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THE MOLECULAR BIOLOGY STORY

C.T. HEDREYDA
No. 3

1859 - Austrian botanist, Gregor Mendel, cross pollinated plants to examine


the patterns of inheritance of several traits:
Mendel suggested that one unit of heredity comes from each parent
which could be dominant or recessive and this led to the Principles of
Inheritance known as Mendelian genetics. Mendel published his work
in 1865 but was not understood till the 1900.

Yellow (dominant) vs Green (recessive)


Smooth (dominant) vs Wrinkled (recessive)
Tall (dominant) vs Dwarf (recessive)

YY - yellow Yy – yellow yy – green

YY x YY YY x yy YY x Yy yy x yy Yy x yy Yy x Yy

Y Y Y y Y Y y y y Y y y Y y Y y

100% YY 100% Yy 50% YY 50%Yy 100% yy , 50%Yy 50%yy, 25%YY


50%Yy25%yy

1869 - Johann Friedrich Miescher, Swiss biochemist, isolated nuclei of


white blood cells called nuclein, now known as nucleic acid
1882 - Walker Flemming, a German cytologist, described threadlike
bodies during cell division. Threadlike bodies were termed as
chromosomes by Waldeyer in 1888.
1903 - Walter Sutton, an American cytologist, described that
chromosomes were carriers of Mendel’s units of heredity or
genes. The term chromosome was given by Wilhelm Johannsen
in 1909.
1920’s – 1930’s Biochemical reactions to many important metabolic
pathways were elucidated. The ultracentrifuge was developed.

1928 - The Transformation Experiments of Fred Griffith


A. S- Strain with polysaccharide capsule R-Strain w/o
capsule Smooth edged colonies in agar plate Rough edged
colonies

HEAT KILLED HEAT KILLED

VIRULENT NON-VIRULENT to mice NON-VIRULENT to mice

B. HEAT KILLED S STRAIN + LIVE R STRAIN = VIRULENT


Bacteria isolated from dead mice were all S. Only live R were used and S were
killed, therefore the live S came from live R which were transformed to S type.
Why? And how?

+ =
Live R non-
Heat killed virulent R virulent strain Live S virulent
strain strains obtained

The R non-virulent strain was transformed to virulent R


strain. Something in the killed S virulent strain was
transferred to the live R strain which resulted in converting R
to the virulent S strain.

C. Questions based on experimental results


 How were the virulent bacteria generated again ?
 Were they generated because the some molecules from the killed virulent
bacteria were taken by the live non-virulent strain and such molecules are
responsible for converting them to the virulent form?
 If so, what in the extract were responsible for the transformation?
 When mouse were subjected to Cell extracts of the virulent (S) strain without
cells + live non-virulent live cells, mouse death occurred.
 Therefore, the extracts from the killed S virulent strains contain the
TRANSFORMING PRINCIPLE, giving rise to the virulent bacteria .

1938 - MOLECULAR BIOLOGY : The term was first used by Warren Weaver, who
later described molecular biology as another branch of science
1944 - The answer to the question on transformation was obtained from the results
of the experiments of Oswald Avery, Colin MacLeod and Maclyn McCarty.

a. Partially purified DNA from the virulent S strain extract was mixed with
live R cells and introduced in mice, resulting in killed mice.
b. When polysaccharide from S strain was added to live R strain and then
introduced to mice, no mice death was observed.
c. The next question : Was the DNA extract used pure and were there no
contaminating proteins or RNA?
d. When the extract was treated with RNAse that destroy RNA, or with a
proteolytic enzyme that destroy proteins, transformation into S virulent
still occurred in both cases.
e. When extract was treated with DNAse that depolymerize DNA, no
transformation was observed.

Therefore, these suggest that the transforming principle was DNA or the
nucleic acid.

1945 – The term molecular biology was used by WILLIAM ASTBURY when he
was referring to the study of chemical and physical structure of
macromolecules
1952 - The Blender Experiment of Alfred Hershey and Martha Chase
The experiment made use of a kitchen blender and showed that the DNA
was introduced into the bacterial cell and DNA was later multiplied and
packaged into new viral particles.

