Lesson 18: Biotechnology and Gene Editing PDF

Lesson 18 In this lesson, we will begin looking at the ways that scientists use biotechnology to work with the genomes of living organisms. Here are the major learning objectives for this lesson. Pause the video for a moment to review these and keep them in mind as you proceed through the lecture....

Lesson 18 In this lesson, we will begin looking at the ways that scientists use biotechnology to work with the genomes of living organisms. Here are the major learning objectives for this lesson. Pause the video for a moment to review these and keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. Until recently, the only method of genetic manipulation was through the use of selective breeding, which is also called artificial selection. This method is still commonly used and involves the controlled mating of individuals of the same species to obtain desired traits. Dog breeding is one example of selective breeding. By mating dogs with the same desired characteristics, such as coat color, There is an increased probability that the offspring will also have the same coat color as the parents. This technique has allowed humans to generate more than 150 recognized dog breeds that vary in size and shape from the tiny Chihuahua to the Great Dane. Selective breeding has also been responsible for the domestication of crops. Ancient civilizations across the globe consistently replanted seeds from wild crops that had yielded the most desirable traits. Over many generations, the selection process generated many of the modern vegetables we eat today. The image on the left shows this progression of the wild mustard plant into cabbage, broccoli, cauliflower, and kale. Selective breeding is typically a long-term process requiring many generations to consistently obtain the desired traits. Sometimes, this results in the inadvertent selection of undesirable or even harmful traits in addition to the desirable ones. For example, approximately 30% of Dalmatian are deaf as a result of a genetic mutation that has been propagated throughout the breeding process. Selective breeding also tends to reduce overall genetic diversity in the organism being bred. Cavendish bananas, which account for 99% of all commercially sold bananas, share the exact same DNA as being seedless. They must grow from the cuttings of the same banana plant. This makes them incredibly vulnerable to disease. If one Cavendish banana is susceptible to a pathogen, all Cavendish bananas will be equally susceptible. In the years since the discovery of the structure of DNA, huge leaps have been made in our understanding of genes and their products. As our understanding increases. So has the development of technology which allows us to manipulate the genome of an organism. Gene editing is the process by which DNA of an organism is modified using technology. While many other terms are sometimes used, such as recombinant DNA technology, biotechnology, and genetic engineering. All of these terms, including gene editing, typically represent the same field of study. Gene editing has many variations and may include taking genes from one organism and inserting them into the genome of another, removing sections of DNA, or even editing individual base pairs within the DNA. Examples of gene editing that we will discuss in more depth during this lecture include molecular cloning and the CRISPR-Cas9 system. The figure on the right shows the exponential increase of the use of gene editing techniques in research. Over the last several years. Gene editing can be used to introduce desirable traits into an organism faster than traditional selective breeding techniques. And it allows researchers to incorporate traits into an organism that are not normally found in that species. Additionally, gene editing can often eliminate the reduction in genetic diversity that is inherent in selective breeding methods. Biologists have developed a wide array of tools and techniques that allow them to work easily with an organism's genome and produce recombinant DNA molecules. That is, molecules that are generated in the laboratory that would not naturally be found in the genome. Many of these techniques utilized enzymes that occur naturally within cells. These enzymes can be isolated and used to manipulate DNA outside of the cellular environment. Restriction enzymes, a common tool used to cut DNA at specific locations, are naturally found in bacteria and help the cell to fight off viral infections. Similarly, the CRISPR-Cas9 system we will discuss in more detail later in this lecture, is also used by bacteria to prevent the re-infection of the bacteria with a virus. Both restriction enzymes and the CRISPR-Cas9 system work via nucleases. That is enzymes that can cut the phosphodiester bonds between nucleotides. Thus acting like molecular scissors. Enzymes involved in DNA synthesis are also commonly used in gene editin​​g. Dna ligase helps seal the gaps in the DNA backbone during DNA synthesis. In the figure to the right, we see a small circular DNA plasmid, which functions like the small circular DNA of prokaryotes, cut with a restriction endonuclease, a new sequence of DNA added shown in green. And finally, the gaps between the new DNA and the plasmid DNA being sealed by ligase. DNA polymerase, The enzyme used to elongate DNA sequences is critical to PCR. The technique use to amplify a sample of DNA from a few strands to millions of copies. ​Here are the major learning objectives for this lesson. Pause the video for a moment to review these, and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives to show how scientists can insert a gene from one species into another. We will use the example of GFP or green fluorescent protein. Gfp is naturally occurring in certain species of bioluminescent jellyfish. The ability to make a protein glow is very useful to researchers since it makes it very easy to track the protein's movements within a live cell. In the following slides, we will walk through the process of molecular cloning, which will allow us to amplify, isolate, and ultimately insert the jellyfish GFP protein into bacterial E coli cells. The first step of molecular cloning is polymerase chain reaction, also known as PCR. During PCR, the DNA of interest. In this example, GFP, is first amplified. This process closely mimics cellular DNA replication. In both processes, the DNA is unwound. The primers are added two template strands. Dna polymerase synthesizes the DNA according to the bases on the template strands. Nevertheless, there are differences in how these steps are carried out. Because PCR is a synthetic process that is performed in a machine called a thermocycler. First, the DNA must be unwound. In a cell, This is done a little at a time by helicase. However, during PCR, all strands of DNA are unwound at once by raising the temperature too close to boiling. This process is called denaturation. Next, the temperature is lowered slightly so that the primers can bind or anneal to the DNA. Rather than using primase, the researchers will design primers that border the gene of interests to amplify only the area of the DNA that contains the gene of interest. Finally, the new DNA must be synthesized. As with cellular DNA replication, DNA polymerase binds to the primer and reads the DNA template, adding the appropriate bases to the new DNA strand, rather than using human DNA polymerase, which will denature at the high temperatures needed to denature the DNA. The DNA polymerase of Thermus aquaticus, a thermophilic bacterium that lives in hot springs, is used. This process is repeated many times, ultimately resulting in millions of copies of a single DNA fragment. In the next step of molecular cloning, the amplified DNA is cut with restriction enzymes, which are a class of endonucleases. It is very important to choose restriction enzyme cutting sites that do not cut into the gene of interest in order to preserve the function of the gene of interest. In the figure to the right, three restriction enzyme sites are mapped on the GFP, DNA. EcoR1, Bam H1, and Hind III. Using either BamH1 or HindIII will cut into the GFP gene. So EcoR1 is the correct restriction enzyme to use based on this data. Once the DNA is cut with the proper restriction enzyme, multiple fragments of DNA with known sizes are generated. Restriction enzymes do not cut the DNA bluntly, but rather leave overlapping ends, often called sticky ends, which allow for easy reassembly of the DNA in later steps. before the fragment of DNA containing the gene of interest. In this case, GFP, can be inserted into the new organism's genome. It must first be separated from the other fragments of DNA leftover from the previous restriction enzyme step. To separate fragments of DNA and identify the fragment containing the gene of interest. Gel electrophoresis is used. Gel Electrophoresis involves moving DNA fragments through a gelatinous material filled with many small pores via electric current. The DNA sample containing all of the fragments is loaded into a small well at the top of the gel. Then an electric current is applied to the buffer solution that surrounds the gel. Because DNA is negatively charged, it will move through the pores within the gel towards the anode, or Positively charged end. Dna fragments in the sample are separated according to their size. Because it takes longer DNA fragments, more time to thread their way through the pores in the gel. Therefore, when the electric current is removed, the longer pieces of DNA will be near the top of the gel, while the shorter pieces of DNA will be closer to the bottom. A fluorescent dye allows the researcher to visualize the bands of separated DNA fragments, as well as a standard set of DNA fragments, often called a DNA ladder. Because restriction enzyme maps tell us at exactly which base pairs the DNA was cut. The researchers can then identify the fragment of DNA containing the gene of interest by size, by comparing it to the known sizes of DNA on the ladder. This band is then removed from the gel in preparation for inserting it into the genome of the new organism. Next, the gene and the plasmid are combined during an incubation period. Remember that we cut our GFP gene with the restriction enzyme EcoR1, creating unique sticky ends to open the plasmid and allow it to fit together with the GFP gene. The plasmid must be cut with EcoR1 restriction enzyme. These sticky ends in the opening of the plasmid pair with the sticky ends of the gene of interest, allowing them to fit together like pieces of a puzzle. Finally, the gap in the phosphodiester backbone must be sealed with ligase. Now, the result in GFP plasmid is a recombinant DNA molecule. Finally, the newly synthesized plasmid can enter the E coli bacteria in a process called transformation. This is not an easy process and it is important to verify that the gene of interest has in fact entered the new cell. In this example, it will be easy if the bacterial cells take up the GFP gene because they will now be bioluminescent as the picture on the right. But more frequently, the gene of interest is not easily identifiable. Thus, many plasmids have a selection marker, often an antibiotic resistance gene. By treating the cells with that antibiotic, the scientist ensures that any cells which did not uptake the plasmid will die as they are not naturally antibiotic resistant. And any cells still surviving must contain the plasmid and also express the new gene of interests. Once the new plasmid is located inside the bacterial cells, it needs to be able to divide so that as the cells reproduce, new generations of the cell also contain the plasmid. The origin of replication is located on the plasmid to ensure the plasmids ability to replicate. While the example used to illustrate molecular cloning showed the GFP gene from a jellyfish being inserted into a population of bacterial cells. The techniques of molecular cloning, as well as more advanced gene editing techniques, can be used to alter the genomes of multicellular organisms as well. Such as the mice to the right, which carry the GFP gene as part of a study on HIV. Often we call genetically altered animals transgenic organisms. While there are many names for organisms generated by gene editing, you are probably most familiar with the term genetically modified organism or GMO. Gmo is a term commonly used to refer to crops and other foods that have been genetically modifying. One of the most famous GMOs is glyphosate resistant soy crops. Glyphosate is a powerful, bio-degradable herbicide that kills most growing plants, but has no impact on animal populations. While useful for killing weeds. Also traditionally kill off soybean crops as well. Molecular cloning helped to generate glyphosate resistant plants by inserting a gene that causes the soybean plants to synthesized 20 times the normal levels of the enzyme that glyphosate inhibits, thus protecting them from glyphosate induced death. This allows farmers to kill the weeds surrounding the soy crops without damaging the soy crops themselves. Currently, 90% of soy crops grown in the US are glyphosate resistant. To insert a new gene into a plant. The edited gene is transformed into a special plasmid called a tumor inducing or TI plasmid. In nature, the TI plasmid infects the host plant by inserting its DNA into the plant genome, as seen in the figure below. When the plasmid containing the gene of interest is put back into the bacterial plant pathogen Agrobacterium tumefaciens. The plant pathogen will infect plant cells and insert the gene of interests directly into the plant genome. Plants grown from the infected cells will then express the gene of interest. ​Here are the major learning objectives for this lesson. