Punnett square

A Punnett square showing a typical test cross

The Punnett square is a diagram that is used to predict an outcome of a particular cross or breeding experiment. It is named after Reginald C. Punnett, who devised the approach, and is used by biologists to determine theprobability of an offspring's having a particular genotype. The Punnett square is a tabular summary of every possible combination of one maternal allele with one paternal allele for each gene being studied in the cross.[1] These tables give the correct probabilities for the genotype outcomes of independent crosses where the probability of inheriting copies of each parental allele is independent. The Punnett Square is a visual representation of Mendelian inheritance.

Monohybrid cross

In this example, both organisms have the genotype Bb. They can produce gametes that contain either the B or the b allele. (It is conventional in genetics to use capital letters to indicate dominant alleles and lower-case letters to indicate recessive alleles.) The probability of an individual offspring's having the genotype BB is 25%, Bb is 50%, and bb is 25%.

Paternal

B b

Maternal

B BB Bb

b Bb bb

It is important to note that Punnett squares give probabilities only for genotypes, not phenotypes. The way in which the B and b alleles interact with each other to affect the appearance of the offspring depends on how the gene products (proteins) interact (see Mendelian inheritance). For classical dominant/recessive genes, like that which determines whether a rat has black hair (B) or white hair (b), the dominant allele will mask the recessive one. Thus, in the example above, 75% of the offspring will be black (BB or Bb) while only 25% will be white (bb). The ratio of the phenotypes is 3:1, typical for a monohybrid cross.

Dihybrid cross

Main article: Dihybrid cross

More complicated crosses can be made by looking at two or more genes. The Punnett square works, however, only if the genes are independent of each other, which means that having a particular allele of gene A does not alter the probability of possessing an allele of gene B. This is equivalent to stating that the genes are not linked, so that the two genes do not tend to sort together during meiosis.

The following example illustrates a dihybrid cross between two heterozygous pea plants. R represents the dominant allele for shape (round), while r represents the recessive allele (wrinkled). A represents the dominant allele for color (yellow), while a represents the recessive allele (green). If each plant has the genotype RrAa, and since the alleles for shape and color genes are independent, then they can produce four types of gametes with all possible combinations:RA, Ra, rA, and ra.

RA Ra rA ra

RA RRAA RRAa RrAA RrAa

Ra RRAa RRaa RrAa Rraa

rA RrAA RrAa rrAA rrAa

ra RrAa Rraa rrAa rraa

Since dominant traits mask recessive traits, there are nine combinations that have the phenotype round yellow, three that are round green, three that are wrinkled yellow, and one that is wrinkled green. The ratio 9:3:3:1 is typical for a dihybrid cross.

Tree method

Another way to solve dihybrid and multihybrid crosses is to use the tree method, although it does not display the genotypes of the gametes correctly.

This method is particularly advantageous when crossing homozygous organisms.

Probability of Inheritance

The value of studying genetics is in understanding how we can predict the likelihood of inheriting particular traits. This can help plant and animal breeders in developing varieties that have more desirable qualities. It can also help people explain and predict patterns of inheritance in family lines.

One of the easiest ways to calculate the mathematical probability of inheriting a specific trait was invented by an early 20th century English geneticist named Reginald Punnett . His technique employs what we now call a Punnett square. This is a simple graphical way of discovering all of the potential combinations of genotypes that can occur in children, given the genotypes of their parents. It also shows us the odds of each of the offspring genotypes occurring.

Setting up and using a Punnett square is quite simple once you understand how it works. You begin by drawing a grid of perpendicular lines:

Next, you put the genotype of one parent across the top and that of the other parent down the left side. For example, if parent pea plant genotypes were YY and GG respectively, the setup would be:

Note that only one letter goes in each box for the parents. It does not matter which parent is on the side or the top of the Punnett square.

Next, all you have to do is fill in the boxes by copying the row and column-head letters across or down into the empty squares. This gives us the predicted frequency of all of the potential genotypes among the offspring each time reproduction occurs.

In this example, 100% of the offspring will likely be heterozygous (YG). Since the Y (yellow) allele is dominant over the G (green) allele for pea plants, 100% of the YG offspring will have a yellow phenotype, as Mendel observed in his breeding experiments.

In another example (shown below), if the parent plants both have heterozygous (YG) genotypes, there will be 25% YY, 50% YG, and 25% GG offspring on average. These percentages are determined based on the fact that each of the 4 offspring boxes in a Punnett square is 25% (1 out of 4). As to phenotypes, 75% will be Y and only 25% will be G. These will be the odds every time a new offspring is conceived by parents with YG genotypes.

An offspring's genotype is the result of the combination of genes in the sex cells or gametes (sperm and ova) that came together in its conception. One sex cell came from each parent. Sex cells normally only have one copy of the gene for each trait (e.g., one copy of the Y or G form of the gene in the example above). Each of the two Punnett square boxes in which the parent genes for a trait are placed (across the top or on the left side) actually represents one of the two possible genotypes for a parent sex cell. Which of the two parental copies of a gene is inherited depends on which sex cell is inherited--it is a matter of chance. By placing each of the two copies in its own box has the effect of giving it a 50% chance of being inherited.

If you are not yet clear about how to make a Punnett Square and interpret its result, take the time to try to figure it out before going on.

Are Punnett Squares Just Academic Games?

