binding sites.
 5' flanking region: A region adjacent to 5' end of the gene. It is not transcribed into RNA; it contains the promoter. May contain enhancers or
other protein binding sites.
 3' untranslated region: The three prime untranslated region (3' UTR) is a particular section of messenger RNA (mRNA). It follows the
coding region. It is a region of the DNA which is transcribed into mRNA and becomes the 3' end or the message, Several regulatory sequences
are found in the 3' UTR. The 3' untranslated region may affect the translation efficiency of the mRNA or the stability of the mRNA. It also has
sequences which are required for the addition of the poly(A) tail to the message (including one known as the "hexanucleotide", AAUAAA).
 5' untranslated region: The five prime untranslated region (5' UTR), also known as the leader sequence, is a particular section of messenger
RNA (mRNA) and the DNA that codes for it. It is a region of a gene which is transcribed into mRNA. It starts at the site (where transcription
begins) and ends just before the start codon (usually AUG) of the coding region. It usually contains a ribosome binding site (RBS), in bacteria also
known as the Shine Dalgarno sequence (AGGAGGU). In prokaryotic mRNA the 5' UTR is normally short. Some viruses and cellular genes have
unusual long structured 5' UTRs which may have roles in gene expression. Several regulatory sequences may be found in the 5' UTR.
 Reverse Transcription: Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA.

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References & Online Further Reading
 Robert F. Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition. ISBN 978-0-07-110216-2
 Innis,David H. Gelfand,John J. Sninsky PCR Applications: Protocols for Functional Genomics: ISBN:0123721865
 Daniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: AACC Press, 1996, ISBN 0-915274-
84-1.
 William B. Coleman,Gregory J. Tsongalis: Molecular Diagnostics: For the Clinical Laboratorian: ISBN 1588293564...
 Robert F. Mueller,Ian D. Young. Emery's Elements of Medical Genetics: ISBN. 044307125X
 Daniel P. Stites,Abba T. Terr. Basic Human Immunology: ISBN. 0838505430
 Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology
of the cell. ISBN. 9780815341055
 http://www.pubmedcentral.nih.gov/
 http://www.biomedcentral.com/1471-2105/2/8/abstract. Elena Rivas and Sean R Eddy Noncoding RNA gene
detection using comparative sequence analysis
BMC Bioinformatics 2001, 2:8doi:10.1186/1471-2105-2-8
 www.medscape.com
 http://www.medterms.com/script/main/art.asp?articlekey=4026
 www.emedicine.com
 www.ebi.ac.uk/2can good introduction to bioinformatics and molecular biology
 http://www.genomicglossaries.com/
 http://www.gene.ucl.ac.uk/nomenclature/guidelines.html defines the nomenclature for human genes
 http://www.accessexcellence.org
 http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Codons.html
 http://www.web-books.com/MoBio/
 http://www.expasy.org
 http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html
 Cell & Molecular Biology online: http://www.cellbio.com/recommend.html
 http://www.ornl.gov/sci/techresources/Human_Genome/glossary/glossary.shtml%20
 http://www.genome.gov/10000715
 http://www.ncbi.nlm.nih.gov/About/primer/mapping.html
 http://www.lilly.com/research/discovering/targets.html
 http://www.informatics.jax.org/expression.shtml
 www.wikipdia.com
 http://www.biology.arizona.edu/cell_bio/tutorials/pev/page2.html
 http://www.genome.ou.edu/protocol_book/protocol_index.html

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