Genes and Genetic Engineering

Contents:
Specification
DNA
  • Nucleotides
  • DNA Structure
  • DNA Function
  • RNA
  • Replication
  • Transcription
  • Translation
  • Mutations
The Cell Cycle
  • DNA and Chromosomes
  • The Cell Cycle and Mitosis
  • Asexual Reproduction
  • Sexual Reproduction

Genetic Engineering

  • Techniques

  • Applications

These notes may be used freely by A level biology students and teachers, and they may be copied and edited. I would be interested to hear of any comments and corrections. Neil C Millar, Head of Biology, Heckmondwike Grammar School, High Street, Heckmondwike WF16 0AH (10/9/00)

Module 2 Specification

DNA

Structure. DNA is a stable polynucleotide. The double-helix structure of the DNA molecule in terms of: the components of DNA nucleotides; the sugar-phosphate backbone; specific base pairing and hydrogen bonding between polynucleotide strands (only simple diagrams of DNA structure are needed; structural formulae are not required). Explain how the structure of DNA is related to its functions.

Replication. The semi-conservative mechanism of DNA replication, including the role of DNA polymerase.

Transcription. The structure of RNA. The production of mRNA in transcription, and the role of RNA polymerase. Explain how the structure of RNA is related to its functions.

Translation. The roles of ribosomes, mRNA and its codons, and tRNA and its anticodons in translation.

Genetic Code. How DNA acts as a genetic code by controlling the sequence of amino acids in a polypeptide. Codons for amino acids are triplets of nucleotide bases. Candidates should be able to explain the relationship between genes, proteins and enzymes.

Mutations

New forms of alleles arise from changes (mutations) in existing alleles.

Mutations occur naturally at random. High energy radiation, high energy particles and some chemicals are mutagenic agents.

Reproduction

Genes and Chromosomes

Genes are sections of DNA which contain coded information that determines the nature and development of organisms. A gene can exist in different forms called alleles which are positioned in the same relative position (locus) on homologous chromosomes.

Mitosis

Mitosis increases cell number in growth and tissue repair and in asexual reproduction. During mitosis DNA replicates in the parent cell, which divides to produce two new cells, each containing an exact copy of the DNA of the parent cell. Candidates should be able to name and explain the stages of mitosis and recognise each stage from diagrams and photographs.

Asexual Reproduction and Cloning

Genetically identical organisms (clones) can be produced by using vegetative propagation, and by the splitting of embryos. Given appropriate information, candidates should be able to explain the principles involved in:

Meiosis

During meiosis, cells containing pairs of homologous chromosomes divide to produce gametes containing one chromosome from each homologous pair. In meiosis the number of chromosomes is reduced from the diploid number (2n) to the haploid number (n). (Details of meiosis not required.)

Sexual Reproduction and Gametes

Sexual reproduction involves gamete formation and fertilisation. In sexual reproduction DNA from one generation is passed to the next by gametes. When gametes fuse at fertilisation to form a zygote the diploid number is restored. This enables a constant chromosome number to be maintained from generation to generation.

Differences between male and female gametes in terms of size, number produced and mobility.

Sexual Life Cycles

Candidates should be able to interpret life cycles of organisms in terms of mitosis, meiosis, fertilisation and chromosome number.

Genetic Engineering

In genetic engineering, genes are taken from one organism and inserted into another.

Genetically Modified Microbes

Microorganisms are widely used as recipient cells during gene transfer. Bacteria containing a transferred gene can be cultured on a large scale in industrial fermenters. Useful substances produced by using genetically engineered microorganisms include antibiotics, hormones and enzymes. (Details of manufacturing processes not required.)

Genetically Modified Animals

How animals can be genetically engineered to produce substances useful in treating human diseases, as exemplified by genetically engineering sheep to produce alpha-1-antitrypsin which is used to treat emphysema and cystic fibrosis.

Gene Therapy and Cystic Fibrosis

In gene therapy healthy genes may be cloned and used to replace defective genes. In cystic fibrosis the transmembrane regulator protein, CFTR, is defective. A mutant of the gene that produces CFTR results in CFTR with one missing amino acid. The symptoms of cystic fibrosis related to the malfunctioning of CFTR.

