Chapter 1 Lecture - Winonacourse1.winona.edu/ssegal/Molecular Course...8/22/11 2 Introduc=on:$Molecular$Biology$PreO History • If$Molecular$Biology$studies$how$heredity$...

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The Origins of Molecular Biology: A Mendelian and Darwinian View of the World

Introduc=on: The Big Ques=on

•  Q: Why did the field of Molecular Biology come into being?

Introduc=on: The Big Ques=on

•  Q: Why did the field of Molecular Biology come into being?

•  A: The simplest answer is that the field of Molecular Biology came into being as a way to explain mechanis=cally how heredity works! –  For example, you may want to know how eye color is inherited

–  You may want to know how, on a molecular level, eye color forms (How the pigment is actually produced)

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Introduc=on: Molecular Biology Pre-‐History

•  If Molecular Biology studies how heredity works, and how traits are expressed then the field of Molecular Biology must have its roots in Gene=cs

•  How old is the field of gene=cs? How long have humans been studying heredity?


•  How old is the field of gene=cs? How long have humans been studying heredity?

•  About 10,000-‐12,000 years ago humans began to manipulate animals and plants, to domes=cate them –  Plants include wheat, barley, len=ls, peas –  Animals include dogs, sheep and goats

•  Humans were able to quickly understand the concept of heredity (create breeds that were beUer suited to agricultural produc=on by ma=ng individual organisms with desirable traits)


•  In terms of crops, humans have selected for varie=es with significantly beUer viability –  Crop variants that produce

more fruit/vegetable –  Crops that are more resistant

to pests

•  Using Molecular Biology: –  Clone genes that allow for pest

resistance etc. –  Gene=c Modifica=on: Insert

that into the genome of a plant using Agrobacterium (round-‐up ready/Bt crops)

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•  Besides domes=ca=on, understanding how heredity works also is extremely important for public health

•  Humans have known for long periods of =me that inbreeding generally results in expression of deleterious traits (generally due to more efficient transmission of deleterious gene variants)

•  Let’s take the example of the Romanov’s –  Tsar Nicholas Romanov II was the Czar of Russia from –  The family included his wife Alexandria as well as

four daughters

•  On August 12, 1904 Tsar Nicholas II and Alexandria had their first son, Alexis

•  Alexis was clumsy as a young child and fell oben. When he cut or scraped himself, he bled profusely, and bruises caused uncontrollable internal bleeding

•  Alexis was suffering from a disease called hemophilia, which ran through the Royal Families of Europe through the 19th century



•  At the =me of the Romanov’s, it was known that the disorder ran in within families –  It was unknown what the mechanism of

inheritance of the disease was –  It was unknown what genes were implicated

•  Today, through Molecular Biology, we know that hemophiliacs contain a defec=ve variant of the Cloeng Factor VIII gene on the X chromosome

•  Today, hemophilia is not a life threatening disease and that blood transfusions are unnecessary

•  Today, using molecular biology in vitro, we can produce the Cloeng Factor VIII protein, which can be given to pa=ents

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Introduc=on: Molecular Biology-‐Where We Go From Here

•  Define Molecular Biology –  Ini=al Terms – Modern Terms

•  Understand the links between Gene=cs and Molecular Biology – How the field of Molecular Biology grew out of Gene=cs

– How we came to learn which molecule contains the gene=c informa=on

Introduc=on: Molecular Biology-‐Where We Go From Here

•  Define Molecular Biology –  Ini=al Terms – Modern Terms

•  Understand the links between Gene=cs and Molecular Biology – How the field of Molecular Biology grew out of Gene=cs

– How we came to learn which molecule contains the gene=c informa=on

Introduc=on: The General Defini=on of Molecular Biology

•  The term Molecular Biology was coined by Dr. Warren Weaver in 1938

•  Warren Weaver was a civil engineer and mathema=cian by trade

•  Weaver was a great advocate for science and was responsible for suppor=ng grants for gene=cs and molecular biology

•  He defined molecular biology as the study of biological phenomena at the molecular level (ini=al defini=on) –  This defini=on covers a wide range of

phenomena –  This defini=on is inaccurate in that it does

not explain

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Introduc=on: The Beginnings of Molecular Biology

•  Molecular Biology: Study how the processes of heredity, evolu=on as well as how basic cellular func=ons work (More Modern Defini=on)

•  Heredity can be defined as the study of the passage of traits from parent to offspring –  Abstract Concepts of Gene=cs –  Eye color –  Hair color –  Body paUern –  Disease or Disease predisposi=on