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. Molecular cloning allows us to move genes from one organism to another. But a more recent technology called CRISPR, Cas9 enables us to directly and more rapidly edit the genes of living cells up to the single nucleotide level of precision. This revolutionary tool was first discovered by Dr. Carpentier and Dr. Doudna, who were studying the ability of microbes to prevent reinfection from viruses, a bacterial immune response. In 2028, years after publishing their discovery, they would win a Nobel Prize for their work. Let's take a deeper look into how bacterial immune responses have revolutionized the world of gene editing in such a short time. First, let us look at how CRISPR and Cas9 work in bacteria. When the bacterium is first infected with viral DNA, the bacterium collect small fragrance of the viral DNA and incorporates them into the bacterial genome. Essentially keeping a library of viral DNA for future reference. This library of viral DNA is called CRISPR or clustered, regularly interspaced, short palindromic repeats. Now, the bacterium can recognize future viral infections if the new virus shares the same DNA sequences as the viral DNA in the bacterium's CRISPR library. When the new virus tries to infect the bacterium, the bacterium will make RNA from the CRISPR viral DNA sequences. And this so-called guide RNA serves as a guide for the second half of the CRISPR system, a protein called Cas9. If CRISPR acts as the library carrying the knowledge of where to cut, Cas9 acts as the scissors. More precisely, Cas9 is a nuclease or a protein capable of cutting the phosphodiester bonds of DNA. Cas9 will cut the new viral DNA at the precise location indicated by the guide RNA provided by the CRISPR library, preventing the new virus from damaging the bacteria. Thus, the CRISPR-Cas9 system is often considered the immune system of the bacteria. In the same way that your adaptive immune cells recognize previous pathogens and work to prevent re-infection. Now, let us look at how the CRISPR-Cas9 system can be adapted by scientists to accurately and relatively easily edit virtually any genome. If a scientist knows what part of the cell's genome they want to edit, they simply need to design and inject the guide RNA that matches the location where the Edit should take place, as well as the Cas9 protein to read the guide RNA and precisely cut the DNA. Once the DNA is cut, there are many ways to use the CRISPR system. Unwanted DNA can be cut out and then the cell can seal the backbone using DNA ligase, essentially deleting unwanted material. Alternately, new DNA can be injected at the same time as the guide RNA and the Cas9 protein. And the cell will incorporate the new DNA into the place of the cut DNA. Adding new DNA allows scientists to repair broken genes or to introduce new mutations instead. These are just a few of the many ways CRISPR can be used to alter a genome directly within the organism. While the CRISPR-Cas9 system has revolutionized gene editing across multiple disciplines, from agriculture to industry. One of the fields most likely to be changed by CRISPR is the field of medicine. Gene therapy is the process of editing the cells genes, either by directly editing the genome of living cells within the body, or by extracting and editing the genome of cells before transplanting them back into the body. Gene therapy specifically targets diseases that have genetic origins. E.g. several ongoing clinical trials using CRISPR technology are targeting sickle-cell anemia, which is caused by a point mutation that results in misshapen red blood cells. By using CRISPR to edit blood stem cells, that is, cells within the body that generate new blood cells and then transplanting them back into the body. All red blood cells produced from the modified stem cells are able to synthesized functional hemoglobin. Another example of gene therapy is the use of CRISPR to prevent HIV infection. Hiv typically infects a specific type of immune cell called a T cell, by binding to one of its surface proteins called CCR5 entering the cell and ultimately causing T cell death. However, some individuals have a mutation in CCR5 preventing HIV from infecting the T-cells. In clinical trials, researchers are taking immune stem cells, that is the cells within the body that generate new immune cells and inducing the CCR5 mutation before transplanting the stem cells back into patients, thus preventing T cell death in HIV positive individuals. Although gene therapy is a promising technique for curing genetic diseases, it is not without risks or ethical concerns. In addition, the high cost of gene therapy may limit who has access to this technology. When discussing gene editing and gene therapy in particular, it is important to distinguish between gene Editing methods that alter somatic cells versus those that alter germ line cells. Somatic cells are any cell that cannot make gametes, also known as sperm and eggs. In the prior two examples of sickle-cell anemia and HIV resistant clinical trials. Somatic cells were removed from the patient, edited via crisper and then transplanted back into the patient. The edited genes will be found in the transplanted cells, as well as the cells they generate, such as the T-cells in the HIV example, but will not be found in any other cell of the body. Thus, any benefits or risks of gene editing to somatic cells remains contained within the individual and will not be passed to the next-generation. Germline cells are cells that make gametes, the zygote, the fertilized egg, which will go on to make every other cell in the body, is a germline cell that is a desirable target for gene editing technology. If the DNA of the zygote is altered, the alterations will be found in every cell of the adult body, including the reproductive cells that will make gametes. Thus, any benefit or risk of gene editing to germline cells impacts not only the individual, but also their offspring. In this case, evolution may be altered as the edited genes are passed down and alter the alleles present in subsequent generations. While gene editing on germ cells that will impact future generations is common in non-human organisms, including food crops and model organisms. The scientific community, as well as the majority of governments, largely agree that editing humans zygotes presents substantial risks to the embryo and also raises many ethical questions. When humans use gene editing to alter the allele frequency of future generations of plants or animals. The impacts can be hard to predict. Each organism forms a point in the complex web of the planet's ecosystem. Altering or removing an organism from that ecosystem can impact other organisms and the surrounding environment. Earlier in this lecture, we discussed genetically modified soybean and corn crops that are resistant to the weed killer glyphosate. Since GMO crops resistant to glyphosate have become widespread. Many weed species that have persistently been exposed to glyphosate have also evolved to develop glyphosate resistance, including the weedy sunflower and several species of weedy grasses. In addition, the ability of domesticated crops to out cross with wild plants albeit infrequently has led to the resistance gene passing on to other plant populations. Scientists estimate that over 200 wild plant species are now resistant to one or more herbicides as a result of gene editing in crop species. To further illustrate the potential impacts of gene editing to the planet. Let us look at current experiments to eradicate mosquito populations. Anopheles mosquitoes are the primary transmitter of malaria, which is responsible for killing over 400,000 people per year. By eradicating the anopheles mosquito, either by inhibiting reproduction or decreasing survival. Scientists hope to positively impact the planet by eliminating malaria. While these experiments aimed to positively impact the planet, there may be other unintended side effects as well. In the initial experiments trying to make mosquitoes infertile, scientists found that the experimental mosquito population mutated to overcome the infertility problem. Finally, by removing mosquitoes from the ecosystem, we may find too late that mosquitoes play a critical role that cannot be filled by other species. Lesson 19 In the last lesson, you learned about techniques used to artificially manipulate genetic material. In this lesson, we will focus on genomics, the branch of biology dealing with mapping, annotating, and analyzing whole genomes. Here are the major learning objectives for this lesson. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. The technological advances of the past 50 years have led to a dramatic increase in the understanding of genomes. The first genes were isolated in the mid 1970s, and the first complete genome, the bacteria Haemophilus influenza, was sequenced by the mid-1990s. By 2003, the human genome had been sequenced. Today it is possible to sequence an entire genome, billions of nucleotide base pairs in length in a matter of hours. The field of genomics integrates classical and molecular genetics to better understand genomes. In the figure to the right, we see that the study of genomics employs a number of sub-disciplines to study the structure, function and relatedness of genomes. The processes of mapping, sequencing, annotating, and analyzing genomes will be discussed in this lecture. We frequently use maps in our daily lives to orient ourselves and find our way. Maps are produced at different levels of resolution depending on whether you need to find your way across the country or navigate a town. The maps used in genomics also have differing levels of resolution, which depending on the situation, may be of help to a researcher. To categories of maps used in genomics are genetic maps, physical maps. Genetic maps are derived from recombination frequency, and linkage analysis, which has been described in other lessons. These maps provide relative locations of genes or genetic markers. Physical maps provide precise position in the genome and often have a resolution at the nucleotide level. Physical maps use landmarks within the DNA sequence as markers. Common landmarks include enzyme cutting sites and small segments of the actual nucleotide sequence. Unlike genetic maps, physical maps show us the absolute location of a marker. The ultimate form of a physical map is the placement of many genetic markers on a complete genetic sequence. However, there are many genomes on Earth that have yet to be sequenced. The distances between markers are measured in base pairs and large maps use kilobase pairs as a measurement. Restriction maps were one of the first types of physical maps generated. These maps are created by digesting DNA with one or more restriction enzymes and mapping the location of the enzyme cuts sites. The figure at the right demonstrates how these maps are created. Restriction maps not only provide the location of cut sites, but researchers can utilize restriction enzymes that recognize these sites to cut and remove important pieces of DNA for study. There are other times when relatively high resolution is not necessary. Chromosome maps are used by researchers who study whole chromosomes or genes which can translocate or move to other chromosomes. These types of maps use various staining techniques which identify regions of interests and can be viewed using a microscope. Although these maps have low resolution, they allow researchers to view entire chromosomes or even genomes at a glance. Recent advances have allowed scientists to combine the high resolution advantage of restriction maps with the ability to view large pieces of DNA, similar to chromosome mapping. The sequence tagged site, or STS, is a small stretch of DNA, found at only one location in the genome, that can be amplified using PCR. Researchers utilize these unique sites to identify the location of a DNA fragment in the genome or piece together fragments by analyzing overlapping STS sites. The presence or absence of an STS site on a fragment helps researchers to organize the fragments into a contiguous sequence or contig, as shown in this figure. Here are the major learning objectives for this lesson. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. As mentioned previously, the highest resolution physical map is the base pair sequence. Recent advances in sequencing technology have made it possible to sequence very large genomes quickly and affordably. All sequencing methods rely on the polymerase chain reaction, electrophoresis, and the use of chain terminating nucleotides. The earliest forms of these chain terminating nucleotides are called dideoxynucleotides. Recall from earlier lessons that during replication, nucleotides are added to the hydroxyl group of the three-prime carbon to form a phosphodiester bond. Dideoxynucleotides lack this hydroxyl group, so they act as chain terminators when incorporated into the DNA molecule during PCR. Dideoxynucleotides can have any of the four DNA bases attached, and as long as the researcher knows which of the four die deoxynucleotides is terminating the sequence, he or she can know the base at that location in the genome sequence. Original manual sequencing reactions created by Fred Sanger utilized multiple replication reactions to accomplish this. This was time-consuming and labor-intensive. Fortunately, there have been many advances in sequencing. The image to the right demonstrates an automated version of sequencing. Researchers begin with a template strand of unknown sequence. This sequence is subjected to a polymerase chain reaction, as has been described in earlier lessons. In addition to the normal nucleotide building blocks, researchers incorporate for different dideoxynucleotides corresponding to each nucleotide base, and each is labeled with a different color fluorescent tag. As DNA polymerase synthesizes a new DNA strand, it will randomly incorporate one of the die deoxynucleotides. The DNA fragments created are separated in a single capillary tube using electrophoresis. The smallest fragments which correspond to the five-prime end of the synthesized molecule, will migrate fastest and the largest fragments will migrate slowest. As the fragments pass through the tube. A laser and photo-detector work in tandem to identify the fluorescent dideoxynucleotides. This type of automated sequencing revolutionized the field of genomics. The next-generation sequencing technologies appeared in the last decade and have the advantages of being faster, cheaper, and able to sequence larger fragments of DNA in a single reaction. In this version of next-generation DNA sequencing, the large DNA sample is first fragmented and the single-stranded fragments are attached to a solid surface using a special adapter. Using PCR, multiple copies of each fragment are created. This step will ensure that all fragments are sequenced multiple times using the following method. Synthesis begins as DNA polymerase recognizes a universal primer attached to the fragment and incorporates nucleotides into the complimentary DNA. Similar to other methods of sequencing, special nucleotides will terminate synthesis, fluoresce identifiable colors, and be detected with a laser. What sets next-generation sequencing apart is that these chain terminating nucleotides can be reversed or changed back into normal nucleotides. This enables each fragment to be sequenced multiple times with new reversible chain terminating nucleotides added each time. The advantages of this method of sequencing have dramatically increased the number of genomes sequenced each year. Regardless of the type of sequencing technology used, most genomes are too large to be sequenced in a single-step. As a result, most approaches require breaking the genome into manageable fragments, sequencing those fragments, and then assembling a contig by matching overlapping sequences. Two general approaches to this are the clone-contig method and the shotgun method. In the clone-contig method, a genome is fragmented into large pieces of DNA called clones. Although the sequence of the clones is not yet known, they can be arranged in order based on physical mapping landmarks like STS sites. The clones are then broken into smaller fragments of appropriate size for sequencing. These sequences are used to generate the clone sequences, and then the clone sequences are combined to create a large contiguous segment of DNA called a contig. The shotgun method does not rely on any genetic or physical maps. Instead, the whole genome is fragmented into manageable pieces for sequencing, and computer software is used to assemble all the pieces based on overlapping nucleotide regions. Both methods are often used to sequence one genome. The human genome project is a good example of one that utilized both methods. In 2000, the first draft of the human genome was published. This accomplishment was the result of more than 20 years of work, and a collaboration between the US government and private biotech company called Celera. The use of genetic maps, clone-contig and shotgun sequencing methods were all employed. Since this time, more accurate and extensively annotated sequences have replaced the draft sequence. Perhaps one of the most surprising findings from the project was that the number of genes was only about 20,000, which was far fewer than the 100,000 genes that was predicted. As can be seen from the figure, organismal complexity is not a simple function of genome size or gene number. Here are the major learning objectives for this lesson. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. A complete DNA sequence is of little importance without knowing the number, location, and kind of genes present and how these genes contribute to phenotype. The process of genome annotation assigns this kind of information to the genomic sequences. The DNA sequence, and it's continually evolving annotations are typically recorded in searchable online databases. Researchers use these databases to look for DNA sequences of a known "signature". An example would be a sequence that codes for a start codon followed by a number of amino acids and eventually a stop codon. This potential gene sequence is referred to as an open reading frame. As researchers learn more about the functions of these genes, the databases can be updated. One of the most prominent databases is GenBank, an annotated collection of publicly available DNA sequences. Using the Basic Local Alignment Search Tool or BLAST. Researchers can compare the sequence of an unknown gene of interest to the database. Finding known genes of the same or similar sequence can provide insights about function. DNA sequences that are used to produce protein or transcribed into functional RNAs, such as transfer RNA or ribosomal RNA, are called coding sequences and are annotated as such in the online databases. A significant amount of the DNA in genomes, particularly in eukaryotic genomes, does not code for proteins. This DNA is called non-coding DNA and may include as much as 99% of the DNA in the human genome. The table depicted here categorizes the DNA annotated in the human genome. As you can see, there are multiple categories of non-coding DNA. Introns, structural DNA, simple sequence repeats, and non-coding RNA involved in gene regulation have been discussed in past lessons. The categories of segmental duplications and pseudo-genes are somewhat self-explanatory even if their evolutionary history is not so clear. Transposable elements represent a diverse group of DNA sequences that can move from one chromosome to another, oftentimes copying themselves as they move. With all these categories of non-coding DNA. A natural question is, what is the function of this DNA? The presence and prevalence of non-coding DNA in genomes has been known for years, but the importance of this DNA has remained unclear. Conducted 2003-2012, the Encyclopedia of DNA Elements, or ENCODE project, was a collaborative effort which sought to identify all functional elements in the human genome. The conclusion of this work suggested that 80% of DNA in the human genome is functional. However, this begs the question, what is meant by functional? In addition to the coding regions, traditionally regarded as functional, the ENCODE researchers also deemed that any sequences with reproducible "biological activity", we're also functional. Examples of these sites are depicted in the figure. DNA methylation, chromatin modification, and various sites that can be cut by DNase enzymes are considered functional because biological activity occurs at those sites. Critics of ENCODE's definition of functional DNA believe that "biological activity" alone is not enough to consider a DNA element as functional. By that logic, all DNA that is replicated by the enzyme DNA polymerase could be considered biologically active. Furthermore, they claim that merely locating a site of enzymatic activity is not sufficient proof that the region actually provides a regulatory function or a gene product. Finally, evolutionary biologists argue that ENCODE researchers should only include items as functional if they have been "selected" for through years of evolutionary pressure and provide a benefit to the organism. While the conclusions of the ENCODE project may be controversial, the work done has contributed substantially to the mapping effort of the human genome. Here are the major learning objectives for this lesson. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. The final component of genomics is genome analysis. In short, genome analysis is concerned with identifying the role or purpose of DNA elements in the genome. The primary approaches are comparative genomics, functional genomics, and proteomics. We will look at each of these in turn. Comparative genomics uses information from one genome to learn about a second genome. One hallmark of comparative genomics is synteny. Synteny refers to the conserved arrangements of segments of DNA in related genomes. Synteny can be derived from comparing physical maps, including, but not limited to, sequence data. The image at the right depicts synteny between four grains, rice, sugarcane, corn, and wheat. To date, only the rice and corn genomes have been fully sequenced. Despite this, it is clear that when these genomes are rearranged and compared to one another, there are portions that are conserved. Researchers look at these conserved or syntenous regions of known and unknown sequence to predict gene function, locate similar genes of interest, or predict evolutionary relatedness. The grasses here are believed to have diverged evolutionarily about 50 million years ago. To fully understand how the genome or genotype contributes to the phenotype of an organism. It is important to characterize the RNA products and proteins encoded by each gene. The field of functional genomics uses biotechnology to highlight the connection between the genotype and phenotype. Functional genomics can be broken into three separate but related categories. The study of all RNA molecules produced by the genome, otherwise known as the transcriptome. The study of the proteins produced by the genome, otherwise known as the proteome, and the study of the interactions between proteins. These last two categories comprise a field called proteomics. Let us first study the RNA molecules produced. DNA microarrays are a tool that allows researchers to determine which genes are being turned on or expressed at a particular location or time. The picture to the right shows a microarray chip. Each dot represents a different known gene from that organism, and the different colors indicate the level of gene expression. Knowing if a gene is being expressed during a developmental stage or in response to an environmental stimulus can provide great insight into the function of a gene or which genes may interact with each other. Microarrays represent a powerful tool, but they require that the researcher design a microarray chip consisting of "known" genes believed to be of importance. RNA sequencing or RNA-seq, uses next-generation sequencing to capture ALL the mRNA transcripts being created at a particular time. Using this approach, researchers can collect information on ALL gene expression rather than just suspected genes. It should be noted that just because a cell expresses mRNA does not ensure that the protein products will be translated. As we have seen in other lessons, post-translational regulation may occur. It is therefore important to determine if the proteins encoded by the mRNA molecules are actually being produced. Proteomics is the study of the proteome or collection of proteins encoded in the genome. Alternative splicing and post-transcriptional modification make it tough to predict a protein's structure from the DNA sequences alone. Because of this, the proteome is more difficult to analyze than the transcriptome. Two techniques that are often employed, our mass spectrometry or mass-spec and protein microarrays. The figure to the right follows the process of analyzing proteins using mass-spec. Proteins are first isolated from a cell and then separated according to physical or chemical properties. Purified proteins are then cut into small peptides using protease enzymes. These peptides are further separated, an ionized so that each peptide has a charge. Mass spectrometry calculates the charge to mass ratio of each peptide and compares those values with a database to identify proteins. Alternatively, the protein can be further fragmented and its component amino acids can be determined. Protein microarrays work similar to the DNA microarrays described earlier. However, instead of DNA sequences applied to the chip, antibodies are applied and used to identify the protein in a sample. Analyzing all the data from a proteomic study presents a challenge. The use of computer programming, mathematics, and modeling to analyze large amounts of biological data is called bioinformatics. The application of bioinformatics to proteomics allows the rapid identification of proteins discovered using techniques previously described. In addition, researchers can use known DNA and amino acid sequences to predict protein structure based on chemical properties of amino acids and principles of how these amino acids interact. The image on the left is a computer-generated model of an enzyme. The application of genomics is an area of great potential and ethical considerations. Synthetic biology is considered by many to be the next frontier for biotechnology. In 2010, scientists successfully constructed an entire synthetic bacterial genome and inserted it into a bacterium. The synthetic genome was able to control the bacterial cell Mycoplasma mycoides. The ultimate goal of synthetic biology is to engineer organisms to solve problems. Examples include: enhancing biofuel production and designing organisms to clean up environmental disasters. An immediate impact of genomics in the health field is diagnostics. The identification of genes responsible for genetic disorders is a primary goal. Furthermore, forensic scientists use genomic sequencing, and STS mapping, to identify unknown remains of people and pathogens used as biological weapons. While this technology offers many wonderful developments, the potential for misuse has prompted ethical concerns. In 2005, a group of scientists, including some at the Centers for Disease Control (CDC), informed the world that they had sequenced and reconstructed the 1918 Spanish Influenza virus that killed 20-50 million people. More recently, there has been concern over the continued research on SARS-CoV-2, the virus, which was the causal agent for the COVID-19 outbreak. The question of who owns the rights to use gene sequences has mostly been answered. In 2013, the Supreme Court ruled that it was illegal for the biotech company, Myriad Genetics to patent the sequence for two genes associated with the development of breast cancer, because the genes had not been invented by the company. However, the company was allowed to patent the rights to certain synthetic sequences created using the gene sequences as a reference. Lesson 20 In this lesson, we will learn about the mechanisms by which a single cell develops into a multicellular organism. The primary focus here will be on animal development. Plant development will be covered in a separate course. Here are the major learning objectives for this lecture. Pause the video for a moment to review these, and then keep them in mind as you proceed. After completing the lecture, you should be able to do each of these objectives. Development describes the process by which a single celled fertilized egg, that as a zygote becomes a fully formed adult. These single cells undergo dramatic changes to first become an embryo, and then a multicellular adult, as you can see in the figures below. All of these complex and organized changes are regulated by changes in gene expression over time. Development is comprised of four major processes: cell division, cell differentiation, pattern formation, and morphogenesis. We'll cover each of these processes in greater detail later in the lesson. But for now, note the sequence depicted here represents the order in which the four processes are initiated. But earlier processes, cell division and differentiation, normally continue throughout development and even into adulthood. Scientists have long use model organisms to study animal development. Model organisms tend to be less complex and therefore easier to study than humans. And also avoid ethical concerns associated with experimental manipulation of human embryos. However, because the basic mechanisms of development are highly conserved, scientists are readily able to draw parallels between the development of model animals and the development of humans and animals in general. In the next few slides, we'll introduce a few of the model organisms that had been used to study development and see that different model organisms are useful for understanding different developmental processes. To start, we will look at a species of nematodes or roundworms known as Caenorhabditis elegans or C. elegans, for short. This species is important to our understanding of development because it was the first model organism for which a complete cell lineage was mapped. The cell lineage map, like the one shown in the bottom figure, represents all the cell divisions that occur during the lifetime of an organism and describes the fate of each and every cell in the body. From this information, scientists have learned about the process of cell division, as well as the way that a cell adopts a particular fate. For example, how it becomes a skin cell or a neuron. Commonly known as the fruit fly, Drosophila melanogaster has been used to study the mechanisms of pattern formation in embryos. Studies with Drosophila have shown that development is driven by changes in gene expression. The figures at center and right depict the highly organized expression patterns of different genes in different regions of the developing Drosophila embryo, and what structures each region will become. We'll discuss pattern formation more detail later in this lesson. African clawed frogs, or Xenopus laevis, are also used as developmental models. Their large eggs, which are visible with the naked eye, make them ideal for studying the early cell divisions of the embryo. Additionally, frogs undergo a change in body form from the tadpole stage to the adult stage, which is a well-known example of morphogenesis. A great deal has been learned about mechanisms driving morphogenesis from the study of frog development. While development has certainly been studied in other model organisms, these three, C, elegans, Drosophila melanogaster, and Xenopus laevis, are perhaps most closely associated with our understanding of how development occurs. Here are the major learning objectives for this lecture. Pause the video for a moment to review these, and then keep them in mind as you proceed. After completing the lecture, you should be able to do each of these objectives. The first process of development we will look at in detail is cell division. Cell division is the most obvious process required to transform a single-celled zygote into a multicellular organism, for without cell division, there would be no multicellular organisms consisting of hundreds, millions, or even trillions of cells. During early embryonic development, cell divisions occur very rapidly and with significant alterations to the normal cell cycle. The primary purpose of these divisions is to split the cytoplasm of the zygote into smaller volumes. And as a result, cell division in the early embryo is often termed cell cleavage. The time between cleavages is quite short because there is no cell growth between these divisions. And in fact, early embryonic cells do not exhibit the G1 or G2 phases of the standard cell cycle. Instead, these cells only transition back and forth between S phase, to replicate DNA, and M phase, to divide into daughter cells Later, as shown at right, once a certain number of cell cleavages have occurred and development has progressed, the length of the cell cycle increases as cell growth becomes necessary, and G1 and G2 phases return to the cell cycle. Cell cleavage in an early embryo is the fastest rate of cell division at any point in an organism's lifetime. For example, in zebrafish, another model organism, cleavages occur every few minutes to produce an embryo with more than one thousand cells in less than three hours. In contrast, human intestinal cells, which have amongst the most rapid turnover rates in our bodies, divide only once every 19 hours. The second major developmental process is cell differentiation, which is the production of a specialized, that is, differentiated, cell. To understand differentiation, we must first discuss the role of specific cells called stem cells, as differentiated cells will arise from the division of stem cells. A stem cell is any cell which has not adopted a particular cell fate, and therefore has the potential to become one of a number of different types of cells. When a stem cell divides, one daughter cell will give rise to cells that differentiate, while the other daughter cell replaces the original stem cell and retains the properties of that stem cell. This continual replacement of stem cells is termed self renewal. Differentiation depends on changes in gene expression. Recall that the DNA sequence in every cell of a given organism is the same, but