Chapter 1: The Origins of Molecular Biology: A Mendelian and Darwinian View of the World

I. Introduction

A. Introduction: The Big Question

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

2. A: The simplest answer is that the field of Molecular Biology came into being as a way to explain mechanistically how heredity works!

a. For example, you may want to know how eye color is inheritedb. You may want to know how, on a molecular level, eye color forms (How the pigment is actually produced)

B. Introduction: Molecular Biology Pre-History

1. If Molecular Biology studies how heredity works, and how traits are expressed then the field of Molecular Biology must have its roots in Genetics

2. How old is the field of genetics? How long have humans been studying heredity?

3. How old is the field of genetics? How long have humans been studying heredity?

4. About 10,000-12,000 years ago humans began to manipulate animals and plants, to domesticate them

a. Plants include wheat, barley, lentils, peasb. Animals include dogs, sheep and goats

5. Humans were able to quickly understand the concept of heredity (create breeds that were better suited to agricultural production by mating individual organisms with desirable traits)

6. In terms of crops, humans have selected for varieties with significantly better viability

a. Crop variants that produce more fruit/vegetableb. Crops that are more resistant to pests

7. Over time, molecular biologists were able to determine which genes allow for better viability

8. With the use of Molecular Biology, we can now genetically modify crops by inserting advantageous genes one organism may have into specific crop plants

a. Isolate the DNA encoding our gene b. Insert that into the plant using a “gene gun”

9. Besides domestication, understanding how heredity works also is extremely important for public health

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

11. Let’s take the example of the Romanov’s

a. Tsar Nicholas Romanov II was the Czar of Russia from b. The family included his wife Alexandria as well as four daughters

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

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

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

15. At the time of the Romanov’s, it was known that the disorder ran in within families

a. It was unknown what the mechanism of inheritance of the disease wasb. It was unknown what genes were implicated

16. Today, through Molecular Biology, we know that hemophiliacs contain a defective variant of the Clotting Factor VIII gene on the X chromosome

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

18. Today, using molecular biology in vitro, we can produce the Clotting Factor VIII protein, which can be given to patients

C. Introduction: The General Definition of Molecular Biology

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

2. Warren Weaver was a civil engineer and mathematician by trade

3. Weaver was a great advocate for science and was responsible for supporting grants for genetics and molecular biology

4. He defined molecular biology as the study of biological phenomena at the molecular level

a. This definition covers a wide range of phenomenab. This definition is inaccurate in that it does not explain

5. Today, the modern study of Molecular Biology focuses on the molecular basis of gene expression

6. There are other processes that also fall under molecular biology that are not considered part of the process of gene expression

a. DNA replicationb. Membrane biosynthesisc. Cellular respiration

7. Specifically, if one wants to study gene expression mechanistically 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 function of a gene

a. Structure of DNA and the hereditary information it encodesb. Structure and function of RNAc. Structure and function proteinsd. Mechanisms of DNA replication before cell division

D. Introduction: The Beginnings of Molecular Biology

1. Molecular Biology as a way to explain how genetics and evolution work

a. Answers important biological questions of who we areb. Answers important biology questions of where we come from

2. At the most fundamental level, Genetics is the study of heredity

3. Heredity can be defined as the study of the passage of traits from parent to offspring

a. Eye colorb. Hair colorc. Body patternd. Disease or Disease predisposition

4. Evolution can be defined as the development of more complex organisms from less complex organisms

a. Development of antibiotic resistance in bacteriab. Humans developing from lesser primates apes

E. Introduction: A History Leading To the Development of Molecular Biology

1. Before Molecular Biology, the fields of genetics and evolution were very abstract fields

2. Abstract Cconcepts of Genetics

a. Over time geneticists became easily able to follow, and the predict how certain traits where inheritedb. However, they did not know how these traits were inherited in the way they predicted and why they did not know why their predictions were never quite perfect

3. Abstract Concepts of Evolution

a. They were able to develop extensive fossil/anatomical records and make important comparisons between known living or extinct species

b. They were able to make important predictions about transitional speciesc. As with the geneticists, they also had no idea why some species were so similar and others so different

4. In each case, Molecular Biology was able to provide the mechanistic basis of how the processes of heredity and evolution work