•  Evolu=on can be defined as the development of more complex organisms from less complex organisms –  Abstract Concepts of Evolu=on –  Development of an=bio=c resistance in bacteria –  Humans developing from lesser primates apes

•  Processes that fall under molecular biology necessary for cellular func=on –  DNA replica=on/Segrega=on –  Membrane biosynthesis –  Cellular respira=on

Introduc=on: The General Defini=on of Molecular Biology

•  Our Main Focus in Molecular Biology: Molecular basis of gene expression

•  Specifically, if one wants to study gene expression mechanis=cally on the molecular level, then one follows the different molecules that allow for expression of a gene as well as the molecules that carry out the func=on of a gene –  Structure of DNA and the hereditary

informa=on it encodes –  Structure and func=on of RNA –  Structure and func=on proteins –  Mechanisms of DNA replica=on before

cell division

Historical Perspec=ves on Heredity: An Introduc=on

•  The study of heredity is not just limited to the modern era, but started over 2000 years ago

•  The study of heredity asks one of the fundamental ques=ons of life, how do we have the traits we have?

•  The study of heredity has captured the imagina=on of many scien=sts through out history

•  The study of heredity started long ago in ancient Greece with Aristotle (384-‐322 BC) –  Aristotle proposed the theory of pangenesis –  Pangenesis: Hereditary characteris=cs are

transmiUed by gemmules from individual body parts

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Historical Perspec=ves on Heredity: An Introduc=on

•  Robert Hooke (1635-‐1703) developed the first microscope in 1665 and allowed humans to see cells for the first =me

•  The use of microscopes allowed for the visualiza=on of sperm and eggs

•  Performa=onism: Inside the sperm or egg exists a miniature adult (a homunculus) which enlarges during development

•  Note: performa=onism meant that all traits would be inherited from one parent

Historical Perspec=ves on Heredity: The Age of Mendel

•  It was not un=l the 1860s that mechanisms of heredity started to become truly understood

•  The person responsible for determining the mechanism of heredity was the Austrian monk Gregor Mendel

•  Gregor Mendel was born in 1822 in what is now considered the Czech Republic to a family of farmers

•  Although his family had liUle money, he s=ll received a substan=al educa=on during his childhood

•  In 1843, Mendel was admiUed to the Augus=nian Monastary in Brno, where he was trained as a priest

•  Mendel later went on the become a teacher and scholar

•  Later he went on to further his educa=on at the University of Vienna from 1851-‐1853, where he took courses in Math, Chemistry, Paleontology, Botany and Plant Physiology


•  Most scien=sts during the middle-‐late 1800s sought to follow human traits as they thought (without evidence) that each organism inherited traits in much different manners than other organism

•  The other scien=sts of that =me who did follow human traits followed those that generally are inherited in a more complex manner –  Traits may involve many genes –  These genes may have strong interac=ons with

environmental factors

•  Mendel took a different approach because he decided he could not use people as a system for studying inheritance, he instead bred pea plants

•  Mendel was easily able to isolate different strains of pea plant with very dis=nct characteris=cs –  Seed shape –  Seed color –  Pod shape –  Stem length

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•  Through his work with pea plants he was able to determine how each of these individual traits were inherited from parent to offspring

•  Each trait was controlled by a pair of factors

•  From Mendel’s study of the different traits, and their mul=ple factors, came his law of Independent Segrega=on (Mendel’s second law) –  Each trait is determined by different factors –  Each organism must inherit two factors for

each trait (one from each parent) –  Each parent then must segregate his/her two

factors into separate gametes

•  In Mendel’s experiments, he no=ced that certain factors are dominant to others, which led to postulate a Concept of Dominance

•  Today, we find that each trait is determined by a gene and that each gene can exist in mul=ple forms (factors) called alleles


•  Mendel extended his breeding experiments such that he could follow more than one trait at a =me

•  From the results of these experiments he postulated the law of independent assortment (Mendel’s first law),

•  The first law states that for each trait, the factors will assort independently from one another during gamete forma=on –  Each gamete will have one factor for each

trait –  The presence of a specific factor for one gene

will have no influence on which factor will be present for another gene

•  Upon union of two gametes, each trait will again be represented by two factors


•  Mendel performed his work generally outside the scien=fic community, and thus, his work although published was not exactly viewed favorably (36 years)

•  His work sat idle un=l 1900 when Hugo DeVries, Karl Correns and Erich Von Tschermak independently recreated his work

•  Although Mendel was able to determine how different traits were inherited he had no idea how traits were encoded –  Had no idea that traits were determined by genes –  Had no idea what genes were composed of

•  During the early 1900’s, many other scien=sts determined the mechanisms of inheritance for a large number of traits in a variety of different organisms

