AQA Module 2: Cell Cycle

AQA(B) AS Module 2:

CELL CYCLES: Contents
The Cell Cycle Mitosis Asexual Reproduction: Natural Asexual Reproduction: Artificial Sexual Reproduction Gametes

The Cell Cycle
The life of a cell is called the cell cycle and has three phases:

In different cell types the cell cycle can last from hours to years. E.g. bacterial cells can divide every 30 minutes under suitable conditions, skin cells divide about every 12 hours on average, liver cells every 2 years. The mitotic phase can be sub-divided into four phases (prophase, metaphase, anaphase and telophase). Mitosis is strictly nuclear division, and is followed by cytoplasmic division, or cytokinesis, to complete cell division. The growth and synthesis phases are collectively called interphase (i.e. in between cell division). Mitosis results in two "daughter cells", which are genetically identical to each other, and is used for growth and asexual reproduction. The details of each of these phases follows.

Cell Division by Mitosis

In this animation the stages of mitosis can clearly be seen - it's important to realise that cell division is a continuous process and that the stages flow into each other.

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Interphase
chromatin not visible DNA replicated

Prophase

chromosomes condensed and visible centrioles at opposite poles of cell phase ends with the breakdown of the nuclear membrane chromosomes align along equator of cell spindle fibres (microtubules) connect centrioles to chromosomes

Metaphase

Anaphase

centromeres split, allowing chromatids to separate chromatids move towards poles

Telophase
spindle fibres
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disperse nuclear membranes form

Cytokinesis
(division of cytoplasm)

In animal cells a ring of filaments form round the equator of the cell, and then tighten to split the cell in two. In plant cells a new cell wall is laid down inside the existing cell splitting the cell into two

Asexual Reproduction
Asexual reproduction is the production of offspring from a single parent using mitosis. Therefore the offspring are genetically identical to each other and to their "parent"- i.e. they are clones. Asexual reproduction can be either natural or artificial. METHODS
OF

ASEXUAL REPRODUCTION Artificial Methods cell culture, fermenters cuttings, grafting, tissue culture embryo splitting, somatic cell cloning

Natural Methods MICROBES binary fission, budding, spores, fragmentation vegetative propagation, parthenogenesis budding, fragmentation, parthenogenesis

PLANTS

ANIMALS

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Binary Fission. The simplest and fastest method of asexual reproduction. The nucleus divides by mitosis and the cell splits into two. Budding. A small copy of the parent develops as an outgrowth, or bud, from the parent, and then is released as a separate individual. Spores. These are simply specialised cells that are released from the parent (usually in large numbers) to be dispersed. Each spore can grow into a new individual. Vegetative Reproduction. (note also the name of an artificial technique) This term describes all the natural methods of asexual reproduction used by plants. A bud grows from a vegetative part of the plant (usually the stem) and develops into a complete new plant, which eventually becomes detached from the parent plant. There are numerous forms of vegetative reproduction, including: bulbs (e.g. daffodil) rhizomes (e.g. couch grass) runners (e.g. strawberry) tubers (e.g. potato)

Natural Methods

Many of these methods are also perenating organs, which means they contain a food store and are used for survival over winter as well as for asexual reproduction. Since vegetative reproduction relies entirely on mitosis, all offspring are clones of the parent. Parthenogenesis. This is used by some plants (e.g. citrus fruits) and some invertebrate animals (e.g. honeybees & aphids) as an alternative to sexual reproduction. Egg cells simply develop into adult clones without being fertilised. These clones may be haploid, or the chromosomes may replicate to form diploid cells.

Artificial Methods: (Plants)
Cloning is of great commercial importance, as brewers, pharmaceutical companies, farmers and plant growers all want to be able to reproduce "good" organisms exactly. Natural methods of asexual reproduction can be used for some organisms (such as potatoes and strawberries), but many important plants and animals do not reproduce asexually, so artificial methods have to be used. Cell Culture. Microbes can be cloned very easily in the lab using their normal asexual reproduction. Microbial cells can be isolated and identified by growing them on a solid medium in an

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agar plate, and can then be grown up on a small scale in a liquid medium in a culture flask. Fermenters. In biotechnology, fermenters are vessels used for growing microbes on a large scale. Fermenters must be stirred, aerated and thermostated, materials can added or removed during the fermentation, and the environmental conditions (such as pH, O 2 , pressure and temperature) must be constantly monitored using probes. This will ensure the maximum growth rate of the microbes. Cuttings. A very old method of cloning plants. Part of a plant stem is cut off and simply replanted in wet soil. Each cutting produces roots and grows into a complete new plant, so the original plant can be cloned many times. Rooting is helped if the cuttings are dipped in rooting hormone (auxin). Many flowering plants, such as geraniums are reproduced commercially by cuttings.

Grafting. Another ancient technique, used for plant species that cannot grow roots from cuttings. Instead they can often be cloned by grafting a stem

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cutting onto the lower part of an existing plant.

Tissue Culture (or micropropagation). A more modern way of cloning plants. Small samples of plant tissue are grown on agar plates in the laboratory in much the same way that bacteria are grown. The plant tissue is separated into individual cells, each which can grow into a mass of cells called a callus, and if the correct plant hormones are added these cells can develop into whole plantlets, which can eventually be planted outside, where they will grow into normal-sized plants. Conditions must be kept sterile to prevent infection by microbes. Micropropagation is used on a large scale for many plants including fruit trees, sugar cane and banana. The advantages are: thousands of clones of a good plant can be made quickly and in a small space disease-free plants can be grown from a few disease-free cells the technique works for plants species that cannot be asexually propagated by other means a single cell can be genetically modified and turned into many identical plants Although some animal cells can be grown in culture, they cannot be grown into complete animals, so tissue culture cannot be used for cloning animals.

Embryo Cloning (or Embryo Splitting). The most effective technique for cloning animals is to duplicate embryo cells before they have irreversibly differentiated into tissues. It is difficult and quite expensive, so is
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Artificial Methods: (Animals)

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only worth it for commercially-important farm animals, such as prize cows, or genetically engineered animals. A female animal is fed a fertility drug so that she produces many mature eggs (superovulation). The eggs are then removed from the female’s ovaries. The eggs are fertilised in vitro (IVF) using selected sperm from a prize male. The fertilised eggs (zygotes) are allowed to develop in vitro for a few days until the embryo is at the 16cell stage. This young embryo can be split into 16 individual cells, which will each develop again into an embryo. (This is similar to the natural process when a young embryo splits to form identical twins.) The identical embryos can then be transplanted into the uterus of surrogate mothers, where they will develop and be born normally. Could humans be cloned this way? Almost certainly yes. A human embryo was split and cloned to the stage of a few cells in the USA in 1993, just to show that it is possible. However experiments with human embryos are now banned in most countries including the UK for ethical reasons. Nuclear Transfer. The problem with embryo cloning is that you don’t know the characteristics of the animal you are cloning. By selecting good parents you hope it will have good characteristics, but you will not know until the animal has grown. It would be far better to clone a mature animal, whose characteristics you know. Until recently it was thought impossible to grow a new animal from the somatic cells of an existing animal (in contrast to plants). However, techniques have gradually been developed to do this most recently with sheep (the famous "Dolly") in 1996. The cell used for Dolly was from the skin of the udder, so was a fully differentiated somatic cell. This cell was fused with a unfertilised egg cell which had had its nucleus removed. This combination of a diploid nucleus in an unfertilised egg cell was a bit like a zygote, and it developed into an embryo. The embryo was implanted into the uterus of a surrogate mother, and developed into an apparently normal sheep, Dolly.