Why is it important for you to know about Punnett squares? The answer is that they can be used as predictive tools when considering having children. Let us assume, for instance, that both you and your mate are carriers for a particularly unpleasant genetically inherited disease such as cystic fibrosis . Of course, you are worried about whether your children will be healthy and normal. For this example, let us define "A" as being the dominantnormal allele and "a" as the recessive abnormal one that is responsible for cystic fibrosis. As carriers, you and your mate are both heterozygous (Aa). This disease only afflicts those who are homozygous recessive (aa). The Punnett square below makes it clear that at each birth, there will be a 25% chance of you having a normal homozygous (AA) child, a 50% chance of a healthy heterozygous (Aa) carrier child like you and your mate, and a 25% chance of a homozygous recessive (aa) child who probably will eventually die from this condition.

If both parents are carriers of the recessive allele for a disorder, all of their children will face the following odds of inheriting it: 25% chance of having the recessive disorder 50% chance of being a healthy carrier 25% chance of being healthy and not have the recessive allele at all

If a carrier (Aa) for such a recessive disease mates with someone who has it (aa), the likelihood of their children also inheriting the condition is far greater (as shown below). On average, half of the children will be heterozygous (Aa) and, therefore, carriers. The remaining half will inherit 2 recessive alleles (aa) and develop the disease.

If one parent is a carrier and the other has a recessive disorder, their children will have the following odds of inheriting it: 50% chance of being a healthy carrier 50% chance having the recessive disorder

It is likely that every one of us is a carrier for a large number of recessive alleles. Some of these alleles can cause life-threatening defects if they are inherited

from both parents. In addition to cystic fibrosis, albinism, and beta-thalassemia are recessive disorders.

Some disorders are caused by dominant alleles for genes. Inheriting just one copy of such a dominant allele will cause the disorder. This is the case with Huntington disease, achondroplastic dwarfism, and polydactyly. People who are heterozygous (Aa) are not healthy carriers. They have the disorder just like homozygous dominant (AA) individuals.

If only one parent has a single copy of a dominant allele for a dominant disorder, their children will have a 50% chance of inheriting the disorder and 50% chance of being entirely normal.

Punnett squares are standard tools used by genetic counselors. Theoretically, the likelihood of inheriting many traits, including useful ones, can be predicted using them. It is also possible to construct squares for more than one trait at a time. However, some traits are not inherited with the simple mathematical probability suggested here. We will explore some of these exceptions in the next section of the tutorial.

Genetic engineering

Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome usingbiotechnology. New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or "knocked out", using a nuclease. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations.

An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). The first GMOs were bacteria in 1973; GM mice were generated in 1974. Insulin-producing bacteria were commercialized in 1982

and genetically modified food has been sold since 1994. Glofish, the first GMO designed as a pet, was first sold in the United States December in 2003.[1]

Genetic engineering techniques have been applied in numerous fields including research, agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and medicines such as insulin and human growth hormone are now manufactured in GM cells, experimental GM cell lines and GM animals such as mice or zebrafish are being used for research purposes, and genetically modified crops have been commercialized.

Definition[

Genetic engineering alters the genetic makeup of an organism using techniques that remove heritablematerial or that introduce DNA prepared outside the organism either directly into the host or into a cell that is then fused or hybridized with the host. This involves using recombinant nucleic acid(DNA or RNA) techniques to form new combinations of heritable genetic material followed by the incorporation of that material either indirectly through a vector system or directly through micro-injection, macro-injection and micro-encapsulation techniques.

Genetic engineering does not normally include traditional animal and plant breeding, in vitro fertilisation, induction of polyploidy, mutagenesis and cell fusion techniques that do not use recombinant nucleic acids or a genetically modified organism in the process.[4] However the European Commission has also defined genetic engineering

broadly as including selective breeding and other means of artificial selection.[5] Cloning and stem cell research, although not considered genetic engineering,[6] are closely related and genetic engineering can be used within them.[7]Synthetic biology is an emerging discipline that takes genetic engineering a step further by introducing artificially synthesized genetic material from raw materials into an organism.[8]

If genetic material from another species is added to the host, the resulting organism is calledtransgenic. If genetic material from the same species or a species that can naturally breed with the host is used the resulting organism is called cisgenic.[9] Genetic engineering can also be used to remove genetic material from the target organism, creating a gene knockout organism.[10] In Europe genetic modification is synonymous with genetic engineering while within the United States of America it can also refer to conventional breeding methods.[11][12] The Canadian regulatory system is based on whether a product has novel features regardless of method of origin. In other words, a product is regulated as genetically modified if it carries some trait not previously found in the species whether it was generated using traditional breeding methods (e.g., selective breeding, cell fusion, mutation breeding) or genetic engineering.[13][14][15] Within the scientific community, the term genetic engineering is not commonly used; more specific terms such as transgenic are preferred.

Genetically modified organisms[edit]

Main article: Genetically modified organism

Plants, animals or micro organisms that have changed through genetic engineering are termed genetically modified organisms or GMOs.[16] Bacteria were the first organisms to be genetically modified. Plasmid DNA containing new genes can be inserted into the bacterial cell and the bacteria will then express those genes. These genes can code for medicines or enzymes that process food and other substrates.[17][18] Plants have been modified for insect protection, herbicide resistance, virus resistance, enhanced nutrition, tolerance to environmental pressures and the production of edible vaccines.[19] Most commercialised GMO's are insect resistant and/or herbicide tolerant crop plants.[20] Genetically modified animals have been used for research, model animals

and the production of agricultural or pharmaceutical products. They include animals with genes knocked out, increased susceptibility to disease, hormones for extra growth and the ability to express proteins in their milk.[21]

History

Humans have altered the genomes of species for thousands of years through artificial selection and more recently mutagenesis. Genetic engineering as the direct manipulation of DNA by humans outside breeding and mutations has only existed since the 1970s. The term "genetic engineering" was first coined byJack Williamson in his science fiction novel Dragon's Island, published in 1951,[22] one year before DNA's role in heredity was confirmed by Alfred Hersheyand Martha Chase,[23] and two years before James Watson and Francis Crick showed that the DNA molecule has a double-helix structure.