•  In the early 1900’s Thomas Hunt Morgan postulated sex-‐linkage for which he won a Nobel Prize

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Historical Perspec=ves on Heredity: The Chromosomal Theory of Heredity •  One of the main difficul=es in the

acceptance of Mendel’s work is the lack of actual physical evidence

•  August Weisman (1893) was one of the first scien=sts to link behavior of chromosomes to heredity

•  When studied segrega=on of chromosomes, he no=ced that the number of chromosomes in the nuclei of germ cells is halved. Therefore, he postulated that the material of heredity (gene=c informa=on) is located in the nucleus

•  From this, Weisman postulated that The Germ-‐plasm theory –  States that cells in the reproduc=ve organs

carry a complete set of gene=c informa=on, and that this informa=on is passed along to the egg and sperm

–  Perhaps this informa=on is present in the chromosomes

Historical Perspec=ves on Heredity: The Chromosomal Theory of Heredity •  In 1903 Walter SuUon published his paper “The

Chromosomes in Heredity –  Paper focused on the principles in Meiosis –  SuUon saw that there appeared to be two copies for

each chromosome –  During meiosis, each gamete receives only one

member of of the chromosome pair, which appropriately follows Mendel’s law of independent assortment

•  SuUon’s Conclusions –  Chromosomal movement explains Mendel’s second

law (independent segrega=on) –  Proposal of the Chromosomal Theory of Heredity

•  SuUon assumed that genes are part of the chromosome –  Assumed that the seed color gene is found on one

pair of chromosomes –  Explains the 3:1 ra=o when crossing heterozygotes –  He also assumed that seed shape genes were found

on another pair of chromosomes from the seed color genes, which allow for the observed 9:3:3:1 ra=o

•  SuUon’s results were important for two reasons –  Most importantly suggested (but did not prove)

physical evidence for Mendel’s rules regarding segrega=on

–  Linked the study of Gene=cs to the study of cytology and would drive the development of the field of Molecular Biology

Historical Perspec=ves: The Chromosomal Theory of Heredity

•  With the work of SuUon and the Chromosomal Theory of Heredity two ques=ons remained –  What were the chromosomes

composed of? –  What material carried the

informa=on of heredity?

•  Given that the chromosomes appeared to segregate according to Mendel’s laws of independent segrega=on, then the material(s) that compose chromosomes must also be responsible for heredity

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The Beginnings of Molecular Biology: Friedrich Meischer’s Contribu=ons To Determining Which Molecule Holds

Gene=c Informa=on •  At about the same =me that Mendel

was working on his pea plants, Friedrich Meischer (1868) was embarking on studying the chemical makeup of cells

•  Meicher theorized that to determine the material of heredity one must understand the chemical nature of cells

•  Meicher, in order to determine the material of heredity studied the chemistry of pus

•  Pus includes bacteria, which cause an infec=on, as well as many white blood cells, which are called on to fight the infec=on

The Beginnings of Molecular Biology: Friedrich Meischer’s Contribu=ons To Determining Which Molecule Holds

Gene=c Informa=on •  Meicher took the white blood

cells and isolated their nuclei to study what was inside

•  Upon analysis, he expected to find protein inside the nucleus, however, he found the ra=os of carbon and nitrogen to be inconsistent the presence of protein

•  As well, he found that the material was slightly acidic and importantly was high in phosphorus-‐he called this material nuclein

•  With further analysis of nuclein, he found that the three main components of nuclein were phosphate, sugar and a nitrogen containing base

The Beginnings of Molecular Biology: The Controversy Between DNA and Protein Carrying the Informa=on of

Heredity

•  In the early 20th Century the controversy raged which molecule contained the informa=on of heredity –  Nucleic Acid (DNA) –  Protein

•  Due to the chemical nature of each molecule, it was thought that proteins contained the informa=on of heredity –  Proteins are composed of a possible 20 different amino acids –  Each amino acid has its own chemical proper=es –  Within a cell there could be many different varia=ons of protein

•  DNA was thought to be much less complex than protein and thus could not be the material of heredity –  Composed of only four different nitrogenous bases –  Only a few structural varia=ons

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The Beginnings of Molecular Biology: Fredrick Griffith’s Contribu=ons To Molecular Biology

•  In Griffith’s experiments he used two different strains of S. pneumoniae –  Type IIIR –  Type IIIS

•  Griffith first injected the non-‐pathogenic Type IIIR strain and found that the mice survived

•  Next, Griffth injected the pathogenic Type IIIS strain into the mice and they died

•  Griffith treated Type S bacteria with heat, thus killing them

•  When he injected the heat killed type S bacteria, he found that the mice remained healthy

