STBP1013 Fundamentals of Molecular Biology STBP1013 Fundamentals of Molecular Biology

Central dogma of molecular biology

• Discovery of DNA and RNA
• Characteristics of genetic materials
Central Dogma of Molecular Biology
• The genetic code
• Gene organization and structure in eukaryote and
prokaryote

Nucleic acids structure

• DNA helix
• Factors affecting DNA structure,
denaturation/renaturation, effects of alkaline and ions
• Fractionation of nucleic acids
• Techniques in electrophoresis, centrifugation and
electron microscopy

The classical view of the central dogma

The classic view of the central dogma of molecular
biology states that "the coded genetic information hard-
wired into DNA is transcribed into individual transportable
cassettes, composed of messenger RNA (mRNA); each
mRNA cassette contains the program for synthesis of a
particular protein (or small number of proteins)."

It was first stated by Francis Crick in 1956 and

re-stated in a Nature paper published in 1970

The central dogma of molecular

biology is an explanation of the flow
of genetic information within a
biological system.

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 STBP1013 Fundamentals of Molecular Biology

Discovery of DNA and RNA

The discovery of DNA

James Watson and Francis
Crick with their DNA model at
the Cavendish Laboratories in
1953
• Many people believe that American biologist James
Watson and English physicist Francis Crick discovered
DNA in the 1950s, but in reality, this is not the case
• Rather, DNA was first identified in the late 1860s by
Swiss chemist Friedrich Miescher
Rosalind Franklin – • Then, in the decades following Miescher's discovery, other
her experimental data Maurice Wilkins – he is scientists carried out a series of research efforts that
provided a very best known for his work revealed additional details about the DNA molecule,
important piece of at King’s College,
evidence leading to London on the structure including its primary chemical components and the
the solving of the DNA of DNA which falls into ways in which they joined with one another
structure three distinct phases
• Without the scientific foundation provided by these
pioneers, Watson and Crick may never have reached their
groundbreaking conclusion of 1953 - that the DNA
molecule exists in the form of a three-dimensional double
helix

Friedrich Miescher - 1869 Friedrich Miescher - 1869

• First identified what he called as “nuclein” in
the nuclei of human white blood cells, which
we now know as DNA
• His original plan had been to isolate and
characterise the protein components of white
blood cells
• To do this, he had made arrangements for a
local surgical clinic to send him pus-saturated
bandages, which he planned to wash out
before filtering the white blood cells and
extracting their various proteins
• However, during the process, he came across
a substance that had unusual chemical
properties unlike the proteins he was
searching for, with very high phosphorous
content and a resistance to protein
digestion
• He quickly realised that he had discovered a
new substance and sensed the importance of
his findings
• Despite this, it took more than 50 years for
the wider scientific community to appreciate
his work