In 1972 Paul Berg created the first recombinant DNA molecules by combining DNA from the monkey virus SV40with that of the lambda virus.[24] In 1973 Herbert Boyer and Stanley Cohen created the first transgenic organism by inserting antibiotic resistance genes into the plasmid of an E. coli bacterium.[25][26] A year later Rudolf Jaenischcreated a transgenic mouse by introducing foreign DNA into its embryo, making it the worlds first transgenic animal.[27] These achievements led to concerns in the scientific community about potential risks from genetic engineering, which were first discussed in depth at the Asilomar Conference in 1975. One of the main recommendations from this meeting was that government oversight of recombinant DNA research should be established until the technology was deemed safe.[28][29]

In 1976 Genentech, the first genetic engineering company, was founded by Herbert Boyer and Robert Swanson and a year later the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.[30] In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented.[31] The insulin produced by bacteria, brandedhumulin, was approved for release by the Food and Drug Administration in 1982.[32]

In the 1970s graduate student Steven Lindow of the University of WisconsinMadison with D.C. Arny and C. Upper found a bacterium he identified as P. syringae that played a role in ice nucleation and in 1977, he discovered a mutant ice-minus strain. Later, he successfully created a recombinant ice-minus strain.[33] In 1983, a biotech company, Advanced Genetic Sciences (AGS) applied for U.S. government authorization to perform field tests with the ice-minus strain of P. syringae to protect crops from frost, but environmental groups and protestors delayed the field tests for four years with legal challenges.[34] In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment[35] when a strawberry field and a potato field in California were sprayed with it.[36] Both test fields were attacked by activist groups the night before the tests occurred: "The world's first trial site attracted the world's first field trasher".[35]

The first field trials of genetically engineered plants occurred in France and the USA in 1986, tobacco plants were engineered to be resistant to herbicides.[37]The Peoples Republic of China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992.[38] In 1994 Calgeneattained approval to commercially release the Flavr Savr tomato, a tomato engineered to have a longer shelf life.[39] In 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first genetically engineered crop commercialized in Europe.[40] In 1995, Bt Potato was approved safe by the Environmental Protection Agency, after having been approved by the FDA, making it the first pesticide producing crop to be approved in the USA.[41] In 2009 11 transgenic crops were grown commercially in 25 countries, the largest of which by area grown were the USA, Brazil, Argentina, India, Canada, China, Paraguay and South Africa.[42]

In the late 1980s and early 1990s, guidance on assessing the safety of genetically engineered plants and food emerged from organizations including the FAO and WHO.[43][44][45][46]

In 2010, scientists at the J. Craig Venter Institute, announced that they had created the first synthetic bacterial genome, and added it to a cell containing no DNA. The resulting bacterium, named Synthia, was the world's first synthetic life form.[47][48]

Process

Main article: Techniques of genetic engineering

The first step is to choose and isolate the gene that will be inserted into the genetically modified organism. As of 2012, most commercialised GM plants have genes transferred into them that provide protection against insects or tolerance to herbicides.[49] The gene can be isolated using restriction enzymes to cut DNA into fragments and gel electrophoresis to separate them out according to length.[50] Polymerase chain reaction (PCR) can also be used to amplify up a gene segment, which can then be isolated through gel electrophoresis.[51] If the chosen gene or the donor organism's genome has been well studied it may be present in a genetic library. If the DNA sequence is known, but no copies of the gene are available, it can be artificially synthesized.[52]

The gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly. The gene can also be modified at this stage for better expression or effectiveness. As well as the gene to be inserted most constructs contain a promoter and terminatorregion as well as a selectable marker gene. The promoter region initiates transcription of the gene and can be used to control the location and level of gene expression, while the terminator region ends transcription. The selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is needed to determine which cells are transformed with the new gene. The constructs are made using recombinant DNA techniques, such asrestriction digests, ligations and molecular cloning.[53] The manipulation of the DNA generally occurs within a plasmid.

The most common form of genetic engineering involves inserting new genetic material randomly within the host genome.[citation needed] Other techniques allow new genetic material to be inserted at a specific location in the host genome or generate mutations at desired genomic loci capable of knocking outendogenous genes. The technique of gene targeting uses homologous recombination to target desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of

gene targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger nucleases,[54][55] engineered homing endonucleases,[56][57] or nucleases created fromTAL effectors.[58][59] In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout.[60][61]

Transformation

About 1% of bacteria are naturally able to take up foreign DNA but it can also be induced in other bacteria.[62] Stressing the bacteria for example, with a heat shock or an electric shock, can make the cell membrane permeable to DNA that may then incorporate into their genome or exist as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus or through the use of viral vectors. In plants the DNA is generally inserted using Agrobacterium-mediated recombination or biolistics.[63]