different cell types will express different genes. Differentiation is not like turning on a light switch in which all the genes required to produce a particular cell fate are activated at once. Rather, gene expression is gradually altered and thus differentiation progressively restricts the number of fates a cell that descends from a stem cell can adopt. These descendant cells are all related to each other as they can be traced back to the same stem cell and are thus said to be in the same cell lineage. Overall differentiation is best viewed as a gradual process that occurs over multiple cell divisions. Stem cells are categorized by their potency, a term used to describe the range of possible cell fates the descendant cells may adopt. In other words, the potency of a given stem cell restricts the possible cell fates of its descendants. Stem cells can be classified into one of four potency levels. At the highest level are totipotent stem cells. These stem cells are able to form all the tissues of an organism, including all of the extraembryonic tissues that are required to support embryonic development, such as the placenta. Only zygotes and blastomeres, which are the individual cells from the first few cleavages of an embryo, can be considered totipotent. The spontaneous separation of one blastomere from an embryo is responsible for the development of embryonic twins, because that one cell can still divide to form a complete organism and all of the required extra embryonic tissues. Pluripotent stem cells represent the second potency level. These stem cells are able to form all the tissues of an organism, but none of the extra embryonic tissues. Therefore, while a pluripotent stem cell has the potential to develop into a complete organism, it cannot form any of the tissues that are required to help support the embryo during development. Embryonic stem cells are the primary example of a pluripotent stem cell. These cells can be obtained from the inner cell mass of an embryo, a cluster of cells that form on the inner surface at the blastocyst stage, when the embryo resembles a hollow ball. Embryonic stem cells are best known for their use in reproductive and therapeutic cloning, procedures we will discuss later, as well as their potential treat a range of medical conditions. However, their use and study remains controversial as harvesting embryonic stem cells requires destruction of the embryo. Multipotent stem cells have the ability to differentiate into only a few different cell types. A multipotent stem cell cannot divide to form a complete organism. Adult stem cells, like the one in the figure, are multipotent. Adult stem cells are responsible for replacing old, worn-out cells in various tissues throughout the body. The stem cell shown in this example, is able to differentiate into muscle cells, fat cells called adipocytes, and neurons. Unipotent stem cells are only able to differentiate into one type of cell, for example, germ cells, specifically here the spermatogonia, are unipotent stem cells that can only differentiate into sperm. Here are the major learning objectives for this lecture. Pause the video for a moment to review these, and then keep them in mind as you proceed. After completing the lecture, you should be able to do each of these objectives. The third process involved in the development of an organism is pattern formation. Pattern formation creates the body plan of an organism. This process depends on differential gene expression. Different genes are expressed depending on the cell's location within the embryo. Different specific sets of expressed genes as visualized in the Drosophila embryos at left, create different discrete segments or sections that will become different body parts in the correct spatial location. In addition, as shown with this tadpole example, and representative of most higher animals, pattern information establishes the three main body axes, anterior and posterior, meaning head end and hind end. Dorsal and ventral, meaning back and front, and left and right. During pattern formation, each body segment is formed at the correct location through the expression of homeobox containing genes, commonly called Hox genes. Each segment expresses a different Hox gene to give itself a unique identity. For example, the regions expressing the Dfd gene are shown in purple on the embryo and the adult fly, and the expression of the Dfd gene identifies the third segment of the Drosophila embryo as the location of the head. Hox genes are highly conserved. In fact, the same genes are present in both mice and drosophila. The major difference between the two organisms is that mice have multiple copies of the Hox genes, whereas drosophila have only one copy of each gene, as you can see in the figures on the left. Mutation in the Hox genes can have dramatic effects, most notably causing one segment of the embryo to develop as a different segment. The images on the right illustrate what can happen when segments of a Drosophila embryo are mis- identified due to Hox mutations. The top picture shows a fly with two sets of wings, the result of a Hox mutation. The bottom figure shows a wild-type and mutant Drosophila. The mutant on the right has a set of perfectly formed legs in place of antennae. It is important to realize that the legs and the extra set of wings are not malformed in any way. The Hox mutations merely result in the replacement of the correct body part with those associated with another segment. Hox gene mutations and duplications are a major mechanism of evolution because of the dramatic effects that these genetic changes can have on the body plan of an organism. The last major developmental process we'll discuss is morphogenesis or the generation of an organism's body form. This may seem similar to pattern formation, but these two processes are in fact quite different. Pattern formation can be compared to the creation of blueprints for a building. In a set of blueprints, all the major decisions are made regarding the location and size of all the rooms. Likewise, pattern information lays out all the positions of the major body parts in an organism. Morphogenesis, on the other hand, is analogous to the actual construction of the building. According to the plans and the blueprints. In morphogenesis, the construction of the body is achieved by the following mechanisms: cell growth, cell division, cell migration, changes in cell shape, and program cell death, also called apoptosis. Cell growth and division are primarily responsible for increasing the size and number of cells within an organism as it develops. As these two concepts are fairly straightforward, we will spend more time discussing each of the other three mechanisms. In order for the body form of an organism to develop properly, some cells must be able to migrate to different locations. Cells migrate through the extracellular matrix, a mixture of carbohydrates and proteins secreted by cells to provide them with support and protection. In order to migrate, cells must be able to change the way that they interact with the extracellular matrix. They do this by controlling the expression and activity of adhesion proteins, which are found on the external surface of their plasma membrane. In order for a cell to move, these cell adhesion proteins must bind and release the extracellular matrix in a coordinated manner. The importance of cell migration for development has been demonstrated through experiments with drugs that inhibit the ability of cell adhesion proteins to bind to the extracellular matrix. When embryos are exposed to such drugs, the embryos will inevitably fail to complete development. Morphogenesis is also associated with changes in the shape of many cells. These changes are usually required in order for the cell to function properly in the embryo and/or the adult organism. For example, as mentioned previously, certain adult stem cells can differentiate into adipocytes, muscle cells, or neurons, cells which have dramatically different shapes. In each case, cell shape and structure are associated with a given cell's function in the body, and failure to adopt the correct shape will lead to the inability of the cell to perform its appropriate function. The last mechanism of morphogenesis we will consider is a apoptosis, or programmed cell death. Apoptosis is a highly controlled process of cell death that is activated by the expression of genes in a cell death pathway. Apoptosis differs from necrosis, which is cell death caused by injury. Necrotic cells burst to release their contents into the extracellular matrix. Conversely, apoptotic cells are gradually broken down into smaller compartments that are easily ingested and recycled by other cells. During development, apoptosis is often used to "sculpt" appendages or reduce cell numbers. For example, human embryos have webbed fingers and toes at an early stage of development. The cells that make up the webbing, shown in red, undergo apoptosis during the normal course of morphogenesis. The controlled death of these cells thus sculpts the human fingers. Likewise, the cells that make up the tail of a tadpole undergo apoptosis to produce the tailless adult frog. Here are the major learning objectives for this lecture. Pause the video for a moment to review these, and then keep them in mind as you proceed. After completing the lecture, you should be able to do each of these objectives. Our increasing understanding of stem cells and the mechanisms of development led scientists to wonder whether cell differentiation was a reversible process. Since differentiation is the result of changes in gene expression, scientists first theorized and then proved that gene expression can be reset to match that of an undifferentiated stem cell. This idea is called nuclear reprogramming and requires that the epigenetic changes present in a differentiated cell's DNA be reversed. Recall that epigenetic changes are chemical modifications of nucleotide bases and do not change the sequence of the cell's DNA, but they do influence gene expression, and are stable throughout cell divisions. A number of methods have been used to achieve nuclear reprogramming. The most well-known method is somatic cell nuclear transfer, or S- C-N-T. Using this method, the nucleus of a differentiated cell, like the fibroblast shown in the image, is removed from the cell and inserted into an oocyte or zygote from which the original nucleus, shown here in orange, has already been removed in a process known as enucleation. The transcription factors present in the cytoplasm of the oocyte or zygote then reprogram the differentiated fibroblast nucleus back to a stem cell state. This method, which was used to create the first cloned animal, Dolly, is technically challenging, but can produce viable embryos as described in the next slide. A more recent method is called direct reprogramming, and it takes advantage of the fact that some of the transcription factors that are expressed only in stem cells have been identified. By introducing these transcription factors into differentiated cells through a variety of methods, scientists are able to reprogram the nucleus to create an induced pluripotent stem cell, or iPS cell. Because the cells produced by this mechanism are pluripotent rather than totipotent, induced pluripotent stem cells alone are unable to generate a complete embryo or adult animal. Each of these methods demonstrates that differentiation can be reversed, and in the following slides we'll discuss how these methods can be utilized. Nuclear reprogramming, particularly through the process of somatic cell nuclear transfer is closely associated with the idea of cloning, and there are two types of cloning that involve nuclear reprogramming: reproductive cloning and therapeutic cloning. Note that both reproductive and therapeutic cloning require the epigenetic changes to the cell's DNA be reversed, and that the cloning described here is quite different from molecular cloning, which uses the cell's machinery to make copies of short segments of DNA. Reproductive cloning creates