Techniques that might possibly be used to introduce healthy CFTR genes into lung epithelial cells include:

Evaluation of Genetic Engineering

Candidates should be able to evaluate the ethical, social and economic issues involved in the use of genetic engineering in medicine and in food production.

 

Genetics

Genetics is the study of heredity (from the Latin genesis = birth). The big question to be answered is: why do organisms look almost, but not exactly, like their parents? There are three branches of modern genetics:

  1. Molecular Genetics (or Molecular Biology), which is the study of heredity at the molecular level, and so is mainly concerned with the molecule DNA. It also includes genetic engineering and cloning, and is very trendy. This module is mostly about molecular genetics.
  2. Classical or Mendelian Genetics, which is the study of heredity at the whole organisms level by looking at how characteristics are inherited. This method was pioneered by Gregor Mendel (1822-1884). It is less fashionable today than molecular genetics, but still has a lot to tell us. This is covered in Module 4.
  3. Population Genetics, which is the study of genetic differences within and between species, including how species evolve by natural selection. Some of this is also covered in Module 4.

 

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, and yet it is only in the past 50 years that we have begun to understand them. DNA stands for deoxyribonucleic acid and RNA for ribonucleic acid, and they are called nucleic acids because they are weak acids, first found in the nuclei of cells. They are polymers, composed of monomers called nucleotides.

Nucleotides

Nucleotides have three parts to them:

Base:

Adenine (A)

Cytosine (C)

Guanine (G)

Thymine (T)

Uracil (U)

 

 

Nucleotide Polymerisation

Nucleotides polymerise by forming bonds between carbon 3 of the sugar and an oxygen atom of the phosphate. This is a condensation polymerisation reaction. The bases do not take part in the polymerisation, so there is 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 end and a free OH group at the other end.

Structure of DNA

The three-dimensional structure of DNA was discovered in the 1950's by Watson and Crick. The main features of the structure are:

Function of DNA

DNA is the genetic material, and genes are made of DNA. DNA therefore has two essential functions: replication and expression.

Expression can be split into two parts: transcription (making RNA) and translation (making proteins).

These two functions are summarised in this diagram (called the central dogma of genetics).

No one knows exactly how many genes we humans have to control all our characteristics, the latest estimates are 60-80,000. The sum total of all the genes in an organism is called the genome.

The table shows the estimated number of genes in different organisms:

Species

Common name

length of DNA (kbp)*

no of genes

phage l

virus

48

60

Eschericia coli

Bacterium

4 639

7 000

Saccharomyces cerevisiae

Yeast

13 500

6 000

Drosophila melaogaster

fruit fly

165 000

~10 000

Homo sapiens

Human

3 150 000

~70 000

*kbp = kilo base pairs, i.e. thousands of nucleotide monomers.

Amazingly, genes only seem to comprise about 2% of the DNA in a cell. The majority of the DNA does not form genes and doesnít seem to do anything. The purpose of this junk DNA remains a mystery!

RNA

RNA is a nucleic acid like DNA, but with 4 differences:

Messenger RNA (mRNA)

mRNA carries the "message" that codes for a particular protein from the nucleus (where the DNA master copy is) to the cytoplasm (where proteins are synthesised). It is single stranded and just long enough to contain one gene only. It has a short lifetime and is degraded soon after it is used.

Ribosomal RNA (rRNA)

rRNA, together with proteins, form ribosomes, which are the site of mRNA translation and protein synthesis. Ribosomes have two subunits, small and large, and are assembled in the nucleolus of the nucleus and exported into the cytoplasm.

Transfer RNA (tRNA)

tRNA is an "adapter" that matches amino acids to their codon. tRNA is only about 80 nucleotides long, and it folds up by complementary base pairing to form a looped clover-leaf structure. At one end of the molecule there is always the base sequence ACC, where the amino acid binds. 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. The amino acids are attached to their tRNA molecule by specific enzymes. These are highly specific, so that each amino acid is attached to a tRNA adapter with the appropriate anticodon.

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 43 or 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.

There are several interesting points from this code:

Replication - DNA Synthesis

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

  1. Replication starts at a specific sequence on the DNA molecule called the replication origin.
  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.