In Agrobacterium-mediated recombination the plasmid construct contains T-DNA, DNA which is responsible for insertion of the DNA into the host plants genome. This plasmid is transformed into Agrobacterium that contains no plasmids and then plant cells are infected. The Agrobacterium will then naturally insert the genetic material into the plant cells.[64] In biolistics transformation particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will enter the cells and transform them. This method can be used on plants that are not susceptible to Agrobacterium infection and also allows transformation of plant plastids. Another transformation method for plant and animal cells is electroporation. Electroporation involves subjecting the plant or animal cell to an electric shock, which can make the cell membrane permeable to plasmid DNA. In some cases the electroporated cells will incorporate the DNA into their genome. Due to the damage caused to the cells and DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial mediated transformation and microinjection.[65]

As often only a single cell is transformed with genetic material the organism must be regenerated from that single cell. As bacteria consist of a single cell and reproduce clonally regeneration is not necessary. In plants this is accomplished through the use of tissue culture. Each plant species has different requirements for successful regeneration through tissue culture. If successful an adult plant is produced that contains thetransgene in every cell. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Selectable markers are used to easily differentiate transformed from untransformed cells. These markers are usually present in the transgenic organism, although a number of strategies have been developed that can remove the selectable marker from the mature transgenic plant.[66] When the offspring is produced they can be screened for the presence of the gene. All offspring from the first generation will be heterozygous for the inserted gene and must be mated together to produce a homozygousanimal.

Further testing uses PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene. These tests can also confirm the chromosomal location and copy number of the inserted gene. The presence of the gene does not guarantee it will be expressed at appropriate levels in the target tissue so methods that look for and measure the gene products (RNA and protein) are also used. These include northern hybridization, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis. For stable transformation the gene should be passed to the offspring in a Mendelian inheritance pattern, so the organism's offspring are also studied.

Genome editing

Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors." The nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cells endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and nonhomologous end-joining (NHEJ). There are currently four families of engineered

nucleases: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), andCRISPRs.[67][68]

Applications

Genetic engineering has applications in medicine, research, industry and agriculture and can be used on a wide range of plants, animals and micro organisms.

Medicine

In medicine genetic engineering has been used to mass-produce insulin, human growth hormones, follistim (for treating infertility), human albumin, monoclonal antibodies, antihemophilic factors, vaccines and many other drugs.[69][70] Vaccination generally involves injecting weak, live, killed or inactivated forms ofviruses or their toxins into the person being immunized.[71] Genetically engineered viruses are being developed that can still confer immunity, but lack theinfectious sequences.[72] Mouse hybridomas, cells fused together to create monoclonal antibodies, have been humanised through genetic engineering to create human monoclonal antibodies.[73] Genetic engineering has shown promise for treating certain forms of cancer.[74][75]

Genetic engineering is used to create animal models of human diseases. Genetically modified mice are the most common genetically engineered animal model.[76] They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease.[77] Potential cures can be tested against these mouse models. Also genetically modified pigs have been bred with the aim of increasing the success of pig to human organ transplantation.[78]

Gene therapy is the genetic engineering of humans by replacing defective human genes with functional copies. This can occur in somatic tissue or germlinetissue. If the gene is inserted into the germline tissue it can be passed down to that person's descendants.[79][80] Gene therapy has been successfully used to treat multiple diseases, including X-linked SCID,[81] chronic lymphocytic leukemia (CLL),[82] and Parkinson's disease.[83] In 2012, Glybera became the first gene therapy treatment to be approved for

clinical use in either Europe or the United States after its endorsement by the European Commission.[84][85] There are also ethical concerns should the technology be used not just for treatment, but for enhancement, modification or alteration of a human beings' appearance, adaptability, intelligence, character or behavior.[86] The distinction between cure and enhancement can also be difficult to establish. Transhumanistsconsider the enhancement of humans desirable.

Genetic engineering is an important tool for natural scientists. Genes and other genetic information from a wide range of organisms are transformed into bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal, multiply quickly, relatively easy to transform and can be stored at -80 C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research.

Organisms are genetically engineered to discover the functions of certain genes. This could be the effect on the phenotype of the organism, where the gene is expressed or what other genes it interacts with. These experiments generally involve loss of function, gain of function, tracking and expression.

Loss of function experiments, such as in a gene knockout experiment, in which an organism is engineered to lack the activity of one or more genes. A knockout experiment involves the creation and manipulation of a DNA construct in vitro, which, in a simple knockout, consists of a copy of the desired gene, which has been altered such that it is non-functional. Embryonic stem cells incorporate the altered gene, which replaces the already present functional copy. These stem cells are injected into blastocysts, which are implanted into surrogate mothers. This allows the experimenter to analyze the defects caused by this mutation and thereby determine the role of particular genes. It is used especially frequently in developmental biology. Another method, useful in organisms such as Drosophila (fruit fly), is to induce mutations in a large population and then screen the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes.

Gain of function experiments, the logical counterpart of knockouts. These are sometimes performed in conjunction with knockout experiments to more finely establish the function of the desired gene. The process is much the same as that in knockout engineering, except that the construct is designed to increase the function of the gene, usually by providing extra copies of the gene or inducing synthesis of the protein more frequently.

Tracking experiments, which seek to gain information about the localization and interaction of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene, which is a juxtaposition of the wild-type gene with a reporting element such as green fluorescent protein (GFP) that will allow easy visualization of the products of the genetic modification. While this is a useful technique, the manipulation can destroy the function of the gene, creating secondary effects and possibly calling into question the results of the experiment. More sophisticated techniques are now in development that can track protein products without mitigating their function, such as the addition of small sequences that will serve as binding motifs to monoclonal antibodies.