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Sexual Reproduction
Sexual reproduction is the production of offspring from two parent using gametes. The cells of the offspring have two sets of chromosomes (one from each parent), so are diploid. Sexual reproduction involves two stages:

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Meiosis- the special cell division that makes haploid gametes Fertilisation- the fusion of two gametes to form a diploid zygote These two stages of sexual reproduction can be illustrated by a sexual life cycle:

All sexually-reproducing species have the basic life cycle shown on the right, alternating between diploid and haploid forms. In addition, they will also use mitosis to grow into adult organisms, the details vary with different organisms.

In the animal kingdom (including humans), and in flowering plants the dominant, long-lived adult form is diploid, and the haploid gamete cells are only formed briefly.

In the fungi kingdom the long-lived adult form is haploid. Haploid spores undergo mitosis and grow into complete adults (including large structures like mushrooms). At some stage two of these haploid cells fuse to form a diploid zygote, which immediately undergoes meiosis to reestablish the haploid state and complete the cycle.

In the plant kingdom the life cycle shows alternation of generations. Plants have two distinct adult forms; one diploid and the other haploid.

Meiosis
Meiosis is a form of cell division. It starts with DNA replication, like mitosis, but then proceeds with two divisions one immediately after the other. Meiosis therefore results in four daughter cells rather than the two cells formed by mitosis. It differs from mitosis in two important aspects: The chromosome number is halved from the diploid number (2n) to the haploid number (n). This is necessary so that the chromosome number remains constant from generation to generation. Haploid cells have one copy of each chromosome, while diploid cells have homologous pairs of each chromosome. The chromosomes are re-arranged during meiosis to form new combinations of genes. This genetic recombination is vitally important and is a major source of genetic variation. It means for example that of all the millions of sperm produced by a single human male, the probability is that no two will be identical.

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You don’t need to know the details of meiosis at this stage (It's covered in module 4).

Gametes
The usual purpose of meiosis is to form gametes- the sex cells that will fuse together to form a new diploid individual. In all plants and animals the gametes are different sizes. This is called heterogamy. Summary table (you need to learn this) Female gametes (ova or eggs in animals, ovules in plants) are produced in fairly small numbers. Human females for example release about 500 ova in a lifetime. They are the larger gametes and tend to be stationary. They often contain food reserves (lipids, proteins, carbohydrates) to nourish the embryo after fertilisation. Male gametes are produced in very large numbers. Human males for example release about 100 million sperm in one ejaculation. They are the smaller gametes and can move. If they can propel themselves they are called motile (e.g. animal sperm). If they can easily be carried by the wind or animals they are called mobile (e.g. plant pollen). These diagrams of human gametes illustrate the differences between male and female.

Fertilisation
Fertilisation is the fusion of two gametes to form a zygote. In humans this takes place near the top of the oviduct. Hundreds of sperm reach the egg (shown in this photo). When a sperm reaches the ovum cell the two membranes fuse and the sperm nucleus enters the cytoplasm of the ovum. This triggers a series of reactions in the ovum that cause the jelly coat to thicken and harden, preventing any other sperm from entering the ovum. The sperm and egg nuclei then fuse, forming a diploid zygote. In plants fertilisation takes place in the ovary at the base of the carpel. The haploid male nuclei travel down the pollen tube from the pollen grain on the stigma to the ovules in the ovary. In the ovule two fusions between male and female nuclei take place: one forms the zygote (which will become the embryo) while the other forms the endosperm (which will become the food store in the seed). This double fertilisation is unique to flowering

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plants.

The Advantages of Sex
For most of the history of life on Earth, organisms have reproduced only by asexual reproduction. Each individual was a genetic copy (or clone) of its "parent", and the only variation was due to random genetic mutation. The development of sexual reproduction in the eukaryotes around one billion years ago led to much greater variation and diversity of life. Sexual reproduction is slower and more complex than asexual, but it has the great advantage of introducing genetic variation (due to genetic recombination in meiosis and random fertilisation). This variation allows species to adapt to their environment and so to evolve. This variation is clearly such an advantage that practically all species can reproduce sexually. Some organisms can do both, using sexual reproduction for genetic variety and asexual reproduction to survive harsh times.

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DNA_Notes

NUCLEIC ACIDS: Contents
Nucleotides DNA Structure DNA Function RNA Replication Transcription Translation Mutations

DNA:
DNA and its close relative RNA are perhaps the most important molecules in biology. They contains the instructions that make every single living organism on the planet. DNA stands for deoxyribonucleic acid and RNA for ribonucleic acid. They are polymers (long chain molecules) made from nucleotides.

Nucleotides
Nucleotides have three parts to them: a phosphate group, which is negatively charged. a pentose sugar, which has 5 carbon atoms in it. In RNA the sugar is ribose. In DNA the sugar is deoxyribose. a nitrogenous base. There are five different bases (you don't need to know their structures). The bases are usually known by there first letters only, you don't need to learn the full names. The base thymine is found in DNA only and the base uracil is found in RNA only. The Bases: Adenine (A), Thymine (T), Cytosine (C), Guanine (G) and Uracil (U)

Nucleotide Polymerisation:

Nucleotides polymerise by forming bonds between the carbon of the sugar and an oxygen atom of the phosphate. The bases do not take part in the polymerisation, so the chain is held together by a sugar-phosphate backbone with the bases extending off it. This means that the nucleotides can join together in any order along the chain. Many nucleotides form a polynucleotide. A polynucleotide has a free phosphate group at one

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DNA_Notes

end and a free OH group at the other end.

Structure of DNA:
The main features of the three-dimensional structure of DNA are: DNA is double-stranded, so there are two polynucleotide stands alongside each other. The two strands are wound round each other to form a double helix. The two strands are joined together by hydrogen bonds between the bases. The bases therefore form base pairs, which are like rungs of a ladder. The base pairs are specific. A only binds to T (and T with A), and C only binds to G (and G with C). These are called complementary base pairs. This means that whatever the sequence of bases along one strand, the sequence of bases on the other strand must be complementary to it. (Incidentally, complementary, which means matching, is different from complimentary, which means being nice.)

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Function of DNA
DNA is the genetic material, and genes are made of DNA. DNA therefore has two essential functions: replication and expression. Replication means that the DNA, with all its genes, must be copied every time a cell divides. Expression means that the genes on DNA must control characteristics. A gene is a section of DNA that codes for a particular protein. Characteristics are controlled by genes through the proteins they code for, like this:

Expression can be split into two parts: transcription (making RNA) and translation (making proteins). These two functions are shown in this diagram.