32
P labeled DNA

bacteriophage

bacteria

6. 19 6. 1940 – 1953 many


Injection blending incubation
lysis
Labeled
with 32P
1950 – 1953 Rosalind Franklin obtained improved X ray diffraction data using
purified DNA samples. Data suggest a 3.4 nm periodicity and a helical
structure
1953 Linus Pauling and Robert Corey used data of Rosalind Franklin to
suggest a triple helix DNA structure with phosphates at the center
1953 James Watson and Francis Crick used the data of R. Franklin and
Chargaff and proposed the double helix structure of the DNA
1955 The first complete sequence of the peptide hormone insulin was
obtained.
1957 Matthew Messelson & Frank Stahl demonstrated how DNA is
replicated in cells.
1960 The existence of the messenger RNA was confirmed.
The first complete sequence of an enzyme, ribonuclease was
reported.
1962 Nobel Prize for the DNA double helix shared by James Watson,
Francis Crick, Rosalind Franklin, and Maurice Wilkins.
1966 The triplet code of all living organisms was completed.
1960 - 1970s DNA sequencing was already being developed.
1967 DNA ligase, the enzyme that catalyzes the formation of a
phospodiester bond between two DNA chains, or joining of DNA
fragments was first discovered.
1968 Meselson and Yuan first discovered restriction enzymes from E.coli
but the cleavage sites are not clear
1970’s The discovery of restriction enzymes, the enzymes that cut DNA and
ligase, enzymes used to connect double stranded DNA and the
discovery of these enzymes paved the way for the advances in
genetic engineering. Now there are about 900 restriction enzymes
from about 230 different bacterial species.
1971 The Ca Cl2 or heat shock protocol for transformation was developed.
1972 Stanford University with Berg, Jackson, Symons, Mertz, and Davis
described the joining or ligation of DNA molecules from different
sources.
Use of gel electrophoresis, EcoRI restriction enzyme, and ethidium
bromide for staining gels with DNA were developed.
1973 Boyer, Helling, Cohen, and Chang published their work showing the
DNA fragment could be ligated or introduced into a plasmid vector.
Genes or DNA from frog was transferred to E. coli while bacteria could
be transformed with mouse DNA.
1977 Two DNA sequencing methods emerged, the chain termination and
chemical degradation
1980 Patent on methods of cloning and transformation was awarded to
Herbert Boye and and Boye later became the founder of the
biotechnology company Genentech.
Bioinformatics or computer applications in biological science was born.
1982 Human insulin produced by genetically engineered bacteria became
the first product of modern biotechnology that was commercially
released.
1987 A crime suspect in UK was convicted using DNA fingerprinting data.
1990 Chymosin enzyme for milk curdling was commercially produced by a
genetically modified bacteria.
1991 The first transgenic animal, Tracey the sheep, was born with a gene
for a therapeutic protein present in its milk.
1994 The first transgenic plant, Flavr Savr Tomato, with the delayed
ripening trait, was commercialized.
1996 –present Genetically modified crops introduced in the market with insect
resistance, herbicide tolerance
1997 The sheep Dolly, the first cloned animal was born.
1998 More than 300,000 partial to complete protein sequences has been
obtained. Start of the human genome project.
2002 Completed sequencing of the human genome.

At present :
 Area planted into genetically modified crops worldwide is more
than 81 million hectares.
 Complete sequences for a number of microbial, plant, and animals
species has been achieved including H. influenzae, M.
pneumoniae, E. coli. M. tuberculosis, V. cholerae, Methanococcus
jannaschii, Pyrococcus horikoshii, S. cerevisiae, C. elegans, D.
melanogaster, Arabidopsis thaliana, Mus musculus, Homo
sapiens
 Dolly is dead due to early aging.

BIOTECH UPDATES

1. http://www.sciencedaily.com/releases/2012/08/120821114746.htm
Stem Cells Can Become Anything, but Not Without This Protein
How do stem cells preserve their ability to become any type of cell in the
body? And how do they "decide" to give up that magical state and start
specializing?
University of Michigan Medical School team led by Yali Dou, Ph.D., show the
crucial role of a protein called Mof in preserving the 'stem-ness' of stem cells, and
priming them to become specialized cells in mice. Without Mof, embryonic stem
cells lost their self-renewal capability and started to differentiate."
Mof appears to control the process that actually allows cells to determine
which genes it wants to read -- a crucial function for stem-ness. Mof marks the
areas that need to stay open and maintains the potential to become anything," Dou
explains. Its crucial role in many species is hinted at by the fact that the gene to
make Mof has the same sequence in fruit flies and mice.

2. http://www.sciencedaily.com/releases/2012/08/120821094034.htm
Alzheimer Protein Seems to Slow Down Neurotransmitter Production
How abnormal protein deposits in the brains of Alzheimer's patients disrupt
the signalling between nerve cells has now been reported by researchers in Bochum
and Munich, led by Dr. Thorsten Müller from the Medizinisches Proteom-Center of
the Ruhr-Universität, in the journal Molecular and Cellular Proteomics. They
varied the amount of APP protein and related proteins associated with Alzheimer's
disease in cell cultures, and then analysed how this manipulation affected other
proteins in the cell. The result: the amount of APP present was related to the
amount of an enzyme that is essential for the production of neurotransmitters and
therefore for communication amongst nerve cells.