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Erwin Chargaff - 1950
• Worked on the chemistry of nucleic acids
• Discovered that
– firstly, in any double-stranded DNA, the
number of guanine units is equal to the
number of cytosine units and the number
of adenine units is equal to the number of
thymine units, and
– secondly, that the composition of DNA
varies between species
• These discoveries are now known as
'Chargaff's Rules'
Discovery of RNA
• Other early biochemical studies showed that RNA was
readily broken down at high pH, while DNA was stable
(although denatured) in alkali
• Nucleoside composition analysis showed first that RNA
contained similar nucleobases to DNA, with uracil
instead of thymine, and that RNA contained a number of
minor nucleobase components, e.g. small amounts of
pseudouridine and dimethylguanine
Oswald Avery - 1944
• Identified DNA as the substance responsible
for heredity
• He worked for many years with the bacterium
responsible for pneumonia, pneumococcus,
and had discovered that if a live but harmless
form of pneumococcus was mixed with an
inert but lethal form, the harmless bacteria
would soon become deadly
• To find out which substance was responsible
for the transformation, he combined forces
with Colin MacLeod and Maclyn McCarty and
began to purify 20 gallons (~75L) of bacteria
• He soon noted that the substance did not seem
to be a protein or carbohydrate but rather a
nucleic acid, and with further analysis, it was
revealed to be DNA
Discovery of RNA
• When first studied in the early 1900s, the chemical and
biological differences between RNA and DNA were not
apparent, and they were named after the materials from
which they were isolated:
• RNA was initially known as "yeast nucleic acid”
• DNA was "pancreas nucleic acid"
• Using diagnostic chemical tests, carbohydrate chemists
showed that the two nucleic acids contained different
sugars, whereupon the common name for RNA became
"ribose nucleic acid"
Discovery of RNA
• Messenger RNA (mRNA) carries genetic information
that directs protein synthesis
• The concept of mRNA emerged during the late 1950s, and
is associated with Crick's description of his "Central
Dogma of Molecular Biology", which asserted that DNA
led to the formation of RNA, which in turn led to the
synthesis of proteins
• During the early 1960s, sophisticated genetic analysis
of mutations in the lac operon of E. coli and in the rII
locus of bacteriophage T4 were instrumental in defining
the nature of both messenger RNA and the genetic code
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 RNA
§ RNA is one of the three major biological
macromolecules that are essential for all known forms of
life (along with DNA and proteins)
§ The central dogma of molecular biology states that the flow
of genetic information in a cell is from DNA through RNA to
proteins: “DNA makes RNA makes protein”
RNA
§ RNA is the “DNA photocopy” of the cell
- when the cell needs to produce a certain protein, it
activates the protein’s gene (the portion of DNA that
codes for that protein) and produces multiple copies of
that piece of DNA in the form of messenger RNA,
or mRNA
- the multiple copies of mRNA are then used to translate
the genetic code into protein through the action of the
cell’s protein manufacturing machinery, the ribosomes
- thus, RNA expands the quantity of a given protein
that can be made at one time from one given gene, and
it provides an important control point for regulating
when and how much protein gets made
RNA
§ In a number of clinically important viruses, RNA (rather
than DNA) carries the viral genetic information
§ RNA also plays an important role in regulating cellular
processes – from cell division, differentiation and growth
to cell aging and death
§ Defects in certain RNAs or the regulation of RNAs have
been implicated in a number of important human
diseases, including heart disease, some cancers, stroke
and many others
RNA
§ Proteins are the workhorses of the cell
- they play leading roles in the cell as enzymes, as
structural components, and in cell signaling, to
name just a few
§ DNA is considered the “blueprint” of the cell
- it carries all of the genetic information required for the
cell to grow, to take in nutrients, and to propagate
RNA
§ For many years RNA was believed to have only three
major roles in the cell:
- as a DNA photocopy (mRNA)
- as a coupler between the genetic code and the protein
building blocks (tRNA)
- as a structural component of ribosomes (rRNA)
§ In recent years, however, we have begun to realize that
the roles adopted by RNA are much broader and much
more interesting
§ We now know that RNA can also act as enzymes
(called ribozymes) to speed chemical reactions
RNA vs DNA
• The chemical structure of RNA is very similar to that of
DNA, but differs in three main ways:
– Unlike double-stranded DNA, RNA is a single-
stranded molecule in many of its biological roles and
has a much shorter chain of nucleotides. However,
RNA can, by complementary base pairing, form
intrastrand double helixes, as in tRNA.
– While DNA contains deoxyribose, RNA contains ribose
(RNA is less stable than DNA because it is more
prone to hydrolysis)
– In DNA, A=T and in RNA, A=U (uracil is an
unmethylated form of thymine)
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 RNA vs DNA
§ Like DNA, most biologically active RNAs (e.g. mRNA,
tRNA, rRNA, snRNAs and other non-coding RNAs) contain
self-complementary sequences that allow parts of the
RNA to fold and pair with itself to form double helices
§ Analysis of these RNAs has revealed that they are highly
structured
§ Unlike DNA, their structures do not consist of long double
helices, but rather collections of short helices packed
together into structures akin to proteins
§ In this fashion, RNAs can achieve chemical catalysis, like
enzymes e.g. determination of the structure of the
ribosome (an enzyme that catalyzes peptide bond
formation), revealed that its active site is composed
entirely of RNA
Conclusion
• DNA is an informational macromolecule that encodes the
complete set of instructions (the genome) that are
required to assemble, maintain, and reproduce every living
organism.
• Proteins are responsible for catalyzing the myriad
biochemical reactions that are required to provide food
and energy for every organism, and for all forms of
movement. In addition proteins carry out all of the other
functions of any given organism e.g. photosynthesis in
plants, or neural function, vision, and structure (skin,
tendons, exoskeleton, etc.) in animals.
• RNA is multifunctional, its primary responsibility is to