No one knows exactly how many genes we humans have to control all our characteristics, the latest estimates are 6080,000. The sum total of all the genes in an organism is called the genome. Genes only seem to comprise about 2% of the DNA in a cell. The majority of the DNA does not form genes and doesn’t seem to do anything. The purpose of this junk DNA remains a mystery!

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RNA
RNA is a nucleic acid like DNA, but with 4 differences: RNA has the sugar ribose instead of deoxyribose RNA has the base uracil instead of thymine RNA is usually single stranded RNA is usually shorter than DNA

Messenger RNA (mRNA)
mRNA carries the "message" that codes for a particular protein from the nucleus (where DNA is) to the cytoplasm (where proteins are synthesised). It is single stranded and just long enough to contain one gene only.

Ribosomal RNA (rRNA)
A structural molecule part of ribosomes - details are not required

Transfer RNA (tRNA)
tRNA matches amino acids to their codon. tRNA is only about 80 nucleotides long, and it folds up by complementary base pairing to form a cloverleaf structure. At one end of the molecule there is an amino acid binding site. On the middle loop there is a triplet nucleotide sequence called the anticodon. There are 64 different tRNA molecules, each with a different anticodon sequence complementary to the 64 different codons on mRNA.

The Genetic Code
The sequence of bases on DNA codes for the sequence of amino acids in proteins. But there are 20 different amino acids and only 4 different bases, so the bases are read in groups of 3. This gives 64 combinations, more than enough to code for 20 amino acids. A group of three bases coding for an amino acid is called a codon, and the meaning of each of the 64 codons is called the genetic code.

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There are several interesting points from this code (which by the wat you do not need to know): The code is degenerate, i.e. there is often more than one codon for an amino acid. The degeneracy is on the third base of the codon, which is therefore less important than the others. One codon means "start" i.e. the start of the gene sequence. It is AUG. Three codons mean "stop" i.e. the end of the gene sequence. They do not code for amino acids. The code is only read in one direction along the mRNA molecule.

Replication - DNA Synthesis
DNA is copied, or replicated, before every cell division, so that one identical copy can go to each daughter cell. The double helix unzips and two new strands are built up by complementary base-pairing onto the two old strands.

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1. Replication starts at a specific sequence on the DNA molecule. 2. An enzyme unwinds and unzips DNA, breaking the hydrogen bonds that join the base pairs, and forming two separate strands. 3. The new DNA is built up from the four nucleotides (A, C, G and T) that are abundant in the nucleoplasm. 4. These nucleotides attach themselves to the bases on the old strands by complementary base pairing. Where there is a T base, only an A nucleotide will bind, and so on. 5. The enzyme DNA polymerase joins the new nucleotides to each other by strong covalent bonds, forming the sugar-phosphate backbone. 6. A winding enzyme winds the new strands up to form double helices. 7. The two new molecules are identical to the old molecule. The Meselson-Stahl Experiment This replication mechanism is sometimes called semi-conservative replication, because each new DNA molecule contains one new strand and one old strand. There was an alternative theory which suggested that a "photocopy" of the original DNA was made, leaving the original DNA conserved (conservative replication). The proof that the semiconservative method was the correct method came from an experiment performed by Meselson and Stahl using the bacterium E. coli together with the technique of density gradient centrifugation, which separates molecules on the basis of their density.

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Transcription - RNA Synthesis
DNA never leaves the nucleus, but proteins are synthesised in the cytoplasm, so a copy of each gene is made to carry the "code" from the nucleus to the cytoplasm. This copy is mRNA, and the process of copying is called transcription.

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1. The start of each gene on DNA is marked by a special sequence of bases. 2. The RNA molecule is built up from the four ribose nucleotides (A, C, G and U) in the nucleoplasm. The nucleotides attach themselves to the bases on the DNA by complementary base pairing, just as in DNA replication. However, only one strand of RNA is made. 3. The new nucleotides are joined to each other by covalent bonds by the enzyme RNA polymerase 4. The initial mRNA contains some regions that are not part of the protein code. These are called introns 5. The introns are cut out by enzymes 6. The result is a shorter mature RNA. 7. The mRNA diffuses out of the nucleus through a nuclear pore into the cytoplasm.

Translation - Protein Synthesis
1. A ribosome attaches to the mRNA at an initiation codon (AUG). The ribosome encloses two codons.

2. met-tRNA diffuses to the ribosome and attaches to the mRNA initiation codon by complementary base pairing.

3. The next amino acid-tRNA attaches to the adjacent mRNA codon (leu in this case).

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4. The bond between the amino acid and the tRNA is cut and a peptide bond is formed between the two amino acids.

5. The ribosome moves along one codon so that a new amino acidtRNA can attach. The free tRNA molecule leaves to collect another amino acid. The cycle repeats from step 3.

6. The polypeptide chain elongates one amino acid at a time, and peels away from the ribosome, folding up into a protein as it goes. This continues for hundreds of amino acids until a stop codon is reached.

A single piece of mRNA can be translated by many ribosomes simultaneously. A group of ribosomes all attached to one piece of mRNA is called a polysome.

Post-Translational Modification
In eukaryotes, proteins often need to be altered before they become fully functional. Modifications are carried out by other enzymes and include: chain cutting, adding sugars (to make glycoproteins) or lipids (to make lipoproteins). These changes occur in the Golgi Apparatus

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Mutations
Mutations are changes in genes, which are passed on to daughter cells. DNA is a very stable molecule, and it doesn't suddenly change without reason, but bases can change when DNA is being replicated. Normally replication is extremely accurate but very occasionally mistakes do occur (such as a T-C base pair). Changes in DNA can lead to changes in cell function like this:

There are basically three kinds of gene mutation, shown in this diagram:

The actual effect of a single mutation depends on many factors: A substitution on the third base of a codon may have no effect because the third base is less important (e.g. all codons beginning with CC code for proline). If a single amino acid is changed to a similar one, then the protein structure and function may be unchanged, but if an amino acid is changed to a very different one, then the structure and function of the protein will be very different. If the changed amino acid is at the active site of the enzyme then it is more likely to affect enzyme function than if it is part of the supporting structure. Additions and Deletions are Frame shift mutations and are far more serious than substitutions because more of the protein is altered. If a frame-shift mutation is near the end of a gene it will have less effect than if it is near the start of the gene If the mutation is in a gene that is not expressed in this cell (e.g. the insulin gene in a red blood cell) then it won't matter. Some proteins are simply more important than others. For instance non-functioning receptor proteins in the tongue may lead to a lack of taste but is not life-threatening, whereas non-functioning haemoglobin is fatal. Some cells are more important than others. Mutations in somatic cells (i.e. non-reproductive body cells) will only affect cells that derive from that cell, so will probably have a small local effect like a birthmark (although they can cause widespread effects like diabetes or cancer). Mutations in germ cells (i.e. reproductive cells) will affect every single cell of the resulting organism as well as its offspring. These mutations are one source of genetic variation. As a result of a mutation there are three possible phenotypic effects: Most mutations have no observable (phenotypic) effect. Of the mutations that have a phenotypic effect, most will have a negative effect. Most of the proteins in cells are enzymes, and most changes in enzymes will stop them working. When an enzyme stops working, a metabolic block can occur, when a reaction in cell doesn't happen, so the cell's function is changed. An example of this is the genetic disease phenylketonuria (PKU), caused by a mutation in the gene for an enzyme. This causes a metabolic block in the pathway involving the amino acid phenylalanine, which builds up, causing mental retardation.
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Very rarely a mutation can have a beneficial phenotypic effect, such as making an enzyme work faster, or a structural protein stronger, or a receptor protein more sensitive. Although rare beneficial mutations are important as they drive evolution.