The Beginnings of Molecular Biology: Fredrick Griffith’s Contribu=ons To Molecular Biology

•  The last experiment he did was he mixed the heat killed type S bacteria with the live type R bacteria

•  He then injected this mixture, and found that the mice became sick and died

•  He concluded that there was a transfer of some component from the dead pathogenic (Type S) bacteria to the live non-‐pathogenic Type R bacteria to make it become pathogenic

•  He called this component the transforming principle, that when transmiUed from the dead S to the live R bacteria allowed them to become pathogenic

•  Griffith was not able to determine the true chemical nature of the transforming principle

The Beginnings of Molecular Biology: In vitro Experiments Based on Griffith’s work

•  In 1931, Henry Dawson showed that the mouse was not needed for transforma=on –  He heat killed the pathogenic type S bacteria and then mixed it with the the live type R

bacteria –  Instead of injec=ng the mixture into mice, he plated the mixture on agar plates –  He found some type R colonies and some type S colonies on his plates

•  In 1933, Lionel Alloway showed that a cell-‐free extract prepared from broken type S bacteria could also be used for transforma=on of live type R cells to type S cells

•  In 1941 Oswald Avery, Colin MacLeod and Maclyn McCarty took Griffith’s experiment further to determine the true chemical nature of the transforming principle –  Took mixtures and incubated them with different degrada=ve enzymes –  DNase –  RNase –  Protease

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The Beginnings of Molecular Biology: In vitro Experiments Based on Griffith’s work

Historical Perspec=ves: The Hershey-‐Chase Experiment

•  Even with the results of Avery, MacLeod and McCarty, the controversy about whether DNA or protein contained the informa=on of heredity, was s=ll raging

•  Hershey and Chase performed what is now recognized as the sen=nel experiment, which put the controversy to rest

•  In order to determine whether protein or DNA was being inserted into the host cell, Hershey and Chase needed to find a way to label each type of molecule

•  Hershey and Chase used the T2 bacteriophage, in their experiments

•  T2 bacteriophage is a virus that infect E. coli. Viruses are unable to reproduce on their own, they need to reproduce use a host cell

•  At the =me, it was known that a virus had an outer protein coat, and inside this protein coat was DNA

•  When a T2 bacteriophage infects and E. coli, it aUaches to the outside, and then injects its gene=c material into the E. coli cell. Once injected, the cell uses this gene=c material to make new virus

•  What Hershey and Chase wanted to do was to figure out what got inserted into the host cell because that must be the gene=c material

The Beginnings of Molecular Biology: The Hershey-‐Chase Experiment

•  They knew protein contained sulfur, whereas DNA did not and DNA contained phosphorus whereas proteins did not

•  They labeled proteins with a radioac=ve form of sulfur (35S)

•  They labeled DNA with a radioac=ve form of phosphorus (32P)

•  Next they created Bacteriophage that had either their DNA labeled with 32P or their protein labeled with 35S

•  They then took their phage that either contained radioac=ve protein or radioac=ve DNA and infected E. coli with them

•  Upon infec=on, the viruses would bind to the outside of the E. coli cell and insert their gene=c material

•  Next, they took their mixture containing infected E. coli and used a blender to lightly shear off whatever was leb that was bound to the outside of the cell

•  They then centrifuged their sample to pellet the bacteria. This leaves any part of the phage that was not inserted into the cell leb in solu=on (supernatant)

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The Beginnings of Molecular Biology: The Hershey-‐Chase Experiment

•  When they looked at sample in which the phage contained radioac=ve protein (35S), they found that the radioac=vity was found in the supernatant and not in the bacterial pellet

•  This suggests that protein is not inserted into the host cell, and thus protein would not be the gene=c material

•  In contrast, when they looked at the sample in which the phage contained radioac=ve DNA (32P), they found that the radioac=vity was found in the bacterial pellet and not in the supernatant

•  This suggests that DNA is being inserted into the host cell, and thus, DNA would be the gene=c material

The Beginnings of Molecular Biology: A Model For the Structure of DNA

•  Previously, it had been shown that DNA is composed of three different components –  Sugar –  Phosphate –  Nitrogenous bases

•  It was known that there were four nitrogenous bases –  Adenine –  Thymine –  Cytosine –  Guanine

•  Quan=ta=ve methods by Erwin Chargaff had shown that the the number of [A] = [T] and the amount of [G] =[C] (However, [G+C] does not equal [A+T]