DNA replication can takes a few hours, and in fact this limits the speed of cell division. One reason bacteria can reproduce so fast is that they have a relatively small amount of DNA.

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. This need not be the case, and alternative theories suggested that a "photocopy" of the original DNA could be made, leaving the original DNA conserved (conservative replication. The evidence for the semi-conservative method came from an elegant experiment performed in 1958 by Meselson and Stahl. They used the bacterium E. coli together with the technique of density gradient centrifugation, which separates molecules on the basis of their density.

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 "message" from the nucleus to the cytoplasm. This copy is mRNA, and the process of copying is called transcription.

  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. The DNA stand that is copied is called the template or sense strand because it contains the sequence of bases that codes for a protein. The other strand is just a complementary copy, and is called the non-template or antisense strand.
  3. The new nucleotides are joined to each other by strong covalent bonds by the enzyme RNA polymerase.
  4. Only about 8 base pairs remain attached at a time, since the mRNA molecule peels off from the DNA as it is made. A winding enzyme rewinds the DNA.
  5. The initial mRNA, or primary transcript, contains many regions that are not needed as part of the protein code. These are called introns (for interruption sequences), while the parts that are needed are called exons (for expressed sequences). All eukaryotic genes have introns, and they are usually longer than the exons.
  6. The introns are cut out and the exons are spliced together by enzymes
  7. The result is a shorter mature RNA containing only exons. The introns are broken down.
  8. 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).

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 acid-tRNA 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, when the ribosome falls apart, releasing the finished protein.

A single piece of mRNA can be translated by many ribosomes simultaneously, so many protein molecules can be made from one mRNA molecule. 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 methyl or phosphate groups to amino acids, or adding sugars (to make glycoproteins) or lipids (to make lipoporteins).

 

 

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:

As a result of a mutation there are three possible phenotypic effects:

The kinds of mutations discussed so far 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:

During the Earth's early history there were far more of these mutagens than there are now, so the mutation rate would have been much higher than now, leading to a greater diversity of life. Some of these mutagens are used today in research, to kill microbes or in warfare. They are often carcinogens since a common result of a mutation is cancer.

DNA and Chromosomes

The DNA molecule in a single human cell is 99 cm long, so is 10 000 times longer than the cell in which it resides (< 100mm). (Since an adult human has about 1014 cells, all the DNA is one human would stretch about 1014 m, which is a thousand times the distance between the Earth and the Sun.) 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. At various times parts of the chromatin will unwind so that genes on the DNA can be transcribed. This allows the proteins that the cell needs to be made.

Just before cell division the DNA is replicated, and more histone proteins are synthesised, so there is temporarily twice the normal amount of chromatin. 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 therefore 100 000 times thicker, so 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

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, and the loci of thousands of human genes are now known. There are on average about 3 000 genes per chromosome, although of course the larger chromosomes have more than this, and the smaller ones have fewer.

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:

 

The Cell Cycle

Cells are not static structures, but are created and die. 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. For example 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

Interphase

  • chromatin not visible
  • DNA, histones and centrioles all replicated

Prophase

  • chromosomes condensed and visible
  • centrioles at opposite poles of cell
  • nucleolus disappears
  • phase ends with the breakdown of the nuclear membrane

Metaphase

  • chromosomes align along equator of cell
  • spindle fibres (microtubules) connect centrioles to chromosomes

Anaphase

  • centromeres split, allowing chromatids to separate
  • chromatids move towards poles, centromeres first, pulled by kinesin (motor) proteins walking along microtubules (the track)

Telophase

  • spindle fibres disperse
  • nuclear membraness from
  • nucleoli form

Cytokinesis

  • In animal cells a ring of actin filaments forms round the equator of the cell, and then tightens to form a cleavage furrow, which splits the cell in two.
 

  • In plant cells vesicles move to the equator, line up and fuse to form two membranes called the cell plate. A new cell wall is laid down between the membranes, which fuses with the existing cell wall.