These kinds of mutation are called point or gene mutations because they affect specific points within a gene. There are other kinds of mutation that can affect many genes at once or even whole chromosomes. These chromosome mutations can arise due to mistakes in cell division. A well-known example is Down syndrome (trisonomy 21) where there are three copies of chromosome 21 instead of the normal two.

Mutation Rates and Mutagens
Mutations are normally very rare, which is why members of a species all look alike and can interbreed. However the rate of mutations is increased by chemicals or by radiation. These are called mutagenic agents or mutagens, and include: High energy ionising radiation such as x-rays, ultraviolet rays, rays from radioactive sources all ionise the bases so that they don't form the correct base pairs. Intercalating chemicals such as mustard gas (used in World War 1), which bind to DNA separating the two strands. Chemicals that react with the DNA bases such as benzene and tar in cigarette smoke.

DNA and Chromosomes
The DNA molecule in a single human cell is about 1m long so in order to fit into the cell the DNA is cut into shorter lengths and each length is tightly wrapped up with histone proteins to form a complex called chromatin. During most of the life of a cell the chromatin is dispersed throughout the nucleus and cannot be seen with a light microscope. Just before cell division the DNA is replicated so there is temporarily twice the normal amount DNA. Following replication the chromatin then coils up even tighter to form short fat bundles called chromosomes. These are about 100 000 times shorter than fully stretched DNA and are thick enough to be seen under the microscope. Each chromosome is roughly X-shaped because it contains two replicated copies of the DNA. The two arms of the X are therefore identical. They are called chromatids, and are joined at the centromere. (Do not confuse the two chromatids with the two strands of DNA.) The complex folding of DNA into chromosomes is shown below.

micrograph of a single chromosome Chromatin DNA + histones at any stage of the cell cycle

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Chromosome compact X-shaped form of chromatin formed (and visible) during mitosis Chromatid single arm of an X-shaped chromosome Since the DNA molecule extends from one end of a chromosome to the other, and the genes are distributed along the DNA, then each gene has a defined position on a chromosome. This position is called the locus of the gene.

Karyotypes and Homologous Chromosomes
If a dividing cell is stained with a special fluorescent dye and examined under a microscope during cell division, the individual chromosomes can be distinguished. They can then be photographed and studied. This is a difficult and skilled procedure, and it often helps if the chromosomes are cut out and arranged in order of size.

This display is called a karyotype, and it shows several features: Different species have different number of chromosomes, but all members of the same species have the same number. Humans have 46. Each chromosome has a characteristic size, shape and banding pattern, which allows it to be identified and numbered. The chromosomes are numbered from largest to smallest. Chromosomes come in pairs, with the same size, shape and banding pattern, called homologous pairs ("same shaped"). So there are two chromosome number 1s, two chromosome number 2s, etc, and humans really have 23 pairs of chromosomes. Homologous chromosomes are a result of sexual reproduction, and the homologous pairs are the maternal and paternal versions of the same chromosome, so they have the same sequence of genes 1 pair of chromosomes is different in males and females. These are the sex chromosomes, and are nonhomologous in one of the sexes. In humans sex chromosomes are homologous in females (XX) and nonhomologous in males (XY). (In birds it is the other way round!) The non-sex chromosomes are sometimes called autosomes, so humans have 22 pairs of autosomes, and 1 pair of sex chromosomes.

Try some comprehension questions

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gene technology notes

Genetic Engineering: Contents
Techniques Restriction Enzymes/DNA Ligase Vectors/Plasmids Gene Transfer Genetic Markers PCR DNA probes Electrophoresis DNA Sequencing Applications Gene Products New Phenotypes Gene Therapy

Genetic Engineering
Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.

Techniques Engineering

of

Genetic

Genetic engineering is a very young discipline, and is only possible due to the development of techniques from the 1960s onwards. These techniques have been made possible from our greater understanding of DNA and how it functions following the discovery of its structure by Watson and Crick in 1953. Although the final goal of genetic engineering is usually the expression of a gene in a host, in fact most of the techniques and time in genetic engineering are spent isolating a gene and then cloning it. This table lists the techniques

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gene technology notes

that we'll look at in detail. TECHNIQUE Restriction Enzymes DNA Ligase Vectors Plasmids Genetic Markers PCR cDNA DNA probes Gene Synthesis Electrophoresis DNA Sequencing

PURPOSE To cut DNA at specific points, making small fragments To join DNA fragments together To carry DNA into cells and ensure replication Common kind of vector To identify cells that have been transformed To amplify very small samples of DNA To make a DNA copy of mRNA To identify and label a piece of DNA containing a certain sequence To make a gene from scratch To separate fragments of DNA To read the base sequence of a length of DNA

Restriction Enzymes
These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases because they cut the bonds in the middle of the polynucleotide chain. Most restriction enzymes make a staggered cut in the two strands, forming sticky ends.

The cut ends are "sticky" because they have short stretches of single-stranded DNA. These sticky ends will stick (or anneal) to another piece of DNA by complementary base pairing, but only if they have both been cut with the same restriction enzyme. Restriction enzymes are highly specific, and will only cut DNA at specific base sequences, 4-8 base pairs long. Restriction enzymes are produced naturally by bacteria as a defence against viruses (they "restrict" viral growth), but they are enormously useful in genetic engineering for cutting DNA at precise places ("molecular scissors"). Short lengths of DNA cut out by restriction enzymes are called restriction fragments. There are thousands of different restriction enzymes known, with over a hundred different recognition sequences. Restriction enzymes are named after the bacteria species they came from, so EcoR1 is from E. coli strain R.

DNA Ligase
This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments. The sticky ends allow two complementary

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restriction fragments to anneal, but only by weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete. DNA ligase completes the DNA backbone by forming covalent bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different sources.

Vectors
In biology a vector is something that carries things between species. E.g. the mosquito is a vector that carries the malaria parasite into humans. In genetic engineering a vector is a length of DNA that carries the gene we want into a host cell. A vector is needed because a length of DNA containing a gene on its own won’t actually do anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down pretty quickly. A vector gets round these problems by having these properties: It is big enough to hold the gene we want It is circular (or more accurately a closed loop), so that it is less likely to be broken down It contains control sequences, such as a transcription promoter, so that the gene will be replicated or expressed. It contain marker genes, so that cells containing the vector can be identified. TYPE
OF VECTOR

MAX

LENGTH OF INSERT

DNA

Plasmid Virus or phage

10 kbp 30 kbp

Plasmids (the common vectors)

most

Plasmids are by far the most common kind of vector, so we shall look at how they are used in some detail. Plasmids are short circular bits of DNA found naturally in bacterial cells. A typical plasmid contains 3-5 genes and there are around 10 copies of a plasmid in a bacterial cell. Plasmids are copied when the cell divides, so the plasmid genes are passed on to all daughter cells. They are also used naturally for exchange of genes
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between bacterial cells (the nearest they get to sex), so bacterial cells will take up a plasmid. Because they are so small, they are easy to handle in a test tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase.