II. Historical Perspectives on Heredity

A. Historical Perspectives on Heredity: An Introduction

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

2. The study of heredity asks one of the fundamental questions of life, how do we have the traits we have?

3. The study of heredity has captured the imagination of many scientists through out history

4. The study of heredity started long ago in ancient Greece with Aristotle (384-322 BC)

a. Aristotle proposed the theory of pangenesisb. Pangenesis: Hereditary characteristics are transmitted by gemmules from individual body cells

5. Robert Hooke (1635-1703) developed the first microscope in 1665 and allowed humans to see cells for the first time

6. The use of microscopes allowed for the visualization of sperm and eggs

7. Performationism: Inside the sperm or egg exists a miniature adult (a homunculus) which enlarges during development

8. Note: performationism meant that all traits would be inherited from one parent

B. Historical Perspectives on Heredity: The Age of Mendel

1. It was not until the 1860s that mechanisms of heredity started to become truly understood

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

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

4. Although his family had little money, he still received a substantial education during his childhood

5. In 1843, Mendel was admitted to the Augustinian Monastery in Brno, where he was trained as a priest

6. Mendel later went on the become a teacher and scholar

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

8. Most scientists 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

9. The other scientists of that time who did follow human traits followed those that generally are inherited in a more complex manner

a. Traits may involve many genes b. These genes may have strong interactions with environmental factors

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

11. Mendel was easily able to isolate different strains of pea plant with very distinct characteristics

a. Seed shapeb. Seed colorc. Pod shaped. Stem length

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

13. Each trait was controlled by a pair of factors

14. From Mendel’s study of the different traits, and their multiple factors, came his law of Independent Segregation (Mendel’s second law)

a. Each trait is determined by different factorsb. Each organism must inherit two factors for each trait (one from each parent)c. Each parent then must segregate his/her two factors into separate gametes

15. In Mendel’s experiments, he noticed that certain factors are dominant to others, which led to postulate a Concept of Dominance

16. Today, we find that each trait is determined by a gene and that each gene can exist in multiple forms (factors) called alleles

17. Mendel extended his breeding experiments such that he could follow more than one trait at a time

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

19. The first law states that for each trait, the factors will assort independently from one another during gamete formation

a. Each gamete will have one factor for each traitb. The presence of a specific factor for one gene will have no influence on which factor will be present for another gene

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

21. Mendel performed his work generally outside the scientific community, and thus, his work although published was not exactly viewed favorably (36 years)

22. His work sat idle until 1900 when Hugo DeVries, Karl Correns and Erich Von Tschermak independently recreated his work

23. Although Mendel was able to determine how different traits were inherited he had no idea how traits were encoded

a. Had no idea that traits were determined by genesb. Had no idea what genes were composed of

24. During the early 1900’s, many other scientists determined the mechanisms of inheritance for a large number of traits in a variety of different organisms

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

C. Historical Perspectives on Heredity: The Chromosomal Theory of Heredity

1. One of the main difficulties in the acceptance of Mendel’s work is the lack of actual physical evidence

2. August Weisman (1893) was one of the first scientists to link behavior of chromosomes to heredity

3. When studied segregation of chromosomes, he noticed that the number of chromosomes in the nuclei of germ cells is halved. Therefore, he postulated that the material of heredity (genetic information) is located in the nucleus

4. From this, Weisman postulated that The Germ-plasm theory

a. States that cells in the reproductive organs carry a complete set of genetic information, and that this information is passed along to the egg and sperm

b. Perhaps this information is present in the chromosomes

5. In 1903 Walter Sutton published his paper “The Chromosomes in Heredity

a. Paper focused on the principles in Meiosisb. Sutton saw that there appeared to be two copies for each chromosomec. During meiosis, each gamete receives only one member of the chromosome pair, which appropriately follows Mendel’s law of independent assortment

6. Sutton’s Conclusions

a. Chromosomal movement explains Mendel’s second law (independent segregation)b. Proposal of the Chromosomal Theory of Heredity

7. Sutton assumed that genes are part of the chromosome

a. Assumed that the seed color gene is found on one pair of chromosomesb. Explains the 3:1 ratio when crossing heterozygotesc. 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 ratio

8. Sutton’s results were important for two reasons

a. Most importantly suggested (but did not prove) physical evidence for Mendel’s rules regarding segregationb. Linked the study of Genetics to the study of cytology and would drive the development of the field of Molecular Biology

9. With the work of Sutton and the Chromosomal Theory of Heredity two questions remained

a. What were the chromosomes composed of?b. What material carried the information of heredity?