 

Asexual Reproduction

Asexual reproduction is the production of offspring from a single parent using mitosis. The offspring are therefore genetically identical to each other and to their "parent"- in other words they are clones. Asexual reproduction is very common in nature, and in addition we humans have developed some new, artificial methods. The Latin terms in vivo ("in life", i.e. in a living organism) and in vitro ("in glass", i.e. in a test tube) are often used to describe natural and artificial techniques. These different methods are summarised in the table.

 

Methods of Asexual Reproduction

 

Natural Methods

Artificial Methods

Microbes

binary fission

budding

spores

fragmentation

cell culture

fermenters

Plants

vegetative propagation

parthenogenesis

cuttings

grafting

tissue culture

Animals

budding

fragmentation

parthenogenesis

embryo splitting

somatic cell cloning

Natural Methods

Binary Fission. This is the simplest and fastest method of asexual reproduction, used by all prokaryotes and by many unicellular organisms such as amoeba. The nucleus divides by mitosis and then the cell simply splits into two equal-sized daughter cells.

Budding. A small copy of the parent develops as an outgrowth, or bud, from the parent, and is eventually released as a independent individual. This method is used by several protoctists, yeasts (fungi) and even by some simple animals.

Spores. These are simply specialised cells that are released from the parent (usually in very large numbers) to be dispersed. Under suitable conditions each cell can grow into a new individual. This method is used by most fungi and by the lower plants (mosses and ferns).

 

Fragmentation. This is when an organism spontaneously breaks into two or more fragments, each of which then develops into a new individual. It is used by some simple animals such as sponges, flatworms and starfish. Do not confuse this with regeneration, where some animals simply regenerate a part of their body if it is cut off.

Vegetative Reproduction. This term describes all the natural methods of asexual reproduction used by plants. A bud grows from a vegetative (i.e. not reproductive) 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. onion, daffodil), corms (e.g. crocus, gladiolus), rhizomes (e.g. iris, couch grass), stolons (e.g. blackberry), runners (e.g. strawberry, creeping buttercup), tubers (e.g. potato, dahlia), tap roots (e.g. carrot, turnip), and tillers (e.g. grasses). 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. the citrus fruits) and some invertebrate animals (e.g. honeybees, aphids, some crustaceans) 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

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 are often quite suitable for some organisms (such as yeast, potatoes and strawberries), but many important plants and animals do not reproduce asexually, so artificial methods have to be used.

Cell Culture. Microbes (bacteria and some fungi) 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 agar plate, and selected strains 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, O2, pressure and temperature) must be constantly monitored using probes. This will ensure the maximum growth rate of the microbes.

Cuttings. This is a very old method of cloning plants. Parts of a plant stem (or even leaves) are 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, & chrysanthemums are reproduced commercially by cuttings.

Grafting. This is another ancient technique, used for plant species that cannot grow roots from cuttings. Instead they can often be cloned by grafting a stem cutting (called a scion) onto the lower part of an existing plant (called the rootstock). One rootstock can take several scions, and need not even be the same species as the scion. The resulting hybrid will produce the flowers and fruits of the scion, but its size will be determined by the rootstock. Almost all fruit trees, such as apples and pears are clones of a few popular varieties grafted onto hardy rootstock.

Tissue Culture (or micropropagation). This is a more modern, and very efficient, way of cloning plants. Small samples of plant tissue, called an explant, can now be grown on agar plates in the laboratory in much the same way that bacteria can be grown. Initially the explant had to be meristem tissue (i.e. undifferentiated buds), but the technique has improved so that any tissue can now be used (e.g. from a leaf). The plant tissue can be 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 fruit trees, ornamental plants and plantation crops such as oils palm, data palm, sugar cane and banana. The advantages are:

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 only worth it for commercially-important farm animals, such as prize cows, or genetically engineered animals. A female animal is fed a fertility drug (FSH) so that she produces many mature eggs (superovulation). The eggs are then surgically removed from the femaleís 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 16-cell 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.

Somatic Cell Cloning (or Nuclear Transfer). The problem with embryo cloning is that you donít 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, first with frogs in the 1970s, and most recently with sheep (the famous "Dolly") in 1996.