One of the most common plasmids used is the R-plasmid (or pBR322). This plasmid contains a replication origin, several recognition sequences for different restriction enzymes (with names like EcoRI), and two marker genes, which confer resistance to different antibiotics (ampicillin and tetracycline).

The diagram below shows how DNA fragments can be incorporated into a plasmid using restriction and ligase enzymes. The restriction enzyme used here (PstI) cuts the plasmid in the middle of one of the marker genes (we’ll see why this is useful later). The foreign DNA anneals with the plasmid and is joined covalently by DNA ligase to form a hybrid vector (in other words a mixture or hybrid of bacterial and foreign DNA). Several other products are also formed: some plasmids will simply re-anneal with themselves to re-form the original plasmid, and some DNA fragments will join together to form chains or circles. Theses different products cannot easily be separated, but it doesn’t matter, as the marker genes can be used later to identify the correct hybrid vector.

Gene Transfer
Vectors containing the genes we want must be incorporated into living cells so that they can be replicated or expressed. The cells receiving the vector are called host cells, and once they have successfully incorporated the vector they are said to be transformed. Vectors are large molecules which do not readily cross cell membranes, so the membranes must be made permeable in some way. There are different ways of doing this depending on the type of host cell. The most important one have the symbol the others are less commonly used Heat Shock. Cells are incubated with the vector in a solution containing calcium ions at 0°C. The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells to take up the vector, though no one knows why. This works well for bacterial and animal cells. Electroporation. Cells are subjected to a high-voltage pulse, which temporarily disrupts the membrane and allows the vector to enter the cell. This is the most efficient method of delivering genes to bacterial cells. Viruses. The vector is first incorporated into a virus, which is then used to infect cells, carrying the foreign gene along with its own genetic material. Since viruses rely on getting their DNA into host cells for their survival they have evolved many successful methods, and so are an obvious choice for gene delivery. The virus must first be genetically engineered to make it safe, so that it can’t reproduce itself
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or make toxins. Three viruses are commonly used: DETAILS (also called phages) are viruses that infect bacteria. They are an effective way of delivering Bacteriophages large genes into bacteria cells in culture. are human viruses that causes respiratory diseases including the common cold. Their genetic material is double-stranded DNA, and they are ideal for delivering genes to living patients in gene therapy. Their DNA is not incorporated into the host’s chromosomes, so it is not replicated, but their genes are expressed. The adenovirus is genetically altered so that its coat proteins are not synthesised, so new virus particles cannot be assembled and the host cell is not killed.. Adenoviruses TYPE OF VIRUS

are a group of human viruses that include HIV. They are enclosed in a lipid membrane and their genetic material is double-stranded RNA. On infection this RNA is copied to DNA and the DNA is incorporated into the host’s chromosome. This means that the foreign genes are replicated into every daughter cell. After a certain time, the dormant DNA is switched on, and the genes are expressed in the host cells. Retroviruses

Plant Tumours. This method has been used successfully to transform plant cells, which are perhaps the hardest to do. The gene is first inserted into the plasmid of a soil bacterium, and then plants are infected with the bacterium. The bacterium inserts the plasmid into the plant cells' chromosomal DNA and causes a "crown gall" tumour. These tumour cells can be cultured in the laboratory. Gene Gun. This technique fires microscopic gold particles coated with the foreign DNA at the cells using a compressed air gun. It is designed to overcome the problem of the strong cell wall in plant tissue. Micro-Injection. A cell is held on a pipette under a microscope and the foreign DNA is injected directly into the nucleus using an incredibly fine micro-pipette. Used where there are only a very few cells available, such as fertilised animal egg cells. Liposomes. Vectors can be encased in liposomes, which are small membrane vesicles (see module 1).

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The liposomes fuse with the cell membrane (and sometimes the nuclear membrane too), delivering the DNA into the cell. This works for many types of cell, but is particularly useful for delivering genes to cell in vivo (such as in gene therapy).

Genetic Markers
These are needed to identify cells that have successfully taken up a vector and so become transformed. With most of the techniques above less than 1% of the cells actually take up the vector, so a marker is needed to distinguish these cells from all the others. A common marker, used in plasmids, is a gene for resistance to an antibiotic such as tetracycline. Bacterial cells taking up this plasmid are resistant to this antibiotic. So if the cells are grown on a medium containing tetracycline all the normal untransformed cells (99%) will die. Only the 1% transformed cells will survive, and these can then be grown and cloned on another plate.

Replica Plating
Replica plating is a simple technique for making an exact copy of an agar plate. A pad of sterile cloth the same size as the plate is pressed on the surface of an agar plate with bacteria growing on it. Some cells from each colony will stick to the cloth. If the cloth is then pressed onto a new agar plate, some cells will be deposited and colonies will grow in exactly the same positions on the new plate. This technique has a number of uses, but the most common use in genetic engineering is to help solve another problem in identifying transformed cells. This problem is to distinguish those cells that have taken up a hybrid plasmid vector (with a foreign gene in it) from those cells that have taken up plasmids without the gene. This is where the second marker gene (for resistance to ampicillin) is used. If the foreign gene is inserted into the middle of this marker gene, the marker gene is disrupted and won't make its proper gene product. So cells with the hybrid plasmid will be killed by ampicillin, while cells with the normal plasmid will be immune to ampicillin. Since this method of identification involves killing the cells we want, we must first make a master agar plate and then make a replica plate of this to test for ampicillin resistance.

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Once the colonies of cells containing the correct hybrid plasmid vector have been identified, the appropriate colonies on the master plate can be selected and grown on another plate.

Polymerase Chain Reaction (PCR)
Genes can be cloned by cloning the bacterial cells that contain them, but this requires quite a lot of DNA in the first place. PCR can clone (or amplify) DNA samples as small as a single molecule. It is a newer technique, having been developed in 1983 by Kary Mullis, for which discovery he won the Nobel prize in 1993. The polymerase chain reaction is simply DNA replication in a test tube. If a length of DNA is mixed with the four nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, then the DNA will be replicated many times.

1. Start with a sample of the DNA to be amplified, and add the four nucleotides and the enzyme DNA polymerase. 2. Normally (in vivo) the DNA double helix would be separated by the enzyme helicase, but in PCR (in vitro) the strands are separated by heating to 95°C for two minutes. This breaks the hydrogen bonds. 3. DNA polymerisation always requires short lengths of DNA (about 20 bp long) called primers, to get it started. In vivo the primers are made during replication by DNA polymerase, but in vitro they must be synthesised separately and added at this stage. This means that a short length of the sequence of the DNA must already be known, but it does have the advantage that only the part between the primer sequences is replicated. The DNA must be cooled to 40°C to allow the primers to anneal to their complementary sequences on the separated DNA strands. 4. The DNA polymerase enzyme can now extend the primers and complete the replication of the rest of
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the DNA. The enzyme used in PCR is derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90°C, so it is not denatured by the high temperatures in step 2. Its optimum temperature is about 72°C, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible. 5. Each original DNA molecule has now been replicated to form two molecules. The cycle is repeated from step 2 and each time the number of DNA molecules doubles. This is why it is called a chain reaction, since the number of molecules increases exponentially, like an explosive chain reaction. Typically PCR is run for 20-30 cycles.