Expression studies aim to discover where and when specific proteins are produced. In these experiments, the DNA sequence before the DNA that codes for a protein, known as a gene's promoter, is reintroduced into an organism with the protein coding region replaced by a reporter gene such as GFP or an enzyme that catalyzes the production of a dye. Thus the time and place where a particular protein is produced can be observed. Expression studies can be taken a step further by altering the promoter to find which pieces are crucial for the proper expression of the gene and are actually bound by transcription factor proteins; this process is known as promoter bashing.

Industrial

Using genetic engineering techniques one can transform microorganisms such as bacteria or yeast, or transform cells from multicellular organisms such as insects or mammals, with a gene coding for a useful protein, such as an enzyme, so that the transformed organism will overexpress the desired protein. One can manufacture mass quantities of the protein by growing the transformed organism in bioreactor equipment

using techniques of industrial fermentation, and then purifying the protein.[88] Some genes do not work well in bacteria, so yeast, insect cells, or mammalians cells, each a eukaryote, can also be used.[89]These techniques are used to produce medicines such as insulin, human growth hormone, and vaccines, supplements such as tryptophan, aid in the production of food (chymosin in cheese making) and fuels.[90] Other applications involving genetically engineered bacteria being investigated involve making the bacteria perform tasks outside their natural cycle, such as making biofuels,[91] cleaning up oil spills, carbon and other toxic waste[92] and detecting arsenic in drinking water.[93]

What is genetic engineering? Genetic engineering is the process of manually adding new DNA to an organism. The goal is to add one or more new traits that are not already found in that organism. Examples of genetically engineered (transgenic) organisms currently on the market include plants with resistance to some insects, plants that can tolerate herbicides, and crops with modified oil content.

Understanding Genetic Engineering: Basic Biology To understand how genetic engineering works, there are a few key biology concepts that must be understood.

CONCEPT #1: What is DNA? DNA is the recipe for life. DNA is a molecule found in the nucleus of every cell and is made up of 4 subunits represented by the letters A, T, G, and C. The order of these subunits in the DNA strand holds a code of information for the cell. Just like the English alphabet makes up words using 26 letters, the genetic language uses 4 letters to spell out the instructions for how to make the proteins an organism will need to grow and live.

Small segments of DNA are called genes. Each gene holds the instructions for how to produce a single protein. This can be compared to a recipe for making a food dish. A recipe is a set of instructions for making a single dish.

An organism may have thousands of genes. The set of all genes in an organism is called a genome. A genome can be compared to a cookbook of recipes that makes that organism what it is. Every cell of every living organism has a cookbook.

CONCEPT #2: Why are proteins important? Proteins do the work in cells. They can be part of structures (such as cell walls, organelles, etc). They can regulate reactions that take place in the cell. Or they can serve as enzymes, which speed-up reactions. Everything you see in an organism is either made of proteins or the result of a protein action.

CONCEPT #3: How is DNA important in genetic engineering? DNA is a universal language, meaning the genetic code means the same thing in all organisms. It would be like if all cookbooks around the world were written in a single language that everyone knew. This characteristic is critical to the success of genetic engineering. When a gene for a desirable trait is taken from one organism and inserted into another, it gives the recipient organism the ability to express that same trait.

How is genetic engineering done? Genetic engineering, also called transformation, works by physically removing a gene from one organism and inserting it into another, giving it the ability to express the trait encoded by that gene. It is like taking a single recipe out of a cookbook and placing it into another cookbook.

The process: Once a goal is in mind

1) First, find an organism that naturally contains the desired trait.

2) The DNA is extracted from that organism. This is like taking out the entire cookbook.

3) The one desired gene (recipe) must be located and copied from thousands of genes that were extracted. This is called gene cloning.

4) The gene may be modified slightly to work in a more desirable way once inside the recipient organism.

5) The new gene(s), called a transgene is delivered into cells of the recipient organism. This is called transformation. The most common transformation technique uses a bacteria that naturally genetically engineer plants with its own DNA. The transgene is inserted into the bacteria, which then delivers it into cells of the organism being engineered. Another technique, called the gene gun method, shoots microscopic gold particles coated with copies of the transgene into cells of the recipient organism. With either technique, genetic engineers have no control over where or if the transgene inserts into the genome. As a result, it takes hundreds of attempts to achieve just a few transgenic organisms.

6) Once a transgenic organism has been created, traditional breeding is used to improve the characteristics of the final product. So genetic engineering does not eliminate the need for traditional breeding. It is simply a way to add new traits to the pool. How does genetic engineering compare to traditional breeding? Although the goal of both genetic engineering and traditional plant breeding is to improve an organisms traits, there are some key differences between them.

While genetic engineering manually moves genes from one organism to another, traditional breeding moves genes through mating, or crossing, the organisms in hopes of obtaining offspring with the desired combination of traits.

genetic engineering

Using the recipe analogy, traditional breeding is like taking two cookbooks and combining every other recipe from each into one cookbook. The product is a new cookbook with half of the recipes from each original book. Therefore, half of the genes in the offspring of a cross come from each parent.

Traditional breeding is effective in improving traits, however, when compared with genetic engineering, it does have disadvantages. Since breeding relies on the ability to mate two organisms to move genes, trait improvement is basically limited to those traits that already exist within that species. Genetic engineering, on the other hand, physically removes the genes from one organism and places them into the other. This eliminates the need for mating and

traditional breeding

allows the movement of genes between organisms of any species. Therefore, the potential traits that can be used are virtually unlimited.