3. http://news.yahoo.com/teen-doing-well-2-years-stem-cell-windpipe-
231123124.html
Teen Doing Well 2 Years After Stem Cell Windpipe Transplant
The windpipe was stripped of the donor's cells down to the inert structure of
collagen. Tissue from the lining of Finn-Lynch's windpipe was implanted in the
new windpipe to kick-start the growth of a lining in the new windpipe. Two years
after he became the first child to receive a stem cell-supported trachea (windpipe)
transplant, a 13-year-old boy is able to breathe normally, has grown about four
inches taller, does not require any anti-rejection drugs and has returned to school.

4. http://news.yahoo.com/european-regulators-back-first-gene-therapy-
drug-123941621--sector.html
European regulators back first gene therapy drug
Use of gene therapy suffered a major setback when an Arizona teenager died
in a gene therapy experiment in 1999 and two French boys with SCID
developed leukemia in 2002. This time, European regulators have
recommended approval of the Western world's first gene therapy drug, after
rejecting it on three previous occasions. The disorder to be treated is
estimated to affect no more than one or two people per million, can cause
acute pancreatitis and death.

5. http://www.biotechdaily.com/genomics_proteomics/articles/294742175/
mirnas_show_promise_for_treatment_of_ovarian_cancer.html
miRNAs Show Promise for Treatment of Ovarian Cancer
Investigators at the Georgia Institute of Technology (Atlanta, GA, USA)
worked with two well-defined mRNA species, MiR-7 and MiR-128. They
conducted a series of controlled experiments in which these two miRNAs--
previously implicated in ovarian cancer onset/progression--were individually
transfected into a well-defined ovarian cancer (HEY) cell line, and the
consequence on global patterns of gene expression was monitored using
microarray technology.

The results published in the August 1, 2012, online edition of the


journal BMC Medical Genomics revealed that the changes in gene expression
induced by the individual miRNAs were functionally coordinated but distinct
between the two miRNAs.

MiR-7 transfection into ovarian cancer cells induced changes in cell


adhesion and other developmental networks previously associated with
epithelial-mesenchymal transitions (EMT) and other processes linked with
metastasis. On the other hand, miR-128 transfection, induced changes in
cell cycle control and other processes commonly linked with cellular
replication. . If we can understand which miRNAs affect which suites of
genes and their coordinated functions, it could allow clinicians to attack
cancer cells on a systems level, rather than going after genes individually”.

6. http://www.macroevolution.net/breast-cancer-vaccine.html
An effective breast cancer vaccine: Attacks sugars specific
to cancer cells

Researchers from the University of Georgia (UGA) and the Mayo


Clinic in Arizona have developed a vaccine that dramatically reduces tumors
in a mouse model. The model mimics 90 percent of human breast and
pancreatic cancer cases --including those resistant to common treatments.
When cells become cancerous, sugars on their surface proteins undergo
changes that distinguish them from healthy cells. Scientists have tried for
decades to enable the immune system to recognize those differences, in order
to destroy cancer cells and not normal cells. Because cancer cells originate
within the body, the immune system usually doesn’t recognize them as it
would foreign cells. In their research, the scientists used unique mice
developed by Sandra Gendler, a cancer researcher at the Mayo Clinic and co-
senior author on the study. Just as humans do, the mice develop tumors that
overexpress a protein known as MUC1 on the surface of their cells. The
tumor-associated MUC1 protein is adorned with a distinctive, shorter set of
carbohydrates that set it apart from healthy cells. A vaccine has been
developed that trains the immune system to distinguish and kill cancer cells
based on their different sugar structures on proteins such as MUC1. Boons,
Gendler and their colleagues are currently testing the vaccine’s effectiveness
against human cancer cells in culture and are planning to assess its toxicity.
If all goes well, they anticipate that phase I clinical trials to test the safety of
the vaccine could begin by late 2013.

7. http://news.discovery.com/tech/coating-kills-superbugs-120511.html
Coating Kills Superbugs
The polymer coating, developed by biomedical engineer Mary Chan of Singapore's
Nanyang Technological University and her colleagues, is already being used on
contact lenses by two manufacturers. When used, the coating kills 99 percent of the
bacteria and fungi it comes in contact with. It could also be used for medical devices
such as catheters, reducing the need for harsh disinfectants and antibiotics, which
helps slow the development of resistance in the bacteria that colonize those surfaces.
Under a microscope, the structure of the polymer coating resembles a sponge.
Positive charges on the surface attract bacteria like a magnetic because their surface
has a negative charge. The pores in the polymer then pull the bacteria in, rupturing
the organism's cellular walls and killing them.