make proteins, according to the instructions encoded
within a cell’s DNA. They control and regulate many
aspects of protein synthesis in eukaryotes.
Timeline of important events in molecular biology
1905-1908
William Bateson and Reginal Crudell Punnett
demonstrated actions of some genes modify action
of other genes: the first time gene regulation was
demonstrated
1910 Thomas Hunt Morgan was the first to recognise genes are
carried on chromosomes: the basis for modern genetics. He
demonstrated the existence of sex-linked genes and
expanded trait linkage using ”crossing-over”
1911* Alfred Sturtevant, mapped the locations of several fruit fly
genes - this was the first genetic map
1926* Thomas Hunt Morgan published the "theory of the gene"
based on Mendelian genetics
1933 A new technique, electrophoresis, was introduced by Arne
Tiselius for separating proteins in solution
Conclusion
• All living organisms are dependent on three types of very
large molecules for essentially all of their biological
functions
• These molecules are DNA, RNA and proteins, and are
classified as biological macromolecules
• Without DNA, RNA and proteins, no known forms of life
could exist
• This is because each molecule plays an indispensable
role in biology
• The simple summary is that DNA makes RNA, and then
RNA makes proteins
Timeline of important events in molecular biology
1859 Charles Darwin published the “Origin of Species”,
introducing that genetic evolution allowed adaption over time
to produce organisms best suited for the environment
1865 Gregor Mendel investigated “traits” passed from parents to
prodigy and coined the term dominant and recessive traits
1869* Friedrich Meisher isolated DNA from nuclei of white blood
cells
1875 Charles Darwin introduced “gemmules” as mechanism of
inheritance
1902* Walter Sutton created the term “gene” to describe “factors”
located on chromosomes: he observed chromosomal
movement during meiosis and developed the chromosomal
theory of heredity
Timeline of important events in molecular biology
1937 Frederick Charles Bawden discovered tobacco mosaic
virus RNA
1944* Barbara McClintock reported transposable elements:
"jumping genes"
1946 Edward Tatum and Joshua Lederberg discovered that
bacteria can exchange genetic material directly through
conjugation
Max Delbruck and Alfred Day Hershey discovered a
combination of genetic material from viruses: genetic
recombination
1950* Erwin Chargaff found that amounts of adenine and thymine
and cytosine and guanine in DNA are always about the
same - this is now called "Chargaff's Rules”
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Timeline of important events in molecular biology
1952* Rosalind Franklin and Maurice Wilkins performed X-ray
crystallography studies of DNA, providing crucial
information that led to the elucidation of the structure of DNA
1953* James Watson and Francis Crick proposed the double-
stranded, helical, complementary, anti-parallel model for
DNA
1955* Frederick Sanger announced the first complete sequence
of a protein, bovine insulin
Arthur Kornberg discovered and isolated DNA polymerase
from E. coli bacteria
1956* Francis Crick and George Gamov worked out the "Central
Dogma" to explain protein synthesis from DNA: the DNA
sequence codes for amino acid sequences and genetic
information flows in one direction - from DNA to mRNA to
protein
Timeline of important events in molecular biology
1972* Paul Berg used a restriction enzyme to cut DNA and ligase
to past two DNA strands together to form hybrid circular
molecule, the first recombinant DNA molecule
First successful DNA cloning experiments
1973* Stanley Cohen and Herbert Boyer first successfully
transfered DNA from one life form into another: a spliced
viral DNA and bacterial DNA to create a plasmid with dual
antibiotic resistance
1974* Allan Maxam and Walter Gilbert (Harvard) and Frederick
Sanger (U.K. Medical Research Council) independently
developed different methods for sequencing DNA
Timeline of important events in molecular biology
1980* Kary Mullis invented the polymerase chain reaction (PCR),
a method for multiplying DNA sequences in vitro
1981 Gordon and Ruddle (Ohio University) made the first
transgenic mice by inserting genes from other animals with
DNA microinjection
Human mitochondral DNA sequenced (16569 bp)
1982* Phage lambda genome sequenced (48,502 bp)
1983 First genetic modifed plant is created; a tobacco plant
resistant to an antibiotic
1984* Alec Jeffreys developed the technique of using sequences of
DNA for identification, called "genetic fingerprinting"
1990* The Human Genome Project begins
1995* Haemophilus influenzae is the first bacterium genome
sequences
Timeline of important events in molecular biology
1959* Francois Jacob and Jacques Monod discovered an important
mechanism behind genetic regulation: mappable control
functions located on chromosomes in DNA sequence -
named "repressor" and "operon"
1961* Marshall Nirenberg, Heinrich Mathaei and Severo Ochoa
cracked the "Genetic Code": a sequence of three
nucleotide bases (codon) determine each of amino acids
1967 Mary Weiss and Howard Green found a technique for
combining human cells and mouse cells grown in one
culture: somatic cell hybridisation
1970* Howard Temin and David Baltimore independently isolated
reverse transcriptase, an enzyme that can make DNA from
RNA
Torbojorn Caspersson and Lore Zech discovered a method
for staining mammalian chromosomes to reveal banding
Timeline of important events in molecular biology
1975* Georges Kohler and Cesar Milstein fused antibody-
producing B lymphocyte cells with tumor cells that are
"immortal" to produce the first monoclonal antibodies
Edwin Southern published the experimental details for the
Southern Blot technique to identify DNA fragments
1977* Bacteriophage FX-174 (5368 bp) was the first complete
genome (DNA) to be sequenced
Richard Roberts’ and Phil Sharp’s labs showed that
eukaryotic genes contain many interruptions, called introns
1978* Genentech successfully produced human insulin using
recombinant DNA technology in E. coli
David Botstein discovered the use of restriction enzymes
produces different fragments from one person to another,
RFLP: restriction fragment length polymorphisms
Timeline of important events in molecular biology
1996* Dolly the sheep is cloned
2003* The Human Genome Project is completed
2005* New DNA sequencing techniques were introduced called
"Next Generation Sequencing" (NGS) techniques
2010 Hamilton Smith and colleagues succeeded in incorporating a
synthetic genome into a bacterium