Breeding is also less precise than genetic engineering. In breeding, half of the genes from each parent are passed on to the offspring. This may include many undesirable genes for traits that are not wanted in the new organism. Genetic engineering, however, allows for the movement of a single, or a few, genes.

DNA, RNA and Protein Synthesis

Structure and function of DNA

DNA molecules are incredibly long, but also very thin. One DNA molecule from the chromosome of a mammal may be about 1 m long when unraveled. However, it has to fit in a nucleus of some 5-6 orders of magnitude smaller and is folded up in chromosomes in a highly organized manner. DNA is a linear polymer that is composed of four different building blocks, the nucleotides. It is in the sequence of the nucleotides in the polymers where the genetic information carried by chromosomes is located. Each nucleotide is composed of three parts: (1) a nitrogenous base known as purine (adenine (A) and guanine (G)) or pyrimidine (cytosine (C) and thymine (T)); (2) a sugar, deoxyribose; and (3) a phosphate group (see pp. 20-22 of Molecular Biotechnology for molecular structures of DNA and its components). The nitrogenous base determines the identity of the nucleotide, and individual nucleotides are often referred to by their base (A, C, G, or T). One DNA strand can be up to several hundred million nucleotides in length. T can form a hydrogen bond with A, and C with G; two DNA strands wind together in an antiparallel fashion in a double-helix.

Inside the cell, the DNA acts like an "instruction manual": in its sequence, it provides all the information needed to function, but the actual work of translating the information into a medium that can be used directly by the cell is done by RNA, ribonucleic acid. The structural difference with DNA is that RNA contains a -OH group both at the 2' and 3' position of the ribose ring, whereas DNA (which stands, in fact, for deoxy-RNA) lacks such a hydroxy group at the 2' position of the ribose. Seehttp://www.ch.cam.ac.uk/magnus/molecules/nucleic/sugars.html. The same bases can be attached to the ribose group in RNA as occur in DNA, with the exception that in RNA thymine does not occur, and is replaced by uracil, which has an H-group instead of a methyl group at the C-5 position of the pyrimidine. The molecular structures of uracil and thymine are compared at http://www.ch.cam.ac.uk/magnus/molecules/nucleic/bases.html. The

RNA has three functions: (a) it serves as the messenger that tells the cell (the ribosomes) what protein to make (messenger RNA; mRNA); (b) it serves as part of the structure of the ribosome, the protein/RNA complex that synthesizes proteins according to the information presented by the mRNA (ribosomal RNA; rRNA); and (c) it functions to bring amino acids (the constituents of the proteins) to the ribosome when a specific amino acid "is called for" by the information on the mRNA to be put in into the protein that is being synthesized; this RNA is called transfer RNA (tRNA).

An important point of emphasis should be that all vegetative cells of one organism contain the same genetic information. Upon division, each daughter cell obtains an "exact" copy of the DNA of the parent (see http://accessexcellence.org/AB/GG/dna_replicating.html). However, the specific genes that are expressed at specific times may be very different between different tissues. These differences in gene expression allow for the regulation of development of the organism, and for the development of different tissues. For the most part, DNA-binding proteins (encoded by the DNA) play an important role in the regulation of expression of genes encoded on the DNA. A very important "chicken-and-egg" problem.....

RNAs

The messenger RNA (mRNA) serves as an intermediate between DNA and protein. Parts of the DNA are "transcribed" into transcripts (single-stranded RNA molecules) that are processed to mRNA. In prokaryotes the transcript generally does not need to be processed, and can serve as mRNA right away. Transcription starts at a specific site on the DNA called a promoter. Each gene or operon has its own promoter(s). Transcription ends at a terminator sequence on the DNA. The transcripts usually are 300-50,000 nucleotides long, and contain the information to make protein. In eukaryotes (organisms with cells containing a nucleus; in fact, any higher organism) generally the transcripts needs to be processed before they can serve as a blueprint for a protein. The processing involves the removal of intervening sequences (introns) in the gene. The introns may be anywhere between 50 and 10,000 nucleotides in length. The coding regions of the mRNA are called exons. There may be up to 100 introns in a single gene. The introns are spliced out by small ribonucleoprotein particles (consisting of RNA and protein), which appear to pull the two ends of the intron together. However, there are also introns that splice out without the need of a protein: the RNA sequence itself appears to contain sufficient information to know where to splice out the intron. In addition to the removal of introns, a poly-A sequence is added to the 3 end of the transcript. The processed transcript is the mRNA, and the information in the mRNA can be used to be "translated" into a protein of specific sequence. However, in prokaryotes introns are rare and mRNA generally does not get processed before translation.

The intron splicing process provides an opportunity to increase the amount of usable genetic information without increasing the genome size of the organism: Alternative splicing of a particular transcript can occur. Alternative splicing means that introns may be recognized in different ways in different molecules of the same primary transcript, and the result is that one gene can give rise to different mRNAs and thereby to different proteins. Note that this process is largely limited to eukaryotes as introns in prokaryotes are rare.

Ribosomal RNAs (rRNAs) are essential components of an important part of the protein synthesis machinery: the ribosomes. In addition to rRNA, there are some 70 different proteins in a ribosome. There are hundreds of copies of rRNA genes per genome, thus making the production of lots of rRNA possible. There are four different rRNAs, each with a different size. Each ribosome contains one molecule of each of the four rRNA types. In prokaryotes, ribosomes bind to the mRNA close to the translation start site. This ribosome binding site is referred to as the Shine-Dalgarno sequence or as the ribosome recognition element. In eukaryotes, ribosomes bind at the 5' end of the mRNA and scan down the mRNA until they encounter a suitable start codon.