a genetically identical copy of an individual organism. This process is outlined in the figure and begins with somatic cell nuclear transfer. Here the nucleus from an adult mammary gland is inserted into an enucleated oocyte from a different animal. The resulting fused cell is allowed to develop into an embryo in the laboratory, and is then implanted into a surrogate, or foster, mother. If all goes well, the resulting progeny is genetically identical to the individual from which the differentiated nucleus came. Therapeutic cloning, on the other hand, does not result in a complete individual. Rather, it produces patient-specific tissues that can be used in medical treatments. Tissues generated by therapeutic cloning could eliminate the risk of immune rejection that is inherent in organ transplants, because the body will identify the transplanted tissues as belonging to itself. The process of therapeutic cloning is similar to the initial steps of reproductive cloning. That is, the nucleus of a differentiated cell is removed and inserted into an enucleated human oocyte. The cell is then allowed to develop into an embryo. However, rather than implanting the embryo into a surrogate mother, the embryonic stem cells that comprise the inner cell mass of the embryo, shown in blue, are harvested and cultured in the laboratory. These pluripotent stem cells are then developed into healthy tissues that can be transplanted back into the individual to replace diseased or damaged cells. Cloning techniques have a great number of potential uses. Reproductive cloning has the potential to allow for reintroduction of extinct species. For example, some scientists have proposed attempting to clone woolly mammoths by using DNA from frozen tissues and the egg cell of a closer related species, most likely an elephant. Reproductive cloning can also be used to replicate important lines of livestock, a valuable tool for many farmers and livestock breeders. And that was the driving force for experiments that led to Dolly. As previously mentioned, therapeutic cloning has the potential to provide patients with rejection-free organs and tissues, and could also be utilized to provide treatments for individuals with autoimmune diseases. To date, however, therapeutic and reproductive cloning procedures are still not common. This is due to technical difficulties and related low rates of success, as well as ethical concerns with the use of human embryos, or the reintroduction of extinct species into an environment that is quite different for when they existed on Earth. Because of ethical concerns associated with human embryos and therapeutic cloning, there is now considerable focus on induced pluripotent stem cells, which are not derived from embryos. But here there are still significant technical hurdles to overcome before treatments with these cells become routine. Lesson 21 In this lesson, we will learn about the mechanisms of evolutionary change. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. Evolution is an important and recurring theme in biology. The term evolution is often used in a non-scientific sense to refer to any entity changing over time. In biology, we define evolution as change in populations of organisms over time. The specific manner in which these populations change is through shifts in allele frequencies within the populations over time. It is critical to remember that evolution happens at the population level over time. Individuals do not evolve during their lifetime. In order for changes to occur in a population, there must be genetic variation, the presence of different alleles in a population. You should recall from previous lessons that alleles are different versions of the same gene. You saw several examples involving the alleles for white and purple pea flowers during our discussion of inheritance, there are many processes that lead to changes in allele frequencies in populations over time. You will learn about them throughout this lecture. Another way to define evolution is to save that it is the result of any process that changes the genetic composition of a population over time. While the underlying cause of genetic variation is differences in the genetic code, it can be studied at different levels within populations. Examples of this are variation in morphology, proteins, genes, and genomes. These lupines display polymorphic variation. This population has individuals with many different alleles resulting in a range of flower colors. An individual's genotype contributes to its phenotype. You should recall that a genotype is the genetic constitution or makeup of an individual. While the phenotype refers to the apparent characteristics of an individual. The phenotype is the result of the individual's genotype. It's inherited characteristics as well as environmental factors. Climate, nutrition, and the presence or absence of predators, are only a few environmental factors that can affect an individual's phenotype. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. A primary focus in evolutionary biology is understanding the mechanisms of evolution or the factors that cause allele frequencies to change over time in a population. We will focus on mutation, gene flow, genetic drift, and selection. Nonrandom mating does not affect allele frequencies in the same way as the other processes, so we will not cover it in this lesson. These mechanisms act both over short scales from generation to generation and over eons to create the vast diversity of life on Earth. Mutation is any change in the base sequence of DNA. It is the ultimate source of genetic variation and gives rise to different alleles. Despite their importance for genetic variation, mutations are rare and not the primary cause of changes in allele frequency within a population. Genes mutate so infrequently once per a 100 thousand cell divisions, that the other four processes have a bigger role in evolutionary change. Gene flow is the movement of alleles from one population to another. This can occur when an individual moves to join a new population, or when gametes in immature stages of plants are carried to new locations via wind or other organisms. Gene flow can occur when individuals mate with members of other populations. Gene flow can lead to new alleles being introduced into a population or a shift in allele frequencies. Genetic drift is defined as change in allele frequencies due to sampling error, often called random chance. Genetic drift has the largest effect in small populations. Much in the same way that sampling error can impact the results of an experiment. Given sufficient time, genetic drift alone can cause substantial changes to allele frequencies. If only a small number of individuals produce offspring, the alleles carried by the offspring may not represent the parent generation. Genetic drift can cause two small populations that become isolated from one another to significantly differ genetically, even without natural selection acting on them. Harmful alleles can build up while beneficial ones are lost simply due to a small population size. Founder effect is the phenomenon in which a few individuals disperse and give rise to a new population. Because it is unlikely that a few individuals will carry all the alleles found in the original population. The new population will display a different allele frequency or even lack certain alleles altogether. If the founding individuals happened to carry rare alleles, the new population might have a much higher frequency of an allele, considered to be rare among the original population. In this example, the island was colonized only by birds with alleles for red feathers. Founder effect is common among plants since a single seed can disperse and give rise to a whole population. Islands such as the Galapagos are excellent places to observe the founder effect since they're typically colonized by a small number of individuals who float or fly in from the original group. Bottleneck effect refers to a situation in which a population loses genetic variation from a drastic reduction in size, only a small number of individuals are contributing gametes to the next-generation. Therefore, the alleles they carry may not represent all the alleles found in the parent population. Common causes are disease, natural disasters, or changes in the environment. If most of the population is wiped out, it is possible that the surviving individuals will not carry all the alleles found in the starting population. Bottleneck effect is often a problem in populations of endangered animals. Even if the population rebounds in number of individuals, the genetic variability will still be lower than it once was. The northern elephant seals population suffered from bottleneck effect when the population was reduced to only 20 individuals in 1890. Even though the population rebounded to tens of thousands of seals after protective measures were implemented. Much of the original genetic variation has been lost. Selection is the process of organisms leaving differential numbers of progeny based on phenotype and behavior. The work of Charles Darwin resulting from his expedition on the HMS Beagle, notably his book, On the Origin of Species, is quite well-known and laid the foundation for the modern concept of evolution. He suggested natural selection is the mechanism of evolution. This excerpt from the introduction of On the Origin of Species summarizes Darwin's ideas about natural selection. As many more individuals of each species are born than can possibly survive. And as consequently, there is a frequently recurring struggle for existence. It follows that any being, if it vary, however slightly in any manner profitable to itself, under the complex and sometimes varying conditions of life will have a better chance of surviving and thus be naturally selected. From the strong principle of inheritance, any selected variety will tend to propagate its new and modified form. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. There are three conditions that must be met for evolution to occur by natural selection. Individuals in a population must be variable, there must be differences in the rates of survival and reproduction between individuals, individuals who are more successful at surviving and reproducing are said to have higher fitness. Finally, there must be a genetic basis for the variation, meaning it can be passed from parent to offspring. To summarize, survival and reproduction are not random. Individuals with the most favorable variations, those who are better at surviving and reproducing, often said to be more fit, are naturally selected. Natural selection occurs when environmental conditions affect which individuals produce the most offspring. Types of natural selection include, but are not limited to, selection to avoid predators, selection to match climatic conditions, selection for pesticide resistance, and selection for microbial resistance. The outcome of natural selection depends on both fitness and allele frequency. If a fitness effect is strong enough, it can act quickly on a population, even when the differences in allele frequency are small. Selection is often very complex and interactions with other mechanisms can lead to a variety of outcomes. When evolutionary forces interact, they can either work together or in opposition. For example, mutations and genetic drift often counter selection. Gene flow can either promote or prevent evolutionary change. If less favorite alleles are continuously being introduced into a population through migration, it can hinder their removal through selection. Artificial selection occurs when a human deliberately selects for certain characteristics in a population. Examples are short legs on a breed of dog or fruit color on a pepper plant. Selective breeding is another name for this process. It is important to remember that natural selection and evolution are not the same thing. Natural selection is the process that leads to evolution. Evolution refers to historical record of change through time. It is the outcome of natural selection. This slide summarizes the four processes discussed in the lecture and shows how they work together to change the genetic composition of a population. A mutation gave rise to the blue insect phenotype. In a small population, genetic drift can lead to the loss of this mutation. Additionally, it is possible that predators are able to spot blue caterpillars better, leading to natural selection favoring the green and maroon individuals. Migration or gene flow is another way the allele frequencies of the insect populations could be changed. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. You may have heard of survival of the fittest, but there is more to fitness in the evolutionary sense than merely surviving. When evolutionary biologists say fitness, they're referring to reproductive success. Survival, mating success, and the number of offspring per mating are all components of fitness. Selection favors phenotypes with the greatest fitness. Predicting fitness can be challenging since so many variables are involved. Water striders are a good example. Large water striders tend to lay more eggs per day, but they also die at a younger age than smaller ones, which means they have fewer chances to lay eggs. These two opposing directions of selection actually cancel each other out. And the intermediate sized water striders produced the most offspring. In this example, natural selection favors the mice with darker coat color since they're able to blend in with the environment, which lowers their chances of being eaten by predators such as owls. Traditionally, scientists have made their observations about evolution based on evidence from the past, but it is possible to observe natural selection occurring in populations with short lifespans. The effects of the Industrial Revolution in England lead to phenotypic changes in the peppered moth population and allowed scientists to actively observe changes in allele frequencies. The tree bark was light-colored before the Industrial Revolution. Light-colored peppered moths were more common since they could blend in with the bark and avoid predation. As soot from the factories darkened all the tree bark, the allele frequency shifted and dark colored moths became more prevalent. After factory emissions were regulated, the trees eventually returned to their natural state with light bark and the peppered moth populations experienced yet another allele shift to match. Guppy color variation has been used to study natural selection both in the lab and in the field. Bright coloration and spots make guppies a target for predation. When guppies are raised in tanks without predators for several generations, they display these features. When predators are placed in the tank, selection favors dull guppies because they can blend in with the environment and hide from predators. The bright colored guppies are eaten and do not pass their alleles on to the next generation. Laboratory experiments are a wonderful tool, but ideally, such experiments should be duplicated in nature to make sure the results truly reflect something that happens in the wild. A similar study was set up using a waterfall as a barrier between two pools of guppies. Guppies from the lower pool containing predators were moved to the upper pool, which did not contain predators to see if the same results would be observed. After several generations, the guppies in the non predator pool did begin to display brighter colors and spots, indicating that natural selection from predation plays a role in guppy coloration. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. The rediscovery of Mendel's work led Godfrey H. Hardy and Wilhelm Weinberg to investigate the reasons why populations maintain multiple alleles and independently develop what is now known as the Hardy-Weinberg principle. The initial question they sought to answer was why the original proportions of genotypes in a population remained constant over many generations, as opposed to the dominant phenotype eventually taking over. They concluded that the proportions of genotypes in a population will remain constant as long as these conditions are met. No mutations take place, no genes are transferred to or from other sources, in other words, no individuals are migrating into or out of the population, mating is random, meaning that individuals do not choose their mates based on phenotype or genotype, the population is very large and no selection is occurring. When the proportion of genotypes does not change, the population is said to be in Hardy-Weinberg equilibrium. If a population is in Hardy-Weinberg equilibrium, there are no evolutionary forces acting on it. The Hardy-Weinberg principle can be used to detect whether evolutionary processes are underway in a population. If there are no evolutionary processes occurring in a population it is in Hardy-Weinberg equilibrium. The allele and genotype frequencies will not change throughout the generations. If the frequencies are changing over time, further examination may they lead to hypotheses about the cause. If a study found that each new generation of cats had a greater proportion of homozygotes, several assumptions can be made. It's possible that natural selection was favoring homozygotes. Individuals could be choosing genetically similar mates. Or homozygote individuals were immigrating into the population or that the heterozygotes were leaving. Aside from helping us better understand the natural world, evolutionary studies have direct application to many key disciplines. In the medical field, knowledge of evolution is critical for understanding antibiotic resistance and infectious diseases. Evolutionary knowledge is also important for conservation efforts since defining species of concern is crucial. Additionally, understanding why species go extinct and how extinction might be prevented, as well as why certain problems affect small populations can help save species from extinction. In the agricultural sciences, artificial selection, that is evolution driven by human selection, plays an important role in crop development. Pesticide resistance and the use of genetically modified organisms are also major issues that involve principles of evolution. Lesson 22 In this lesson, we will discuss species concepts, mechanisms of speciation, and the pace of evolution. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. Defining a species is a challenging endeavor. Biologists still struggle to agree on a single species concept that applies to all situations. There are two phenomena that all species concepts must account for. A species concept must consider the distinctiveness of species which occur together in a single location, as well as the connections that occur between populations of a single species living in different locations. Groups of species can be organized into two major categories, sympatric and allopatric. Sympatric species live in the same geographic location and often utilize different parts of the habitat. Allopatric species live in different geographic locations and are often separated by some sort of physical barrier, such as a body of water or mountain range. When different species live in the same area but remain distinct, we refer to them as sympatric. Sympatric species usually have visible morphological differences. If you go for a walk in a garden, you will see many different butterflies on the flowers. All these butterflies are living in the same place, but their morphologies are unique and you can see that they are different species. Unfortunately for biologists, this isn't the case with all sympatric species. Some organisms may look the same but have different mating calls or behaviors. The organisms can recognize members of their own species easily, but we might have trouble telling them apart. Some species of frogs, for example, such as the one pictured here, looks similar to other species but have distinct mating calls. A single species may not always be found in a continuous range. When populations of the same species are found in different areas, they often have distinct differences. These populations are often classified as subspecies. If the two populations are found close together, you would expect to find intermediate individuals. Members of different subspecies must be able to produce fertile offspring together to be considered members of the same species. You may be wondering how similar species living in the same area can remain distinct. Despite living in the same range, they only exchange genetic material with members of the same species. We will discuss some of the mechanisms responsible for this later. Conversely, members of geographically distant populations must still experience some gene flow to continue to be the same species. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. Populations are said to be reproductively isolated when individuals will not mate with each other, cannot mate with each other or produce sterile offspring. Reproductive isolation is a critical aspect of the process of speciation. It is important to remember that you must have disruption in gene flow before reproductive isolation can occur. Reproductive isolation is an umbrella term describing many different mechanisms. Some mechanisms are referred to as pre-zygotic because they prevent a zygote from being formed. Others are referred to as post-zygotic because they impact hybrid zygotes if the pre-zygotic mechanisms fail to prevent fertilization. We will discuss each of these mechanisms in greater detail in the next few slides. Species that occur in the same area may use different areas of the environment and not encounter each other frequently, if at all. This is called ecological isolation. For example, lions and tigers are technically capable of producing hybrid offspring. The figure shows a lion tiger hybrid produced in captivity. Even though the ranges of the two species overlap in India, hybridization does not occur in nature because each species utilizes different parts of the habitat and they simply do not encounter each other very often. In the case of behavioral isolation, differences in courtship rituals and mating calls prevent different species from accidentally mating. Blue footed boobies have elaborate mating dances. There are several closely-related species in the same region that they have different foot colors and unique mating rituals that help them find the proper species to mate with. Different species of lace wings pictured in the right figure, have unique mating calls that help them attract mates of the same species. Temporal isolation is another important mechanism of reproductive isolation. Species capable of hybridizing in the laboratory may not hybridize in nature because they have different growing or breeding seasons. Structural differences in the reproductive systems of some plants and animal species prevent mating from occurring. Many closely-related insect groups have specialized reproductive organs that are not functional between different species. Flowers often have unique structures or pollinators that limit the transfer of pollen between species. Mechanisms that prevent the fusion of gametes are important for animals that shed gametes directly into the water and plants that rely on wind or pollinators to transfer pollen. Species that undergo internal fertilization may also have mechanisms that prevent sperm and pollen from different species from fertilizing eggs. All of the mechanisms discussed previously were pre-zygotic isolation mechanisms. They prevent the formation of a zygote from occurring. If mating does occur and results in fertilization there are still mechanisms that prevent fertile offspring from being produced. We call these postzygotic isolation mechanisms. In some situations the embryos are not viable and do not survive. We call this hybrid inviability. If the hybrids do survive it is unlikely that they will be fertile. This is referred to as hybrid infertility. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. The biological species concept was proposed by Ernst Mayr in 1942. He defined a species as groups of actually or potentially inter-breeding natural populations which are reproductively isolated from other such groups. In short, to be considered the same species, individuals must be capable of producing fertile offspring. The biological species concept is not perfect. Sometimes individuals we have classified as separate species will mate and produce fertile intermediate offspring. The figure shows the morphology of four distinct populations of milk snakes. The morphotypes are quite unique and they live in distinct geographic regions. Based on this, one might assume they are all separate species. Careful observation of the snakes has revealed that they do interbreed where the populations crossover and produce fertile offspring with intermediate morphotypes. For this reason, the snake populations have been classified as subspecies. In-depth studies of all closely-related species are required to investigate whether they are interbreeding and producing fertile offspring in order to classify them under the biological species concept. Obviously, it is not feasible to carry out such studies for all populations of species. There are other pitfalls that can complicate the biological species concept. For example, hybridization is incredibly common between plant species, making it difficult to classify them using the definitions of the biological species concept. In both plants and animals, it is difficult to apply the principle to geographically isolated populations since interbreeding cannot be observed. Additionally, the biological species concept cannot be applied to asexual organisms since they do not mate. This limits its utility to sexually reproducing organisms. The phylogenetic species concept proposes that the term species should only be applied to groups of populations that have been evolving independently from other groups of populations. Let's review what a phylogeny is since it is the basis of the phylogenetic species concept. Phylogenies are often referred to as evolutionary trees. They represent hypotheses about patterns of relationships among or between species based on comparison of shared traits. In the phylogenetic species concept species are identified by examining phylogenies from phylogenetic analyses. The phylogenetic species concept solves two problems with the biological species concept. It does not require knowledge about the ability of allopatric populations to interbreed. And it can be applied to both sexually and asexually reproducing species. Despite the problems it solves, the phylogenetic species concept still has drawbacks. Using this concept may lead to defining every slightly different population as a new species, even though they can still produce fertile offspring. Species may not always be found in a single clade, contrary to strict definitions of the phylogenetic species concept. In the figure here, you can see that all five plant populations were initially members of the same species. Population C experienced recent evolutionary changes that the other populations did not, causing it to be differentiated and reproductively isolated. This population would be considered a separate species by all species concepts. The problem is that population C now interrupts the clade, which would normally be used to define the other four populations as a single species. So what is a species? In summary, defining a species is not simple and there is still much debate about the criteria for classifying organisms. Species formation is a continuous process. During the process, populations may only be partially reproductively isolated. If reproductive isolation mechanisms have not fully evolved and members of the two populations meet and began breeding, the differences they have developed will disappear over time and speciation will not occur. If the evolution of reproductive isolation mechanisms is advanced when the populations meet, breeding will not be successful and they will remain separate species. If hybrids are partially sterile, selection will favor alleles preventing hybridization since non-hybrid meetings results in more offspring. This is why it is said that selection may favor reproductive isolation. The continual improvement of pre-zygotic isolation mechanisms is called reinforcement. European flycatchers presents an excellent example of reinforcement. The pied and collared flycatchers display similar morphological features when they do not live in the same range. In regions where the two species are sympatric, they have evolved different coloration that makes it easier to find the proper species to mate with, preventing accidental hybridization. The development of different morphologies acts as a pre-zygotic isolation mechanism. This is an example of reinforcement since the mechanism continues to improve when species lives sympatrically; therefore, helping ensure that gene flow is not occurring. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. Reinforcement does not always result in speciation. When partially isolated populations come together, gene flow will occur to some degree if any of the resulting hybrids are fertile. If they don't produce any fertile offspring, reproductive isolation is already complete. Surviving hybrids serve as a conduit for gene flow. Adaptive change in mating signals can result from selective forces in new environments leading to reproductive isolation. Let's use anoles as an example. Male anoles use colorful dewlaps, the flap of skin under their neck to attract mates. The color of the dewlap can't match the plants that the anole lives on, or it will be hard for the females to see. When an anole moves to new environments, selection favors dewlaps that do not match the surroundings. As a result, different colors of dewlaps lead to speciation since female preference for certain color serves as a means of reproductive isolation. Random changes in the allele frequency of a population can also cause reproductive isolation. Genetic drift, founder effects and population bottlenecks can all lead to reproductive isolation. Given a long enough period of time, any isolated population will diverge due to genetic drift. If divergence leads to traits which cause reproductive isolation speciation will occur. Allopatric species are geographically isolated from each other. If a population is broken into smaller populations due to geographic changes, allopatric speciation may occur since gene flow stops. Examples shown in the figure include an ocean separating a colonizing population from the ancestral one, a river or stream cutting through a population, and extinction of intermediate populations leading to isolation. Sometimes geographic isolation is an important prerequisite for speciation. In little paradise king fisher species, we see that there's little variation between species living on the mainland since the population is large and gene flow is occurring constantly. Among the different species that developed on isolated islands, we see a great deal of variation. This variation may ultimately lead to speciation of gene flow ceases. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. Adaptive radiation describes a group of species which have recently evolved from a common ancestor by adapting to different parts of the environment. Adaptive radiation can lead to diversity. The figure shows how adaptive radiation occurs on an island archipelago. Initially an ancestral species colonize an island. Gradually members of the population colonize other islands and allopatric speciation occurs. Each new species has evolved in response to different environmental variables on each of the islands. Eventually members of these new species will move to the other islands. Because they are specialized to certain conditions and habitats, there will be less competition between the species and they can coexist together since they use different resources and parts of the environment. Cichlids in Lake Victoria are a case of recent and rapid evolution. Molecular dating studies have shown that the cichlid population has only lived in the lake for about 200 thousand years. That is a rather short period of time in terms of deep history. The lake has undergone many fluctuations in its water-level leading to isolated populations. This has contributed to periods of rapid evolution. The cichlids have an unusual secondary set of functional jaws that may have been instrumental in their evolutionary radiation. Having a second set of jaws has allowed the primary set to have evolutionary flexibility, leading to modification for diverse functions, that assist with life in the different habitats. There are two hypotheses to explain the relationship between speciation and the evolutionary change that occurs within a species over time. Gradualism and punctuated equilibrium. With gradualism, small changes occur very slowly and accumulate over thousands and millions of years, leading to major changes. With punctuated equilibrium, species experience periods of little to no change, punctuated by bursts of evolutionary change occurring over relatively short periods of time. The figure provides a visual example to contrast the two pieces of evolution. Gradualism and punctuated equilibrium represent two ends of the spectrum, but they are not mutually exclusive. Evidence suggests that some groups have evolved in a gradual manner, some through punctuated equilibrium, and others have had both gradual and punctual episodes in their evolutionary history. Lesson 23 In this lesson, we will examine how scientists reconstruct evolutionary relationships and make hypotheses. Here are the major learning objectives for this lecture. Pause the video for a moment to review these and then keep them in mind as you proceed through the lecture. After completing the lecture, you should be able to do each of these objectives. Understanding the ancestor, descendant relationships of life on Earth is a challenging feat. If the fossil record were perfect and presented a complete collection of fossils through time, we would be able to easily trace these relationships. Unfortunately, the fossil record is far from complete. Scientists have found other types of evidence to use in the formation of hypotheses about evolutionary relationships. We will discuss these throughout the lesson. The field of phylogenetic systematics, often referred to simply as systematics, deals with the reconstruction and study of evolutionary relationships. Scientists in this field are called systematists. They construct evolutionary trees called phylogenies to present hypotheses about relationships among species. Branching diagrams are used to depict evolutionary relationships. Charles Darwin illustrated the idea of species descending from a single ancestor with a branching tree diagram. An early tree diagram from his notebook can be seen on the left. This type of diagram is called a phylogeny. Sometimes these diagrams are referred to as cladograms. A cladogram is a type of phylogeny that does not include any type of scale for the evolutionary changes that depicts. We will simply use the term phylogeny in this lecture. The figure on the right presents three variations of the same phylogeny. All three figures show the exact same relationship between the primates, even though the branches are arranged in slightly different configurations. It is crucial to interpret a phylogeny by looking at how recently sets of species shared a common ancestor, rather than looking at the arrangement of species across the top of the tree, since this order may change without affecting the actual relationships. Take a moment to compare the three versions. You should be able to see that they depict the same relationships. Phylogenies are essentially hypotheses about the way groups of organisms are related based on the best available data. There are multiple sources of data that can be used. DNA and protein sequence data is the most common source of information for generating phylogenies. But morphology, physiology, and behavioral data can also be used. Let's take a moment to familiarize ourselves with so