Lecture 2 HORT250 F97

HORT 250 - Biotechnology in Agriculture

Lecture 2 - DNA as the hereditary material

The identification and selection of organisms for specific purposes and applications is central to agriculture. The production of food and fibre by cultivating plants and raising animals has depended on such selection since the origins of agriculture, some 10,000 years ago. Although the process I outline below may not have been articulated by our ancestors, this is the scheme they followed to develop plants and animals that are used by humans:

Identify a specific problem or need.
Identify an organism that can address this problem.
Select varieties of that species that are better suited for this task.

This model would apply to the domestication and improvement of both crop plants and farm animals. It also applies to modern problems such as the development of microbial strains for antibiotic production. The implicit assumption in all of these examples is that the individual plants in the wheat population, or cows in the herd, or strains of the Penicillium fungus, are not identical. There must be variation within the population, the same sort of variation that is the basis for natural selection and evolution. This variation is also called genetic diversity.

Plant and animal breeders have been using this variation and genetic diversity within species to select crop plants and livestock with improved performance. Breeders can generate varieties with new combinations of genes by making specific crosses between individuals with desirable characteristics. Screening the offspring of such crosses results in identification of new, improved, elite lines. (see diagram of basic crossing and selection scheme) This approach utilises the available genetic diversity within a species. And this approach has been spectacularly successful in many ways. As an example of how successful breeding and selection has been, let's look at average corn yields in the US over last 120 years.

from 1870 to 1935, corn yield in the US averaged a little less than 30 bushels per acre, with little evidence for any sustained yield increase during that 65 year period.
from 1935 to 1960, yields increased from 35 to 60 bushels per acre. This coincided with the introduction of hybrid corn.
from 1970 until 1996, yields increased from 60 to more than 120 bushels per acre, an average increase of almost 2 bushels per acre per year.

The graph below illustrates the increased yield for corn in Indiana over the last 70 years.

While we cannot say that all of this increase in yield is the result of breeding and selection, it is the biggest factor contributing to this phenomenal accomplishment. Other factors that have played a role include improvements in weed control, fertilizers, efficient planting and harvesting. In fact, if we still had the same yields as in 1900, we would have to increase the acreage planted to corn by more than 300 million acres to produce the same amount of corn that we now harvest each year.

The fact that we can feed our current world population, and have some surplus, is testament to the power of breeding and selection, as well as other aspects of agricultural production. But there are limitations to this methodology and those limitations include:

the size of the gene pool, the available genetic diversity
the ability to create new genetic combinations by standard sexual breeding methods.

For most cultivated species there are germplasm collections that contain both domesticated and wild samples of the species which have been collected over many years from many locations. These are collections of genes and unique combinations of genes that cannot be recreated by other means and are an invaluable resource. When a breeder is faced with a new problem, such as how to breed plants with resistance to a new pathogen, he or she surveys the germplasm collection to identify individuals within the collection that are resistant to that pathogen. Using breeding and selection, this trait - resistance to a pathogen - can be incorporated into elite varieties that are otherwise excellent but are susceptible to the pathogen.

What can be done if no varieties in the collection can be found with the desired disease resistance or other specific trait? In many cases, plant breeders can expand the genetic boundaries of their crop by looking at related species, usually within the same genus. For tomato (Lycopersicon esculentum), genes for resistance to many pathogens have been identified in related species (L. hirsutum and others). Many Lycopersicon species form fertile interspecific hybrids. Therefore, wide crosses can be made and genetic traits transferred between species, although success with this approach is far from routine. Another approach is to generate new genetic variation by mutagenesis - deliberately exposing the organism to chemicals or radiation that will cause genetic alterations (a popular theme of many sci-fi movies and TV shows from the 1950's and 60's). The vast majority of these alterations will be deleterious resulting in plants or animals with undesirable traits. However, a small fraction may have desirable properties, and these can be selected. Unfortunately, this strategy, known as mutation breeding, has resulted in relatively few advances in plant improvement but has been quite successful and widely used with microbes.

If the genetic information for a particular trait is not present in either the narrow gene pool (within the species) or the wider gene pool (in closely related species that can be hybridised), and cannot be generated by mutagenesis, what can be done to improve or modify the organism? The revolutionary aspect of biotechnology is the ability to produce organisms that possess genes from non-traditional sources. These are genes that a plant or animal breeder could not incorporate into a maize plant or a pig by traditional breeding; or genes that a microbiologist could not select in the laboratory on a petri dish.

The ability to isolate, manipulate, and transfer genes between species expands the gene pool that is available and can be utilised in the genetic modification and improvement of plants, animals and microbes. I will spend most of the class looking at the methods that are used to produce genetically modified organisms, and the applications of this technology in agriculture.