The technique used to create Dolly is similar to embryo cloning, but has one crucial difference. 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 sure enough it developed into an embryo. The embryo was implanted into the uterus of a surrogate mother, and developed into an apparently normal sheep, Dolly. It took hundreds of attempts to achieve success with Dolly, but once the technique is improved it will be possible to combine this technique with embryo cloning to make many clones of an adult animal. Dollyís "mother" was just an ordinary sheep, but in the future prize animals (or genetically engineered ones) could be cloned in this way.

 

 

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:

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, but 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 dominant, long-lived adult form is haploid. Haploid spores undergo mitosis and grow into complete, differentiated 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. In the simpler plants (mosses and liverworts) the haploid form is larger than the diploid form, while in the higher plants (ferns and conifers) the diploid form is larger.

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:

You donít 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 some algae and fungi the gametes are roughly the same size. This is called isogamy. There are no male and female sexes, but there can be + and - strains, who reproduce together.

In all plants and animals the gametes are different sizes. This is called heterogamy.

These are the male gametes, and they are produced in very large numbers. Human males for example release about 108 sperm in one ejaculation.

It is this difference in gametes that actually defines the sex of an individual. Those individuals that produce small mobile gametes are the males, and those that produce the larger gametes are the females. In some species (such as most flowering plants) the same individual organisms can produce both male and female gametes, so they do not have distinct sexes and are called hermaphrodites. In other species (such as mammals) there are two distinct sexes, each producing their own gametes. These are called unisexual.

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 and use their tails to swim through the follicle cells (shown in this photo). When they reach the jelly coat surrounding the ovum they bind to receptors and this stimulates the rupture of the acrosome membrane in the sperms, releasing digestive enzymes, which make a path through the jelly coat. 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 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.

 

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 of Genetic Engineering

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 that we shall look at in detail.

 

Technique

Purpose

1

Restriction Enzymes

To cut DNA at specific points, making small fragments

2

DNA Ligase

To join DNA fragments together

3

Vectors

To carry DNA into cells and ensure replication

4

Plasmids

Common kind of vector

5

Gene Transfer

To deliver a gene to a living cells

6

Genetic Markers

To identify cells that have been transformed

7

Replica Plating

To make exact copies of bacterial colonies on an agar plate

8

PCR

To amplify very small samples of DNA

9

cDNA

To make a DNA copy of mRNA

10

DNA probes

To identify and label a piece of DNA containing a certain sequence

11

Shotgun

To find a particular gene in a whole genome

12

Antisense genes

To stop the expression of gene in a cell

13

gene Synthesis

To make a gene from scratch

14

Electrophoresis

To separate fragments of DNA

15

DNA Sequencing

To read the base sequence of a length of DNA

 

1. 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. Some restriction enzymes cut straight across both chains, forming blunt ends, but most 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 with complementary sequences. 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, called recognition sequences.

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, and HindIII is from Haemophilis influenzae.

 

2. 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 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.

3. Vectors

In biology a vector is something that carries things between species. For example the mosquito is a disease vector because it 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 wonít actually do anything inside a host cell. Since it is not part of the cellís normal genome it wonít be replicated when the cell divides, it wonít be expressed, and in fact it will probably be broken down pretty quickly. A vector gets round these problems by having these properties:

Many different vectors have been made for different purposes in genetic engineering by modifying naturally-occurring DNA molecules, and these are now available off the shelf. For example a cloning vector contains sequences that cause the gene to be copied (perhaps many times) inside a cell, but not expressed. An expression vector contains sequences causing the gene to be expressed inside a cell, preferably in response to an external stimulus, such as a particular chemical in the medium. Different kinds of vector are also available for different lengths of DNA insert:

Type of vector

Max length of DNA insert

Plasmid

10 kbp

Virus or phage

30 kbp

Bacterial Artificial Chromosome (BAC)

500 kbp

4. Plasmids

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 usually around 10 copies of a plasmid in a bacterial cell. Plasmids are copied separately from the main bacterial DNA when the cell divides, so the plasmid genes are passed on to all daughter cells. They are also used naturally for exchange of genes between bacterial cells (the nearest they get to sex), so bacterial cells will readily 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 PstI and 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 (weíll 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 doesnít matter, as the marker genes can be used later to identify the correct hybrid vector.