Transfer RNA (tRNA) carries amino acids to the ribosomes, to enable the ribosomes to put this amino acid on the protein that is being synthesized as an elongating chain of amino acid residues, using the information on the mRNA to "know" which amino acid should be put on next. For each kind of amino acid, there is a specific tRNA that will recognize the amino acid and transport it to the protein that is being synthesized, and tag it on to the protein once the information on the mRNA calls for it.

All tRNAs have the same general shape, sort of resembling a clover leaf. Parts of the molecule fold back in characteristic loops, which are held in shape by nucleotide-pairing between different areas of the molecule. There are two parts of the tRNA that are of particular importance: the aminoacyl attachment site and the anticodon. The aminoacyl attachment site is the site at which the amino acid is attached to the tRNA molecule. Each type of tRNA specifically binds only one type of amino acid. The anticodon (three bases) of the tRNA base-pairs with the appropriate mRNA codon at the mRNA-ribosome complex. This temporarily binds the tRNA to the mRNA, allowing the amino acid carried by the tRNA to be incorporated into the polypeptide in its proper place. Thus, the sequence of the codon (three bases) in the mRNA dictates the amino acid to be put in in the protein at a specific site. The "dictionary" of codons coding for amino acids is called the genetic code. A summary of the amino acids that the 64 possible codons encode can be found at http://molbio.info.nih.gov/molbio/desk.html (choose "Table of Standard Genetic Code" for a codon table, and "Amino Acid Structure and Properties" for information regarding the amino acids). The three codons for which there is no matching tRNA (UAA, UGA, and UAG) serve as "stop-translation" signals at which the ribosome falls off.

Protein synthesis

After having discussed DNA and the various RNAs, the stage has been set for protein synthesis. The basic reaction of protein synthesis is the controlled formation of a peptide bond between two amino acids. This reaction is repeated many times, as each amino acid in turn is added to the growing polypeptide. Protein synthesis starts when the mRNA binds to a small ribosomal subunit near a AUG sequence in the mRNA. The AUG codon is called start codon, since it codes for the first amino acid (a methionine) to be made of the protein. The AUG codon base-pairs with the anticodon of tRNA carrying methionine. A large ribosomal subunit binds to the complex, and the reactions of protein synthesis itself can begin. The aminoacyl-tRNA to be called for next is determined by the next codon (the next three bases) on the mRNA. Each amino acid is coded for by one or more (up to six) codons. Of course, it would be more straightforward

to have each amino acid coded for by only one codon, but nature appears to have chosen a more complex route. The reason for this in part is that there are 20 different amino acids, and 4x4x4=64 different combinations possible in a codon. When the ribosome reaches one of the three codons for which there is no matching tRNA, the ribosome falls off and the synthesized protein is released. The degeneracy of the genetic code for certain amino acids could have a function in regulation of translation; any idea how? The process of protein synthesis has been summarized on pages 34-38 of Molecular Biotechnology, and can also be found on the web at http://accessexcellence.org/AB/GG/protein_synthesis.htmltranslation (in conjunction with transcription) and http://accessexcellence.org/AB/GG/dna_molecule.html.

Amino acids represent quite a broad spectrum of different chemical structures. The web address http://www.ch.cam.ac.uk/magnus/molecules/amino/ provides the structure of all amino acids. With the generation of a protein with a specific amino acid sequence using essentially the genetic information present in the DNA, the link between genetic and functional information is complete.

RNA editing

Over the last several years, it has become obvious that the sequence present in DNA does not always dictate literally the sequence of the protein. In a number of instances "RNA editing" has been observed (particularly in the small genomes present in mitochondria and chloroplasts), in which transcripts are chemically modified (for example, some Cs are changed to Us) by enzymes before translation takes place. Thus, the DNA sequence in such cases does not precisely correlate with the sequence of the gene product (the protein). One thus needs to compare sequences from DNA and protein (or from DNA and processed RNA) if one suspects that RNA editing can occur. The function of RNA editing has not been elucidated yet.

Questions, Chapter 3

1. Just to provide yourself with a perspective on how much genetic flexibility there is, calculate how many different sequences of 150 nucleotides long could exist that would code for a short, 50-amino-acid protein. And how many different ways are there to make a short protein of 50 amino acids? What would be the answer if you had a large, 6000 nucleotide long sequence that coded for a large protein of 2000 amino acids?

2. Some prokaryotes grow at very high temperature (70-100 C) and are called thermophiles. Organisms living in the deep sea where the pressure (and thus the boiling point of water) is very high grow at even higher temperatures and are called extremophiles. A group of these prokaryotes, named archaea, have now been found to contain DNA-binding proteins, whereas other thermophilic prokaryotes are found to have a very high GC content (and thus

a low AT content) in their DNA. Can you explain why this would be?

ontrol of protein synthesis

Most of the time when a cell is not dividing, it is performing a series of activities under the control of the DNA in its nucleus. In order to do this, information from certain portions of the DNA in the chromosomes must be taken out into the cytoplasm, to be used to make (synthesise) control proteins (enzymes, etc) for the cell. There are 2 parts to this process: transcription and translation.