Before we can manipulate genes, we must first have an understanding of what genes are made of (DNA) and how genes are encoded in DNA.

Milestones in the discovery of DNA

1865

Mendel publishes the results of his expts. on the inheritance of traits in peas. These experiments were largely ignored by the rest of the scientific community. (Why?)

1869

Meischer reports that DNA is a major component of the nucleus. But the importance of the nucleus to inheritance was not established, and the nucleus is composed of more than just DNA. The Cold Spring Harbor Labs web site has an excellent review of this topic (click on the animation button at the bottom).

1900s

Mendel's results are "rediscovered" simultaneously by others and the science of genetics begins. Laws of heredity are confirmed in plants, and in 1902 are also demonstrated to apply to animals.

1910

Genes are shown to reside on chromosomes. First shown for sex-linked genes, then extended to all genes. Within a species, chromosomes are constant in number. But chromosomes are made of both DNA & protein - which of these is responsible for carrying information from generation to generation?

1928

Griffith demonstrates the "transformation" of non-pathogenic bacteria to pathogenic. He performs 4 expts. with a bacterial pathogen (pneumococcus) that can infect and kill mice.

Expt. 1. Inject mice with NON-VIRULENT bacteria

mice survive, no bacteria recovered from mice

Expt. 2. Inject mice with VIRULENT bacteria

mice die and virulent bacteria are recovered from the mice

Expt. 3. Heat-kill the VIRULENT bacteria, then inject these into mice

mice survive, no bacteria recovered; the bacteria need to be living to cause disease

Expt. 4. Mix heat killed VIRULENT bacteria (#3) with NON-VIRULENT bacteria (#1) and inject this mixture into mice. Note that neither of these agents caused disease on their own.

mice die, and VIRULENT bacteria are recovered from the dead mice

We can conclude that the non-virulent bacteria have been transformed by their interaction with the dead bacteria. Griffith referred to this agent that changes the properties of the bacterium from NON-VIRULENT to VIRULENT as the transforming principle. But what is the nature of the transforming principle?

1944

Avery, MacLeod & McCarty separated the various biochemical components of virulent bacteria into: lipids, polysaccharides, proteins, and nucleic acids (RNA and DNA) and asked which component could transform the non-virulent bacteria into virulent, disease causing bacteria. Only the nucleic acid component could transform the bacteria. This contains both RNA & DNA.

They then used enzymes to break down either the RNA or DNA in this mixture of nucleic acids and showed that only DNA was able to transform the bacteria from non-virulent to virulent.

This term "transformation" is now widely used in biotechnology to describe the process of transferring DNA into an organism. For example, we will talk about transformation of bacteria, plants or animals and it will specifically mean transfer of DNA, which will give a new property to the recipient host.

There is an excellent animation describing the experiments of Griffith and Avery et al. at the Cold Spring Harbor Labs DNA Learning Center. I will direct you to this site as a source of information on many aspects of gene structure and expression.

1953

Hershey and Chase showed that DNA and not protein was the genetic material in viruses that infect bacteria, known as "bacteriophage".

1953

Watson & Crick proposed their model for the structure of DNA, the double helix. Again, the CSHL has a good animation describing the structure of DNA.

A timeline of DNA discovery and biotechnology can be viewed at the Access Excellence site from Genentech

The important features of the model of DNA structure proposed by Watson and Crick include:

2 chains, wound around each other in a helix.
The 2 chains are antiparallel - run in opposite orientations.
The backbone of each chain is comprised of a sugar and a phosphate; the backbone is on the outside of the helix.
The bases are attached to the sugar groups and are on the inside of the helix.
Hydrogen bonds between the bases on the 2 chains hold the double helix together.
There are 4 bases (Adenine, Cytosine, Guanine and Thymine)
If there is an A in one strand it always bonds with a T on the opposite strand; likewise a C in one chain always pairs with a G on the other chain.

A large collection of illustrations detailing the structure of DNA can be see in the Graphics Gallery section of the Access Excellenc web site, especially in the section entitled From Gene to Function.

If DNA is the hereditary material, it must be able to perform a number of functions:

1. DNA must be replicated accurately so that there is stable transmission of genes from cell to cell and from generation to generation.

How is accurate, faithful replication achieved? In essence, the two strands of the double helix separate, and each strand serves as a template for precise replication of 2 strands with the sequence of bases in the existing strands determining the sequence of bases in the new strands. The end result is 2 identical copies of the original DNA molecule. (DNA replication is a much more complicated process than this, but for the purposes of this class we have no need to know the details). DNA is also a relatively stable molecule that is not readily broken down, as evidenced by the ability of scientists to recover DNA from samples that are thousands of years old, but not millions of years old as suggested in the Jurassic Park movies.