10. Given that the chromosomes appeared to segregate according to Mendel’s laws of independent segregation, then the material(s) that compose chromosomes must also be responsible for heredity

III. The Beginnings of Molecular Biology

A. The Beginnings of Molecular Biology: Friedrich Meischer’s Contributions To Determining Which Molecule Holds Genetic Information

1. At about the same time that Mendel was working on his pea plants, Friedrich Meischer (1868) was embarking on studying the chemical makeup of cells

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

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

4. Pus includes bacteria, which cause an infection, as well as many white blood cells, which are called on to fight the infection

5. Meicher took the white blood cells and isolated their nuclei to study what was inside

6. Upon analysis, he expected to find protein inside the nucleus, however, he found the ratios of carbon and nitrogen to be inconsistent the presence of protein

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

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

B. The Beginnings of Molecular Biology: The Controversy Between DNA and Protein Carrying the Information of Heredity

1. In the early 20th Century the controversy raged which molecule contained the information of heredity

a. Nucleic Acid (DNA)b. Protein

2. Due to the chemical nature of each molecule, it was thought that proteins contained the information of heredity

a. Proteins are composed of a possible 20 different amino acidsb. Each amino acid has its own chemical propertiesc. Within a cell there could be many different variations of protein

3. DNA was thought to be much less complex than protein and thus could not be the material of heredity

a. Composed of only four different nitrogenous basesb. Only a few structural variations

C. The Beginnings of Molecular Biology: Fredrick Griffith’s Contributions To Molecular Biology

1. Next Griffith treated Type S bacteria with heat, thus killing them

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

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

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

5. 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

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

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

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

1. In 1931, Henry Dawson showed that the mouse was not needed for transformation

a. He heat killed the pathogenic type S bacteria and then mixed it with the live type R bacteriab. Instead of injecting the mixture into mice, he plated the mixture on agar platesc. He found some type R colonies and some type S colonies on his plates

2. In 1933, Lionel Alloway showed that a cell-free extract prepared from broken type S bacteria could also be used for transformation of live type R cells to type S cells

3. In 1941 Oswald Avery, Colin MacLeod and Maclyn McCarty took Griffith’s experiment further to determine the true chemical nature of the transforming principle

4. Purified protein, DNA, and RNA from type S bacteria

a. They then treated live type R cells with either the DNA, RNA or the proteinb. They found that the DNA led to transformation of the live type R to type S bacteria, whereas the other two did not

5. The Beginnings of Molecular Biology: In vitro Experiments Based on Griffith’s work (Supplemental Figure)

D. The Beginnings of Molecular Biology: The Hershey-Chase Experiment

1. Even with the results of Avery, MacLeod and McCarty, the controversy about whether DNA or protein contained the information of heredity, was still raging

2. Hershey and Chase performed what is now recognized as the sentinel experiment, which put the controversy to rest

3. 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

4. Hershey and Chase used the T2 bacteriophage, in their experiments

5. 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

6. At the time, it was known that a virus had an outer protein coat, and inside this protein coat was DNA

7. When a T2 bacteriophage infects and E. coli, it attaches to the outside, and then injects its genetic material into the E. coli cell. Once injected, the cell uses this genetic material to make new virus

8. What Hershey and Chase wanted to do was to figure out what got inserted into the host cell because that must be the genetic material

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

10. They labeled proteins with a radioactive form of sulfur (S35)

11. They labeled DNA with a radioactive form of phosphorus (P32)

12. Next they created Bacteriophage that had either their DNA labeled with P32 or their protein labeled with S35

13. They then took their phage that either contained radioactive protein or radioactive DNA and infected E. coli with them

14. Upon infection, the viruses would bind to the outside of the E. coli cell and insert their genetic material

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

16. They then centrifuged their sample to pellet the bacteria. This leaves any part of the phage that was not inserted into the cell left in solution (supernatant)

17. When they looked at sample in which the phage contained radioactive protein (35S), they found that the radioactivity was found in the supernatant and not in the bacterial pellet

18. This suggests that protein is not inserted into the host cell, and thus protein would not be the genetic material

19. In contrast, when they looked at the sample in which the phage contained radioactive DNA (32P), they found that the radioactivity was found in the bacterial pellet and not in the supernatant