8. http://news.discovery.com/tech/gm-mosquitoes-released-in-malaysia-
to-reduce-dengue-spread.html

GM Mosquitoes Released in Malaysia to Reduce Dengue Spread


In late December, the Institute for Medical Research in
Malaysia released about 6,000 genetically modified (GM)
mosquitoes into the country's Eastern forests. These are
male mosquitoes that carried a “lethal” gene that kills
offspring early on; any survivors are not fit for
reproduction. This was one way of greatly reducing the number of offspring
and hence the number of dengue-carriers in the next generation. Some
groups, including Greenpeace, worry that reducing the population of just one
species will only make it easier for other species to proliferate in the wild --
and Aedes aegypti is not the only dengue carrier.

9. http://news.discovery.com/tech/big-stink-technology-farms-120105.html
Bacteria Tech Halts Big Stink
Sulfur compounds produced industrially are what we
find most nauseating. These compounds can wrinkle human
noses with their cabbage-like stench at less than a part per
billion.A bacteria-filled filter is being perfected to prevent
bad industrial smells from spreading over a whole town.
Bacteria cover corrugated cellulose pads on the inside of an
odor biofilter used by pig farming operations in Denmark.
The filter contains corrugated cardboard filled with bacteria that are placed
inside machines. A large amount of air from the industrial site is then
pumped through the machines hourly, and a water rinse provides optimal
moist conditions. As the air flows through the machine, the bacteria process
the smelliest -- and most toxic -- compounds. With better efficiency and
improved design, the filters are getting close to costing less than a dollar per
pig to remove most odors it produces.

10. http://news.discovery.com/tech/poo-powered-bacteria-power-house-
bio-light-philips-111128.html
Poo-Powered Glowing Bacteria Light Up the House
Bio-light, a greener lighting system that's part of their
Microbial Home (MH) system. It isn't powered by electricity or
sunlight, but by glowing bioluminescent bacteria that thrive on
waste generated in the average home. The bioluminescent
bacteria is housed in hand-blown glass cells, clustered together to form a
lamp that could easily be displayed in a modern art museum. Each cell is
connected to the lamp's reservoir base by thin silicon tubes that pipe
methane gas from composted bathroom solids and vegetable scraps via a
kitchen dodad that digests bio-waste.

11. http://news.discovery.com/tech/tech-tackles-wine-allergies-
120713.htmlTech Tackles Wine Allergies

Some people are allergic to certain wines -- that nice Loire Valley red
gives them a rash or headache, or that California Chardonnay makes them
sneeze. The University of British Columbia's Wine Research Center might
have found a way to solve this problem. Ordinarily winemakers use yeast to
convert the sugar in the grape juice to alcohol. But wine isn't just made from
juice; it also involves something called must, which is the skin and other stuff
from the grapes that get crushed. After the yeast converts the first batch of
sugars to alcohol, there's a secondary fermentation that happens, as bacteria
in the mixture convert malic acid –- which has a harsh taste –- into lactic
acid, which is smoother. In modern commercial winemaking the bacteria is
added deliberately. Hennie van Vuuren, the Director of the Wine Research
Centre at UBC, who led the research, told Discovery News that the bacteria
can present a problem: sometimes they convert chemicals called histidines in
the wine to histamines. Histamines are what give some people allergic
reactions, if they are particularly sensitive to them. (It is not always the
histamines that cause the problem; sulfites can as well, and there are people
who are allergic to the alcohol itself). Van Vuuren said his team took the gene
from the malolactic bacteria that allow the malic acid -- which yeast usually
ignores -- to cross the yeast cell membrane. They also added a bacterial gene
that allows the yeast to digest the malic acid. The result is yeast that
eliminates the need for malolactic bacteria and avoids producing chemicals
that can cause reactions to the wine. Since the yeast is digesting the same
chemicals that the introduced bacteria would, it doesn't affect the taste.

12. http://news.discovery.com/animals/beagle-dog-glows-green-
110801.html
Genetically Modified Beagle Glows

Scientists have created a genetically modified female beagle


that glows fluorescent green when under ultraviolet light.
The process to create the beagle, "Tegon," allows the glow to
be turned on and off by command. Researchers hope such
animals will help scientists track the course of disease via
fluorescence.