2. DNA must be able to carry information, the information that determines the development, structure, function, and reproduction of an organism.

The sequence of bases (A,C,T,G) encodes the information. How could 4 bases have the capacity to carry that amount of information? Binary code (two characters, 0 and1) works in computers. We will cover this in the future.

3. DNA must be capable of variation - the variation that gives rise to different forms of life, as well as the variation that produces differences between individuals.

Again, differences in the sequence and organization of bases account for the variation between species, and between individuals. There are relatively small differences in the DNA sequence between individuals of the same species while the differences between species are greater. Consider that two individual humans differ at about 0.25% of the bases in their genomes, or 1 base out of every 400. For comparison, humans and chimpanzees differ at 2% of their bases, or 1 base out of every 50 .

These relatively small differences in DNA sequence are important as they give rise to the physical differences between individuals, as well as the differences between species.

Because all organisms use DNA as their genetic material, it is feasible to consider moving genes between organisms that would not normally mate or be sexually compatible. This is the basis for much of the biotechnology that we will discuss.

Having just said that all organisms use DNA as their genetic material, there are some exceptions. Many viruses use RNA as their genetic material. Nearly all of the viruses that infect plants contain RNA, not DNA, in the virus particle. Most of these viruses do not use a DNA intermediate in their replication. Among the animal viruses, retroviruses are one group that use RNA. This includes HIV, the virus responsible for AIDS.

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Lecture Schedule

1865	Mendel publishes the results of his expts. on the inheritance of traits in peas. These experiments were largely ignored by the rest of the scientific community. (Why?)
1869	Meischer reports that DNA is a major component of the nucleus. But the importance of the nucleus to inheritance was not established, and the nucleus is composed of more than just DNA. The Cold Spring Harbor Labs web site has an excellent review of this topic (click on the animation button at the bottom).
1900s	Mendel's results are "rediscovered" simultaneously by others and the science of genetics begins. Laws of heredity are confirmed in plants, and in 1902 are also demonstrated to apply to animals.
1910	Genes are shown to reside on chromosomes. First shown for sex-linked genes, then extended to all genes. Within a species, chromosomes are constant in number. But chromosomes are made of both DNA & protein - which of these is responsible for carrying information from generation to generation?
1928	Griffith demonstrates the "transformation" of non-pathogenic bacteria to pathogenic. He performs 4 expts. with a bacterial pathogen (pneumococcus) that can infect and kill mice. Expt. 1. Inject mice with NON-VIRULENT bacteria mice survive, no bacteria recovered from mice Expt. 2. Inject mice with VIRULENT bacteria mice die and virulent bacteria are recovered from the mice Expt. 3. Heat-kill the VIRULENT bacteria, then inject these into mice mice survive, no bacteria recovered; the bacteria need to be living to cause disease Expt. 4. Mix heat killed VIRULENT bacteria (#3) with NON-VIRULENT bacteria (#1) and inject this mixture into mice. Note that neither of these agents caused disease on their own. mice die, and VIRULENT bacteria are recovered from the dead mice We can conclude that the non-virulent bacteria have been transformed by their interaction with the dead bacteria. Griffith referred to this agent that changes the properties of the bacterium from NON-VIRULENT to VIRULENT as the transforming principle. But what is the nature of the transforming principle?
1944	Avery, MacLeod & McCarty separated the various biochemical components of virulent bacteria into: lipids, polysaccharides, proteins, and nucleic acids (RNA and DNA) and asked which component could transform the non-virulent bacteria into virulent, disease causing bacteria. Only the nucleic acid component could transform the bacteria. This contains both RNA & DNA. They then used enzymes to break down either the RNA or DNA in this mixture of nucleic acids and showed that only DNA was able to transform the bacteria from non-virulent to virulent. This term "transformation" is now widely used in biotechnology to describe the process of transferring DNA into an organism. For example, we will talk about transformation of bacteria, plants or animals and it will specifically mean transfer of DNA, which will give a new property to the recipient host. There is an excellent animation describing the experiments of Griffith and Avery et al. at the Cold Spring Harbor Labs DNA Learning Center. I will direct you to this site as a source of information on many aspects of gene structure and expression.
1953	Hershey and Chase showed that DNA and not protein was the genetic material in viruses that infect bacteria, known as "bacteriophage".
1953	Watson & Crick proposed their model for the structure of DNA, the double helix. Again, the CSHL has a good animation describing the structure of DNA.