Transcription

The 2 strands of the DNA molecule are temporarily split by enzymes which cause a short part to be copied into a similarly short section of RNA molecule. The copying is along the same lines as already explained, (A for T, G for C, C for G) except that a different base called U (uracil) replaces T (thymine). Also, RNA is only made of a single strand, and it contains a different sidechain subunit. The RNA copy from one section of DNA, which usually corresponds to a single gene, is called messenger RNA (mRNA). What will be the sequence of bases on the mRNA strand if the strand of DNA to be transcribed has the following base sequence? C A T G A G C G C G A T, > GUA CUC GCG CUA

Transcription: RNA made according to base sequence in DNA

30 base pairs (10 triplets) shown for example - actual genes are usually hundreds or thousands of base pairs in length

The two strands of DNA - shown here in black and grey - separate (under the influence of the enzyme RNA polymerase). Messenger RNA - here red - forms on one - black - strand of DNA. The other strand - grey - does not take part in the process.

The strand of messenger RNA (mRNA) formed then leaves the nucleus and passes into the cytoplasm. The opened-up section of DNA re-forms into a double helix, as before.

Translation

Messenger RNA then passes out of the nucleus and travels to small structures called ribosomes in the cytoplasm. Here the message it contains is interpreted, and a protein is built up, bit by bit, from its individual subunits - amino - acids, which are in the cytoplasm.

There are 20 different amino acids, with rather formidable names. Although they differ greatly in size and chemical properties, they all have a similar section by which they may be linked, to form a polypeptide chain, which will then coil to make a protein. Each section of 3 bases in the messenger RNA strand

is called a triplet, which carries enough information to identify the next amino acid which will be added to the developing polypeptide chain. The actual amino acids that are added as a result of the particular sequence of bases has been found out as a result of experiments. It has been discovered that there are several different triplet codes for each amino acid, as well as special ones to signify the start and end of the polypeptide chain. This base code seems to be the same in practically all living organisms, which confirms its fundamental significance in the organisation of life. It also explains how it is sometimes possible to take sections of DNA corresponding to genes from one organism and transfer them to another organism in which they may still work. This is the basis of genetic engineering.

The genetic code

The genetic codes for each amino acid

RNA triplet codes

amino acid abbreviation RNA triplet codes

amino acid abbreviation

AAA AAG lysine lys GAA GAG glutamic acid glu

AAC AAU asparagine asn GAC GAU aspartic acid asp

ACA ACC ACG ACU

threonine thr GCA GCC GCG GCU

alanine ala

AGA AGG arginine arg GGA GGC GGG GGU

glycine gly

AGC AGU serine ser GUA GUC GUG GUU

valine val

AUA AUG (start)

methionine met UAA UAG UGA

(stop)

AUC AUU isoleucine ile UAC UAU tyrosine tyr

CAA CAG glutamine gln UCA UCC UCG UCU

serine ser

CAC CAU histidine his UGC UGU cysteine cys

CCA CCC CCG CCU

proline pro UGG tryptophan try

CGA CGC CGG CGU

arginine arg UUA UUG leucine leu

CUA CAC CUG CUU

leucine leu UUC UUU phenylalanine phe

The information above is included here for reference only. Do not worry about the details!

Different varieties of another form of RNA (transfer RNA) and a variety of enzymes are involved in recognising the messenger RNA triplet codes, due to the way in which one RNA strand can pair up with a complementary part of another. Working together, these bring in the individual amino acids one by one in the correct order for assembly into the protein. For example, the triplet CCC in messenger RNA pairs up with its counterpart triplet GGG in transfer RNA, which will result in the amino acid proline being added to the polypeptide chain.

So a protein is the final product of the the gene made up of DNA.

The overall process may be summarised as follows:

TRANSCRIPTION TRANSLATION DNA messenger RNA PROTEIN

in nucleus in cytoplasm

Translation: Protein made according to base sequence in RNA

As messenger RNA (mRNA) - red - passes through the ribosome - grey, it causes a protein to be made (synthesised) by joining together various amino acids - green - in a particular order. A different combination of 3 mRNA bases, also called a triplet, codes for each one of the 20 amino acids. Each triplet in mRNA causes a corresponding transfer RNA (tRNA) molecule - blue - to bring in the appropriate amino acid. This occurs because the triplet of 3 bases in mRNA, also called a codon, pairs up inside the ribosome with the corresponding 3 bases in tRNA. also called an anticodon.

When the transfer RNA has delivered the amino acid to the growing polypeptide chain, it leaves the ribosome, returns to the cytoplasm and picks up another amino acid. As the ribosome moves along the mRNA strand, the synthesis process continues until it reaches the stop code which causes amino acid addition to cease. The polypeptide is then released, and it may fold into its final protein structure. The messenger RNA may enter another ribosome and repeat the protein synthesis process, or it may be broken down and its sub-units may be re-used.

During translation, the following stages are taking place: (1) Messenger RNA strand passes through ribosome (2) Triplet CCC codes for the next amino acid to be brought into position in the ribosome (3) Transfer RNA brings in the appropriate amino acid (proline) (4) Amino acid added to polypeptide chain (5) Transfer RNA released to pick up amino acid to be recycled (6) "Used" messenger RNA strand may pass on to another ribosome (7) Process repeats with next triplet code until: (8) Triplet UAA causes translation to stop (9) If it is "long", the polypeptide chain folds into the shape of the final protein

Summary - base conversions

DNA (coding strand)

DNA (non-coding strand)

Messenger RNA (mRNA)

Transfer RNA (tRNA)

A (adenine) T (thymine) U (uracil) A (adenine)

T (thymine) A (adenine) A (adenine) U (uracil)

G (guanine) C (cytosine) C (cytosine) G (guanine)

C (cytosine) G (guanine) G (guanine) C (cytosine)