PCR can be completely automated, so in a few hours a tiny sample of DNA can be amplified millions of times with little effort. The product can be used for further studies, such as cloning, electrophoresis, or gene probes. Because PCR can use such small samples it can be used in forensic medicine (with DNA taken from samples of blood, hair or semen), and can even be used to copy DNA from mummified human bodies, extinct woolly mammoths, or from an insect that's been encased in amber since the Jurassic period. One problem of PCR is having a pure enough sample of DNA to start with. Any contaminant DNA will also be amplified, and this can cause problems, for example in court cases.

Complementary DNA
Complementary DNA (cDNA) is DNA made from mRNA. This makes use of the enzyme reverse transcriptase, which does the reverse of transcription: it synthesises DNA from an RNA template. It is produced naturally by a group of viruses called the retroviruses (which include HIV), and it helps them to invade cells. In genetic engineering reverse transcriptase is used to make an artificial gene of cDNA as shown in this diagram.

Complementary DNA has helped to solve different problems in genetic engineering: It makes genes much easier to find. There are some 70 000 genes in the human genome, and finding one

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gene out of this many is a very difficult (though not impossible) task. However a given cell only expresses a few genes, so only makes a few different kinds of mRNA molecule. For example the b cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can be isolated from these cells and used to make cDNA of the insulin gene.

DNA Probes
These are used to identify and label DNA fragments that contain a specific sequence. A probe is simply a short length of DNA (20-100 nucleotides long) with a label attached. There are two common types of label used: a radioactively-labelled probe (synthesised using the isotope 32 P) can be visualised using photographic film (an autoradiograph). a fluorescently-labelled probe will emit visible light when illuminated with invisible ultraviolet light. Probes can be made to fluoresce with different colours. Probes are always single-stranded, and can be made of DNA or RNA. If a probe is added to a mixture of different pieces of DNA (e.g. restriction fragments) it will anneal (base pair) with any lengths of DNA containing the complementary sequence. These fragments will now be labelled and will stand out from the rest of the DNA. DNA probes have many uses in genetic engineering: To identify restriction fragments containing a particular gene out of the thousands of restriction fragments formed from a genomic library. This use is described in shotguning below. To identify the short DNA sequences used in DNA fingerprinting. To identify genes from one species that are similar to those of another species. Most genes are remarkably similar in sequence from one species to another, so for example a gene probe for a mouse gene will probably anneal with the same gene from a human. This has aided the identification of human genes. To identify genetic defects. DNA probes have been prepared that match the sequences of many human genetic disease genes such as muscular dystrophy, and cystic fibrosis. Hundreds of these probes can be stuck to a glass slide in a grid pattern, forming a DNA microarray (or DNA chip). A sample of human DNA is added to the array and any sequences that match any of the various probes will stick to the array and be labelled. This allows rapid testing for a large number of genetic defects at a time.

Shotguning
This is used to find one particular gene in a whole genome, a bit like finding the proverbial needle in a haystack. It is called the shotgun technique because it starts by indiscriminately breaking up the genome (like firing a shotgun at a soft target) and then sorting through the debris for the particular gene we want. For this to work a gene probe for the gene is needed, which means at least a short part of the gene’s sequence must be known.

Antisense Genes

These are used to turn off the expression of a gene in a cell. The principle is very simple: a copy of the gene to be switch off is inserted into the host genome the "wrong" way round, so that the complementary (or antisense) strand is transcribed. The antisense mRNA produced will anneal to the normal sense mRNA forming double-stranded RNA. Ribosomes can’t bind to this, so the mRNA is not translated, and the gene is effectively "switched off".

Electrophoresis
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This is a form of chromatography used to separate different pieces of DNA on the basis of their length. It might typically be used to separate restriction fragments. The DNA samples are placed into wells at one end of a thin slab of gel (usually made of agarose) and covered in a buffer solution. An electric current is passed through the gel. Each nucleotide in a molecule of DNA contains a negatively-charged phosphate group, so DNA is attracted to the anode (the positive electrode). The molecules have to diffuse through the gel, and smaller lengths of DNA move faster than larger lengths, which are retarded by the gel. So the smaller the length of the DNA molecule, the further down the gel it will move in a given time. At the end of the run the current is turned off.

Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There are three common methods for doing this: The gel can be stained with a chemical that specifically stains DNA, such as ethidium bromide. The DNA shows up as blue bands. The DNA samples at the beginning can be radiolabelled with a radioactive isotope such as 32 P. Photographic film is placed on top of the finished gel in the dark, and the DNA shows up as dark bands on the film. This method is extremely sensitive. The DNA fragments at the beginning can be labelled with a fluorescent molecule. The DNA fragments show up as coloured lights when the finished gel is illuminated with invisible ultraviolet light.

DNA Sequencing
This means reading the base sequence of a length of DNA. Once this is known the amino acid sequence of the protein that the DNA codes for can also be determined, using the genetic code table. The sequence can also be compared with DNA sequences from other individuals and even other species to work out relationships. DNA sequencing is based on a beautifully elegant technique developed by Fred Sanger, and now called the Sanger method. Label 4 test tubes labelled A, T, C and G. Into each test tube add: a sample of the DNA to be sequenced (containing many millions of individual molecules) a radioactive primer (so the DNA can be visualised later on the gel), the four DNA nucleotides and the enzyme DNA polymerase.

In each test tube add a small amount of a special modified dideoxy nucleotide that cannot form a bond and so stops further synthesis of DNA. Tube A has dideoxy A (A*), tube T has dideoxy T (T*), tube C has dideoxy C
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(C*) and tube G has dideoxy G (G*). The dideoxy nucleotides are present at about 1% of the concentration of the normal nucleotides. Let the DNA polymerase synthesise many copies of the DNA sample. From time to time at random a dideoxy nucleotide will be added to the growing chain and synthesis of that chain will then stop. A range of DNA molecules will be synthesised ranging from full length to very short. The important point is that in tube A, all the fragments will stop at an A nucleotide. In tube T, all the fragments will stop at a T nucleotide , and so on. The contents of the four tubes are now run side by side on an electrophoresis gel, and the DNA bands are visualised by autoradiography. Since the fragments are now sorted by length the sequence can simply be read off the gel starting with the smallest fragment (just one nucleotide) at the bottom and reading upwards.

There is now a modified version of the Sanger method called cycle sequencing, which can be completely automated. The primers are not radiolabelled, but instead the four dideoxy nucleotides are fluorescently labelled, each with a different colour (A* is green, T* is red, C* is blue and G* is yellow). The polymerisation reaction is done in a single tube, using PCR-like cycles to speed up the process. The resulting mixture is separated using capillary electrophoresis, which gives good separation in a single narrow gel. The gel is read by a laser beam and the sequence of colours is converted to a DNA sequence by computer program (like the screenshot below). This technique can sequence an amazing 12 000 bases per minute.