5. 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.

1. Bacteriophages (or phages) are viruses that infect bacteria. They are a very effective way of delivering large genes into bacteria cells in culture.

2. Adenoviruses 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 hostís 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.

3. Retroviruses 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 hostís 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 all the host cells.

6. 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. Weíll look at how to do this with bacterial host cells, as thatís the most common technique.

A common marker, used in the R-plasmid, is a gene for resistance to an antibiotic such as tetracycline. Bacterial cells taking up this plasmid can make this gene product and so are resistant to this antibiotic. So if the cells are grown on a medium containing tetracycline all the normal untransformed cells, together with cells that have taken up DNA thatís not in a plasmid (99%) will die. Only the 1% transformed cells will survive, and these can then be grown and cloned on another plate.

7. 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 the normal plasmid. 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.

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.

The R-plasmid with its antibiotic-resistance genes dates from the early days of genetic engineering in the 1970s. In recent years better plasmids with different marker genes have been developed that do not kill the desired cells, and so do not need a replica plate. These new marker genes make an enzyme (actually lactase) that converts a colourless substrate in the agar medium into a blue-coloured product that can easily be seen. So cells with a normal plasmid turn blue on the correct medium, while those with the hybrid plasmid can't make the enzyme and stay white. These white colonies can easily be identified and transferred to another plate. Another marker gene, transferred from jellyfish, makes a green fluorescent protein (GFP).

8. 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 95įC 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 40įC 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 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 90įC, so it is not denatured by the high temperatures in step 2. Its optimum temperature is about 72įC, 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.

9. 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 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.

10. 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:

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:

11. 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 geneís sequence must be known.

 

12. 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 canít bind to this, so the mRNA is not translated, and the gene is effectively "switched off".

13. Gene Synthesis

It is possible to chemically synthesise a gene in the lab by laboriously joining nucleotides together in the correct order. Automated machines can now make this much easier, but only up to a limit of about 30bp, so very few real genes could be made this way (anyway itís usually much easier to make cDNA). The genes for the two insulin chains (xx bp) and for the hormone somatostatin (42 bp) have been synthesisied this way. It is very useful for making gene probes.

14. Electrophoresis

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 made of agarose or polyacrylamide, 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:

15. 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.

  1. 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 (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.

 

 

Applications of Genetic Engineering

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

using genetically modified organisms (usually microbes) to produce chemicals, usually for medical or industrial applications.

  • New Phenotypes

using gene technology to alter the characteristics of organisms (usually farm animals or crops)

  • Gene Therapy

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

Use

Host Organism

Insulin

human hormone used to treat diabetes

bacteria /yeast

HGH

human growth hormone, used to treat dwarfism

bacteria

BST

bovine growth hormone, used to increase milk yield of cows

bacteria

Factor VIII

human blood clotting factor, used to treat haemophiliacs

bacteria

Anti-thrombin

anti-blood clotting agent used in surgery

goats

Penicillin

antibiotic, used to kill bacteria

fungi / bacteria

Vaccines

hepatitis B antigen, for vaccination

yeast

AAT

enzyme used to treat cystic fibrosis and emphysema

sheep

a-glucosidase

enzyme used to treat Pompeís disease

rabbits

DNase

enzyme used to treat CF

bacteria

rennin

enzyme used in manufacture of cheese

bacteria /yeast

cellulase

enzyme used in paper production

bacteria

PHB

biodegradable plastic

plants

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 genetically-engineered gene products.

Type of organism

Advantages

Disadvantages

Prokaryotes

(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.

canít splice introns; no post-translational modification; small gene size

Eukaryotes

can splice introns; 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.

Fungi (yeast, mould)

asexual so can be cloned; haploid, so only one copy needed; can be grown in vats

canít always make animals gene products

Plants

photosynthetic so donít need much feeding; can be cloned from single cells; products can be secreted from roots or in sap.

cell walls difficult to penetrate by vector; slow growing; must be grown in fields; multicellular

Animals

(pharming)

most likely to be able to make human proteins; products can be secreted in milk or urine

multicellular; slow growing

 

Weíll 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 donít 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 wasnít 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.