20. This suggests that DNA is being inserted into the host cell, and thus, DNA would be the genetic material

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

1. Previously, it had been shown that DNA is composed of three different components

a. Sugarb. Phosphatec. Nitrogenous bases

2. It was known that there were four nitrogenous bases

a. Adenineb. Thyminec. Cytosined. Guanine

3. Quantitative methods by Erwin Chargaff had shown that the number of [A] = [T] and the amount of [G] =[C] (However, [G+C] does not equal [A+T]

4. Based off of this work, and by X-ray diffraction analysis on DNA by Maurice Wilkins and Rosalind Franklin, James Watson and Francis Crick were able to determine the 3-D structure of DNA

a. Found that the shape of DNA is in the form of a helix of constant diameterb. Found that the nitrogenous bases were stacked towards the interior of the molecule, with the backbone containing sugar (deoxyribose) and phosphatec. They were able to determine the distance between the stacked bases

5. The Beginnings of Molecular Biology: A Model For the Structure of DNA (Supplemental Figure)

IV. The Gene Is The Basic Unit of Heredity

A. The Gene Is The Basic Unit of Heredity: Introduction

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

2. Instead he was only following hereditary characteristics

a. Seed shapeb. Seed colorc. Plant size

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

4. De Vries stated that the pangen is “smallest particle representing one hereditary characteristic”

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

6. Be aware neither scientist understood physically what a gene was, they only knew that it encoded a specific hereditary characteristic

B. The Gene Is The Basic Unit of Heredity: The Molecular Identity of a Gene

1. Over time, it was determined that the genes were located on chromosomes, which were composed of primarily DNA and associated proteins

2. As early as 1910, Thomas Hunt Morgan and his research group at Columbia University started mapping the exact positions of each discovered gene within the genome

a. Morgan and his group worked with Drosophila melanogasterb. Produced mutant flies with different characteristics (mutant strains)

3. Morgan and his group were able to map their positions by using a series of genetic crosses using his different mutant strains

a. Mapped each new gene with respect to known genesb. Were able to map each gene by determining which genes were linked (on the same chromosome)

4. By 1913 Alfred Sturtyvant, student in Morgans’s lab, produced the first ever physical map locating each known gene of an organism’s genome (Drosophila)

5. At this point in time, a gene was being better defined as a unit that encodes a specific inherited trait

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

1. As the field of molecular biology started to develop, researchers wanted to develop a better molecular definition of a gene

a. What the structure of a gene looks likeb. More importantly, what a gene actually encodes

2. 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

3. Beadle and Tatum worked backwards using specific mutants of the pink bread mold, Neurospora crassa in which specific chemical reactions were blocked

4. Beadle and Tatum followed the biochemical pathway for niacin biosynthesis

a. Considered a water soluble vitaminb. Niacin is a precursor to NADPH

5. They induced mutations into the Neurospora by using X-irradiation and then plated them on minimal medium

6. As long as a metabolic step is not affected by the X-irradiation, the Neurospora should grow on the minimal media

7. However, failure to grow on the media indicates that a mutation has occurred leading to a growth defect

8. These mutants need media with specific supplemented compounds (usually amino acids) to grow-these mutant strains of Neurospora are known as auxotrophs

9. In the Beadle and Tatum experiment, they studied the pathway of niacin synthesis (See Figure 1.5)

10. One can determine the specific nature of the auxotroph by placing the auxotrophic strain supplemented with various intermediates in the pathway of niacin synthesis (Figure out which enzyme within the pathway has been affected)

11. Beadle and Tatum observed a one-to-one correspondence between the genetic mutations and the lack of a specific enzyme required in a biochemical pathway, and they dubbed this the “One Gene – one enzyme hypothesis”

D. The Gene Is The Basic Unit of Heredity: Determining What A Gene Encodes on a Molecular Level (Modern)

1. Eventually, the one gene-one enzyme hypothesis was amended based on several other discoveries in addition to that of Beadle and Tatum

a. The discovery that DNA holds the genetic information by Avery, Macleod and McCarty as well as Hershey and Chase b. The discovery of the DNA double helix by Watson and Crick

2. Watson and Crick further proposed that a gene encodes a protein

a. Not all genes encoding proteins encode enzymesb. Some genes encode structural proteins, signaling proteins as well as others

3. This hypothesis has been amended further, because there are some genes that do not even encode polypeptides, they encode RNAs

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

5. In 1972, Walter Fiers and his colleagues were the first to sequence an entire gene