Thousands of genes have been sequenced using these methods and the entire genomes of several organisms have also been sequenced. A huge project is underway to sequence the human genome, and it delivered a draft sequence in June 2000. The complete 3 billion base sequence should be complete by 2003. This information will give us unprecedented knowledge about ourselves, and is likely to lead to dramatic medical and scientific advances.

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Applications Engineering

of

Genetic

We have now looked at some of the many techniques used by genetic engineers. What can be done with these techniques? By far the most numerous applications are still as research tools, and the techniques above are helping geneticists to understand complex genetic systems. Despite all the hype, genetic engineering still has very few successful commercial applications, although these are increasing each year. The applications so far can usefully be considered in three groups. Gene Products New Phenotypes Gene Therapy using genetically modified organisms (usually microbes) to produce chemicals, usually for medical or industrial applications. using gene technology to alter the characteristics of organisms (usually farm animals or crops) using gene technology on humans to treat a disease

Gene Products
The biggest and most successful kind of genetic engineering is the production of gene products. These products are of medical, agricultural or commercial value. This table shows a few of the examples of genetically engineered products that are already available. PRODUCT Insulin Factor VIII AAT rennin USE human hormone used to treat diabetes human blood haemophiliacs clotting factor, used to treat HOST ORGANISM bacteria /yeast bacteria sheep bacteria /yeast

enzyme used to treat cystic fibrosis and emphysema enzyme used in manufacture of cheese

The products are mostly proteins, which are produced directly when a gene is expressed, but they can also be non-protein products produced by genetically-engineered enzymes. The basic idea is to transfer a gene (often human) to another host organism (usually a microbe) so that it will make the gene product quickly, cheaply and ethically. It is also possible to make "designer proteins" by altering gene sequences, but while this is a useful research tool, there are no commercial applications yet. Since the end-product is just a chemical, in principle any kind of organism could be used to produce it. By far the most common group of host organisms used to make gene products are the bacteria, since they can be grown quickly and the product can be purified from their cells. Unfortunately bacteria cannot not always make human proteins, and recently animals and even plants have also been used to make gene products. In neither case is it appropriate to extract the product from their cells, so in animals the product must be secreted in milk or urine, while in plants the product must be secreted from the roots. This table shows some of the advantages and disadvantages of using different organisms for the production of geneticallyengineered gene products. TYPE OF ORGANISM ADVANTAGES DISADVANTAGES

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Prokaryotes (i.e.Bacteria)

no nucleus so DNA easy to modify; have plasmids; small genome; genetics well understood; asexual so can be cloned; small and fast growing; easy to grow commercially in fermenters; will use cheap carbohydrate; few ethical problems.

can’t splice introns; no posttranslational modification; small gene size

Eukaryotes

can do post-translational modifications; can accept large genes

Do not have plasmids (except yeast); often diploid so two copies of genes may need to be inserted; control of expression not well understood. can’t always make animals gene products cell walls difficult to penetrate by vector; slow growing; must be grown in fields; multicellular

Fungi (yeast, mould)

asexual so can be cloned; haploid, so only one copy needed; can be grown in vats photosynthetic so don’t need much feeding; can be cloned from single cells; products can be secreted from roots or in sap. most likely to be able to make human proteins; products can be secreted in milk or urine

Plants

Animals (pharming)

multicellular; slow growing

We’ll look at some examples in detail.

Human Insulin
Insulin is a small protein hormone produced by the pancreas to regulate the blood sugar concentration. In the disease insulin-dependent diabetes the pancreas cells don’t produce enough insulin, causing wasting symptoms and eventually death. The disease can be successfully treated by injection of insulin extracted from the pancreases of slaughtered cows and pigs. However the insulin from these species has a slightly different amino acid sequence from human insulin and this can lead to immune rejection and side effects. The human insulin gene was isolated, cloned and sequenced in the 1970s, and so it became possible to insert this gene into bacteria, who could then produce human insulin in large amounts. Unfortunately it wasn’t that simple. In humans, pancreatic cells first make pro-insulin, which then undergoes post-translational modification to make the final, functional insulin. Bacterial cells cannot do post-translational modification. Eventually a synthetic cDNA gene was made and inserted into the bacterium E. coli, which made pro-insulin, and the post-translational conversion to insulin was carried out chemically. This technique was developed by Eli Lilly and Company in 1982 and the product, "humulin" became the first genetically-engineered product approved for medical use. In the 1990s the procedure was improved by using the yeast Saccharomyces cerevisiae instead of E. coli. Yeast, as a eukaryote, is capable of post-translational modification, so this simplifies the production of human insulin. However another company has developed a method of converting pig insulin into human insulin by chemically changing a few amino acids, and this turns out to be cheaper than the genetic engineering methods. This all goes to show that genetic engineers still have a lot to learn.

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Bovine Somatotrophin (BST)
This is a growth hormone produced by cattle. The gene has been cloned in bacteria by the company Monsanto, who can produce large quantities of BST. in the USA cattle are often injected with BST every 2 weeks, resulting in a 10% increase in mass in beef cattle and a 25% increase in milk production in dairy cows. BST was tested in the UK in 1985, but it was not approved and its use is currently banned in the EU. This is partly due to public concerns and partly because there is already overproduction of milk and beef in the EU, so greater production is not necessary.

Rennin
Rennin is an enzyme used in the production of cheese. It is produced in the stomach of juvenile mammals (including humans) and it helps the digestion of the milk protein caesin by solidifying it so that is remains longer in the stomach. The cheese industry used to obtain its rennin from the stomach of young calves when they were slaughtered for veal, but there are moral and practical objections to this source. Now an artificial cDNA gene for rennin has been made from mRNA extracted from calf stomach cells, and this gene has been inserted into a variety of microbes. The rennin extracted from these microbes has been very successful and 90% of all hard cheeses in the UK are made using microbial rennin. Sometimes (though not always) these products are labelled as "vegetarian cheese".

AAT ( a-1-antitrypsin) AAT is a human protein made in the liver and found in the blood. As the name suggests it is an inhibitor of protease enzymes like trypsin and elastase. There is a rare mutation of the AAT gene (a single base substitution) that causes AAT to be inactive, and so the protease enzymes to be uninhibited. The most noticeable effect of this in the lungs, where elastase digests the elastic tissue of the alveoli, leading to the lung disease emphysema. This condition can be treated by inhaling an aerosol spray containing AAT so that it reaches the alveoli and inhibits the elastase there. AAT for this treatment can be extracted from blood donations, but only in very small amounts. The gene for AAT has been found and cloned, but AAT cannot be produced in bacteria because AAT is a glycoprotein, which means it needs to have sugars added by post translational modification. This kind of modification can only be carried out by animals (because they have a golgi body), and AAT is now produced by geneticallymodified sheep. In order to make the AAT easy to extract, the gene was coupled to a promoter for the milk protein b-lactoglubulin. Since this promoter is only activated in mammary gland cells, the AAT gene will only be expressed in mammary gland cells, and so will be secreted into the sheep's milk. This makes it very easy to harvest and purify without harming the sheep. The first transgenic sheep to produce AAT was called Tracy, and she was produced in Edinburgh in 1993. This is how Tracy was made: A female sheep is given a fertility drug to stimulate her egg production, and several mature eggs are collected from her ovaries.