Human Growth Hormone (HGH)

HGH is a protein hormone secreted by the pituitary gland, which stimulates tissue growth. Low production of HGH in childhood results in pituitary dwarfism. This can be treated with HGH extracted from dead humans, but as the treatment caused some side effects, such as Creutzfeldt-Jacod disease (CJD), the treatment was withdrawn. The HGH gene has been cloned and an artificial cDNA gene has been inserted into E. coli. A signal sequence has been added which not only causes the gene to be translated but also causes the protein to be secreted from the cell, which makes purification much easier. This genetically engineered HGH is produced by Genentech and can successfully restore normal height to children with HGH defficiency.

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. Traditionally the cheese industry has used rennin obtained from the stomach of young calves when they are 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 such as the bacterium E. coli and the fungus Aspergillus niger. 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 glycoprotein, which means it needs to have sugars added by post translational modification. This kind of modification can only be carried out by animals, and AAT is now produced by genetically-modified 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 by PPL Pharmaceuticals in Edinburgh in 1993. This is how Tracy was made:

  1. 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-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 canít 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 generally commercially-important crops or farm animals, and the object is to improve their quality in some way. This 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 donít need to study any of these in detail, but this table gives an idea of what is being done.

organism

Modification

long life tomatoes

There are two well-known projects, both affecting the gene for the enzyme polygalactourinase (PG), a pectinase that softens 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.

Insect-resistant crops

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 environment) is unnecessary.

virus-resistant crops

Gene for virus coat protein has been cloned and inserted into tobacco, potato and tomato plants. The coat protein seems to "immunise" the plants, which are much more resistant to viral attack.

herbicide resistant crops

The gene for resistance to the herbicide BASTA has been transferred from Streptomyces bacteria to tomato, potato, corn, and wheat plants, making them resistant to Basta. Fields can safely be sprayed with this herbicide, which will kill all weeds, but the crops. However, this means using more agrochemicals, not less.

pest-resistant legumes

The gene for an enzyme that synthesises a chemical toxic to weevils has been transferred from Bacillus bacteria to The Rhizobium bacteria that live in the root nodules of legume plants. These root nodules are now resistant to attack by weevils.

Nitrogen-fixing crops

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.

crop improvement

Proteins in some crop plants, including wheat, are often deficient in essential amino acids (which is why vegetarians have to watch their diet so carefully), so the protein genes are being altered to improve their composition for human consumption.

mastitis-resistant cattle

The gene for the enzyme lactoferrin, which helps to resists the infection that causes the udder disease mastitis, has been introduced to Herman Ė the first transgenic bull. Hermanís offspring inherit this gene, do not get mastitis and so produce more milk.

tick-resistant sheep

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.

Fast-growing sheep

The human growth hormone gene has been transferred to sheep, so that they produce human growth hormone and grow more quickly. However they are more prone to infection and the females are infertile.

Fast-growing fish

A number of fish species, including salmon, trout and carp, have been given a gene from another fish (the ocean pout) which activates the fishís own growth hormone gene so that they grow larger and more quickly. Salmon grow to 30 times their normal mass at 10 time the normal rate.

environment cleaning microbes

Genes for enzymes that digest many different hydrocarbons found in crude oil have been transferred to Pseudomonas bacteria so that they can clean up oil spills.

 

 

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

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.

SCID

Severe Combined Immunodefficiency Disease (SCID) is a rare genetic disease that affects the immune system. It is caused by a mutation in the gene for the enzyme adenosine deaminase (ADA). Without this enzyme white blood cells cannot be made, so sufferers have almost no effective immune system and would quickly contract a fatal infection unless they spend their lives in sterile isolation (SCID is also known as "baby in a bubble syndrome"). Gene therapy has been attempted with a few children in the USA and UK by surgically removing bone marrow cells (which manufacture white blood cells in the body) from the patient, transfecting them with a genetically-engineered virus containing the ADA gene, and then returning the transformed cells to the patient. The hope is that these transformed cells will multiply in the bone marrow and make white blood cells. The trials are still underway, so the success is unknown.

The Future of Gene Therapy

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:

It is important to appreciate the different between somatic cell therapy and germ-line therapy.

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.