•  Based off of this work, and by X-‐ray diffrac=on analysis on DNA by Maurice Wilkins and Rosalind Franklin, James Watson and Francis Crick were able to determine the 3-‐D structure of DNA –  Found that the shape of DNA is in the form of a helix

of constant diameter –  Found that the nitrogenous bases were stacked

towards the interior of the molecule, with the backbone containing sugar (deoxyribose) and phosphate

–  They were able to determine the distance between the stacked bases

The Beginnings of Molecular Biology: A Model For the Structure of DNA

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The Gene Is The Basic Unit of Heredity: Introduc=on

•  As Mendel worked with his pea plants he had no concept of what a gene was

•  Instead he was only following hereditary characteris=cs –  Seed shape –  Seed color –  Plant size

•  In 1889 Hugo de Vries tried to explain Mendel’s factors in his book “Intracellular Pangenesis”

•  De Vries stated that the pangen is “smallest par=cle represen=ng one hereditary characteris=c”

•  About 20 years late Wilhelm Johannsen coined the term gene by shortening the word pangen

•  Be aware neither scien=st understood physically what a gene was, they only knew that it encoded a specific hereditary characteris=c

The Gene Is The Basic Unit of Heredity: The Molecular Iden=ty of a Gene

•  Over =me, it was determined that the genes were located on chromosomes, which were composed of primarily DNA and associated proteins

•  As early as 1910, Thomas Hunt Morgan and his research group at Columbia University started mapping the exact posi=ons of each discovered gene within the genome –  Morgan and his group worked with Drosophila

melanogaster –  Produced mutant flies with different characteris=cs

(mutant strains)

•  Morgan and his group were able to map their posi=ons by using a series of gene=c crosses using his different mutant strains –  Mapped each new gene with respect to known genes –  Were able to map each gene by determining which

genes were linked (on the same chromosome)

•  By 1913 Alfred Sturtyvant, student in Morgans’s lab, produced the first ever physical map loca=ng each known gene of an organism’s genome (Drosophila)

•  At this point in =me, a gene was being beUer defined as a unit that encodes a specific inherited trait

The Gene Is The Basic Unit of Heredity: Determining What A Gene Encodes on a Molecular Level

•  As the field of molecular biology started to develop, researchers wanted to develop a beUer molecular defini=on of a gene –  What the structure of a gene looks like –  More importantly, what a gene actually

encodes

•  In 1941, George Beadle and Edward L. Tatum were the first to demonstrate the link between a gene and a step in a metabolic pathway which is catalyzed by an enzyme

•  Beadle and Tatum worked backwards using specific mutants of the pink bread mold, Neurospora crassa in which specific chemical reac=ons were blocked

•  Beadle and Tatum followed the biochemical pathway for niacin biosynthesis –  Considered a water soluble vitamin –  Niacin is a precursor to NADPH

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•  Beadle and Tatum knew that niacin could be produced star=ng from the amino acid tryptophan

•  Niacin produc=on from tryptophan is not just a one step process, in that there are several intermediates involved –  Kynurenine –  3-‐hydroxyanthranilic acid

•  Beadle and Tatum understood that produc=on of Niacin was a three step process, which involves three different enzymes –  Each enzyme catalyzes a specific step –  Hypothesized that each enzyme was encoded by a

different gene

•  To test this, they induced muta=ons into the Neurospora by using X-‐irradia=on and then plated them on minimal medium, or supplemented media

•  As long as a metabolic step is not affected by the X-‐irradia=on, the Neurospora should grow on the minimal media

•  However, failure to grow on the media indicates that a muta=on has occurred leading to a growth defect


•  Beadle and Tatum observed a one-‐to-‐one correspondence between the gene=c muta=ons and the lack of a specific enzyme required in a biochemical pathway

•  From their work arose the “One Gene – one enzyme hypothesis”


•  Eventually, the one gene-‐one enzyme hypothesis was ameneded based on several other discoveries in addi=on to that of Beadle and Tatum –  The discovery that DNA holds the gene=c informa=on by Avery, Macleod and McCarty as

well as Hershey and Chase –  The discovery of the DNA double helix by Watson and Crick

•  Watson and Crick further proposed that a gene encodes a protein –  Not all genes encoding proteins encode enzymes –  Some genes encode structural proteins, signaling proteins as well as others

•  This hypothesis has been amended further, because there are some genes that do not even encode polypep=des, they encode RNAs

•  Today through the work of many Molecular Biologists the actual structure of the gene has been determined

•  In 1972, Walter Fiers and his colleagues were the first to sequence an en=re gene

Chapter 1 Lecture - Winonacourse1.winona.edu/ssegal/Molecular Course...8/22/11 2 Introduc=on:$Molecular$Biology$PreO History • If$Molecular$Biology$studies$how$heredity$...

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