The eggs are fertilised in vitro.

A plasmid is prepared containing the gene for human AAT and the promoter sequence for b-

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lactoglobulin. Hundreds of copies of this plasmid are microinjected into the nucleus of the fertilised zygotes. Only a few of the zygotes will be transformed, but at this stage you can’t tell which. The zygotes divide in vitro until the embryos are at the 16-cell stage. The 16-cell embryos are implanted into the uterus of surrogate mother ewes. Only a few implantations result in a successful pregnancy. Test all the offspring from the surrogate mothers for AAT production in their milk. This is the only way to find if the zygote took up the AAT gene so that it can be expressed. About 1 in 20 eggs are successful. Collect milk from the transgenic sheep for the rest of their lives. Their milk contains about 35 g of AAT per litre of milk. Also breed from them in order to build up a herd of transgenic sheep. Purify the AAT, which is worth about £50 000 per mg.

New Phenotypes
This means altering the characteristics of organisms by genetic engineering. The organisms are usually commercially-important crops or farm animals. It can be seen as a high-tech version of selective breeding, which has been used by humans to alter and improve their crops and animals for at least 10 000 years. Nevertheless GMOs have turned out to be a highly controversial development. We don’t study any of these in detail, but this table gives an idea of what is being done.

ORGANISM

MODIFICATION There are two well-known projects, both affecting the gene for the enzyme (PG) which softens the fruits as they ripen. Tomatoes that make less PG ripen more slowly and retain more flavour. The American "Flavr Savr" tomato used antisense technology to silence the gene, while the British Zeneca tomato disrupted the gene. Both were successful and were on sale for a few years, but neither is produced any more. Genes for various powerful protein toxins have been transferred from the bacterium Bacillus thuringiensis to crop plants including maize, rice and potatoes. These Bt toxins are thousands of times more powerful than chemical insecticides, and since they are built-in to the crops, insecticide spraying (which is non-specific and damages the

long life tomatoes

Insectresistant crops

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environment) is unnecessary. This is a huge project, which aims to transfer the 15-or-so genes required for nitrogen fixation from the nitrogen-fixing bacteria Rhizobium into cereals and other crop plants. These crops would then be able to fix their own atmospheric nitrogen and would not need any fertiliser. However, the process is extremely complex, and the project is nowhere near success. The gene for the enzyme chitinase, which kills ticks by digesting their exoskeletons, has bee transferred from plants to sheep. These sheep should be immune to tick parasites, and may not need sheep dip.

Nitrogenfixing crops

tickresistant sheep

Gene Therapy
This is perhaps the most significant, and most controversial kind of genetic engineering. It is also the least well-developed. The idea of gene therapy is to genetically alter humans in order to treat a disease. This could represent the first opportunity to cure incurable diseases. Note that this is quite different from using genetically-engineered microbes to produce a drug, vaccine or hormone to treat a disease by conventional means. Gene therapy means altering the genotype of a tissue or even a whole human.

Cystic Fibrosis (you must learn this one!)

Cystic fibrosis (CF) is the most common genetic disease in the UK, affecting about 1 in 2500. It is caused by a mutation in the gene for protein called CFTR (Cystic Fibrosis Transmembrane Regulator). The gene is located on chromosome 7, and there are actually over 300 different mutations known, although the most common mutation is a deletion of three bases, removing one amino acid out of 1480 amino acids in the protein. CFTR is a chloride ion channel protein found in the cell membrane of epithelial (lining) tissue cells, and the mutation stops the protein working, so chloride ions cannot cross the cell membrane. Chloride ions build up inside these cells, which cause sodium ions to enter to balance the charge, and the increased concentration of the both these ions inside the epithelial cells decreases the osmotic potential. Water is therefore retained inside the cells, which means that the mucus secreted by these cells is drier and more sticky than normal. This sticky mucus block the tubes into which it is secreted, such as the small intestine, pancreatic duct, bile duct, sperm duct, bronchioles and alveoli. These blockages lead to the symptoms of CF: breathlessness, lung infections such as bronchitis and pneumonia, poor digestion and absorption, and infertility. Of these symptoms the lung effects are the most serious causing 95% of deaths. CF is always fatal, though life expectancy has increased from 1 year to about 20 years due to modern treatments. These treatments include physiotherapy many times each day to dislodge mucus from the lungs, antibiotics to fight infections, DNAse drugs to loosen the mucus, enzymes to help food digestion and even a heart-lung transplant. Given these complicated (and ultimately unsuccessful) treatments, CF is a good candidate for gene therapy, and was one of the first diseases to be tackled this way. The gene for CFTR was identified in 1989 and a cDNA clone was made soon after. The idea is to deliver copies of this good gene to the epithelial cells of the lung, where they can be incorporated into the nuclear DNA and make functional CFTR chloride channels. If about 10% of the cells could be corrected, this would cure the disease. Two methods of delivery are being tried: liposomes and adenoviruses, both delivered with an aerosol inhaler, like those used by asthmatics. Clinical trials are currently underway, but as yet no therapy has been shown to be successful.

The Future of Gene Therapy

http://www.mrothery.co.uk/genetech/genetechnotes.htm[21-Mar-11 1:52:15 PM]

gene technology notes

Gene therapy is in its infancy, and is still very much an area of research rather than application. No one has yet been cured by gene therapy, but the potential remains enticing. Gene therapy need not even be limited to treating genetic diseases, but could also help in treating infections and environmental diseases: White blood cells have be genetically modified to produce tumour necrosis factor (TNF), a protein that kills cancer cells, making these cells more effecting against tumours. Genes could be targeted directly at cancer cells, causing them to die, or to revert to normal cell division. White blood cells could be given antisense genes for HIV proteins, so that if the virus infected these cells it couldn’t reproduce. It is important to appreciate the different between somatic cell therapy and germ-line therapy. Somatic cell therapy means genetically altering specific body (or somatic) cells, such as bone marrow cells, pancreas cells, or whatever, in order to treat the disease. This therapy may treat or cure the disease, but any genetic changes will not be passed on their offspring. Germ-line therapy means genetically altering those cells (sperm cells, sperm precursor cell, ova, ova precursor cells, zygotes or early embryos) that will pass their genes down the "germ-line" to future generations. Alterations to any of these cells will affect every cell in the resulting human, and in all his or her descendants. Germ-line therapy would be highly effective, but is also potentially dangerous (since the long-term effects of genetic alterations are not known), unethical (since it could easily lead to eugenics) and immoral (since it could involve altering and destroying human embryos). It is currently illegal in the UK and most other countries, and current research is focussing on somatic cell therapy only. All gene therapy trials in the UK must be approved by the Gene Therapy Advisory Committee (GTAC), a government body that reviews the medical and ethical grounds for a trial. Germ-line modification is allowed with animals, and indeed is the basis for producing GMOs.

http://www.mrothery.co.uk/genetech/genetechnotes.htm[21-Mar-11 1:52:15 PM]