快猫短视频s can now manipulate genes with ease. The results include advances in diagnosis of genetic diseases before birth and the possibility of 鈥済ene therapy鈥� in the future
IMAGINE trying to find a needle in a haystack the size of Mount Everest. Impossible? Probably, yet scientists have made giant strides in molecular biology over the past decade enabling them to perform the molecular equivalent of that 鈥渋mpossible鈥� task.
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Each cell in our bodies contains all the genetic information to make an entire human being. That information is carried in the genes that make up our chromosomes. The stuff of genes is deoxyribonucleic acid (DNA), a spiral molecule resembling a ladder whose 鈥渞ungs鈥� are built of pairs of bases.
If we were to unravel the DNA of all the chromosomes in each cell in a person鈥檚 body and join it end to end it would stretch to the Moon and back about 8000 times. Yet, with the new techniques of molecular biology, scientists can now isolate a single gene of perhaps 1000 to 2000 bases from an amount of DNA sufficient to contain more than six million genes of similar size!
Being able to locate and analyse a single gene promises to revolutionise the study of inherited, or genetic diseases. It already enables doctors to diagnose some inherited diseases in human fetuses only eight weeks old. Examples are haemophilia and sickle cell anaemia, two inherited diseases of the blood.
The new techniques are also probing the mystery of how disorders such as cancer and heart disease develop. Eventually, they may even lead to gene therapy, in which people born with faulty genes may be given normal ones.
Inherited diseases
Genes from our parents
GENETIC diseases follow two patterns of inheritance. Virtually all the cells in your body contain 46 chromosomes in their nuclei. The only exceptions are the gametes, the sperm and egg cells, which contain half that number. You will have inherited 23 of those chromosomes from your mother (via an egg cell) and 23 from your father (via a sperm). So all the genes on chromosomes come in two versions, one inherited from each parent.
In some cases a single abnormal gene from one parent is sufficient to cause disease; here the gene is said to be dominant. More commonly, a child must inherit a defective gene from both parents before the disorder shows itself; in this case the genes are called recessive. Healthy persons who have just one defective recessive gene are called carriers. They do not suffer from the disease but could pass it on to their children. We all carry a few potentially harmful recessive genes.
Some diseases, such as haemophilia, are caused by defective genes on the X- chromosome, one of the 鈥渟ex chromosomes鈥�. Cells of females have two X- chromosomes whereas those of males have one X- and one Y-chromosome.
A woman who has the haemophilia gene on both her X-chromosomes will suffer the disease. If she has the defective gene on one of her X-chromosomes, she will not show symptoms of the disease but is said to be a carrier. A male inheriting the defective gene on his X-chromosome will suffer the disease. Such disorders are said to be sex-linked.
Until very recently, doctors could not usually determine whether a fetus was afflicted with a serious genetic disease. All they could do was give some parents a rough idea of the risk of having an affected child. Such genetic counselling is usually feasible only if the parents are known to be carriers. This might be known because they already have one child with an inherited disease, such as cystic fibrosis. Children with cystic fibrosis make excessively thick secretions which can block the air passages of the lungs.
A doctor can tell the parents their chances of producing a second child with cystic fibrosis. This approach is limited by the fact that, in most cases, people do not know that they are carriers until they have produced an affected child. Nor are there any simple diagnostic tests to identify carriers of most genetic diseases.
For some diseases, doctors can now isolate and analyse the specific fetal genes suspected to be defective from as little as 2 millilitres of fetal blood, or two-millionths of a gram of fetal DNA. The advances in molecular biology go hand in hand with new methods of obtaining fetal cells.
Medical scientists developed the first method of obtaining fetal cells about 20 years ago. The technique, called amniocentesis, is carried out 14 to 15 weeks into pregnancy. Amniocentesis is used in prenatal diagnosis, in which doctors diagnose disease in a fetus. It gives the woman the chance to decide whether she wishes to continue her pregnancy.
In amniocentesis, a doctor uses ultrasound to image the membranous bag that encloses the fetus within the womb. By means of a hypodermic needle, the doctor draws up a sample of amniotic fluid. Amniotic fluid surrounds and cushions the fetus while it is in the uterus. The fluid contains some fetal cells which scientists can examine directly for chromosomal abnormalities.
Amniotic fluid contains many chemicals involved in the fetus鈥檚 metabolism. 快猫短视频s can detect a few rare inherited disorders of metabolism by measuring the quantity and turnover of these chemicals in amniotic fluid. They can now extract the DNA from the fetal cells and test for the presence of specific faulty genes.
The newest method of obtaining fetal cells, however, is called chorion villus sampling (CVS). It allows doctors to make prenatal diagnoses of certain diseases after only 8 to 10 weeks of pregnancy. Amniocentesis can provide only a few fetal cells which doctors must then grow in culture to provide enough fetal DNA to analyse, and this can delay results by several weeks. In contrast, CVS can provide enough fetal DNA for direct analysis, and give an answer in a day or two.
After the embryo has implanted itself in the uterus, the chorionic plate begins to grow around the fetus and later forms the placenta which feeds the growing fetus. The chorionic plate is made up of cells derived from the outer layer of the embryo. In CVS, doctors use ultrasound scanning to obtain chorion cells between 8 and 10 weeks of pregnancy. One sample can provide as much as 100 micrograms of fetal DNA.
New research in mammalian development suggests that scientists may, in the future, be able to carry out diagnostic tests in the pre-embryonic鈥� period. This period spans the first two weeks after an egg has been fertilised and before it has begun to implant itself into the lining of the uterus.
Using in vitro fertilisation (IVF), the pre-embryos could be checked for defects before replacing them in the woman鈥檚 uterus. Such preimplantation diagnosis would offer women the chance of starting pregnancy knowing that their children will not inherit the disease in question, rather than finding out during the first or second three months of pregnancy. This is particularly advantageous for women who find abortion unacceptable for ethical or religious reasons.
Given sufficient fetal DNA, how can scientists locate the often small changes that give rise to inherited disease? This is where the new techniques of molecular biology come in.
The two component strands of DNA are bound together by specific pairing between so-called complementary bases 鈥� adenine binds only to thymine, and guanine only to cytosine. When one strand meets its match, they bind together.
快猫短视频s have exploited this fact of life to make gene probes for a number of mutations that give rise to genetic diseases. They isolated one particular intermediary between DNA and protein, called messenger RNA (mRNA) and then made a complementary DNA (cDNA) from it.
Exposed to cellular DNA the cDNA probe recognises and binds to its complementary sequence. In preparing a probe, scientists incorporate radioactive bases that 鈥渓abel鈥� the cDNA and enable them to find it once it has bound to its complementary sequence in the cellular DNA.
To find a particular gene in someone鈥檚 DNA, scientists first chop the long strands of DNA into smaller fragments using bacterial enzymes that cut the DNA in a consistent way. The enzymes are called restriction endonucleases.
Placing the DNA fragments on a special gel exposed to an electric field makes them separate according to size, and form a pattern. The pattern is then 鈥渂lotted鈥� onto a nitrocellulose filter.
The DNA fragments are bound to the filter by baking and then exposed to a radioactively labelled probe. When the filter is placed on an X-ray film the position of the probe, and therefore the fragment containing the altered base sequence, shows up as a dark band. The picture of bands is called a gene map.
Most haemoglobin disorders, and many other genetic diseases caused by a defect in a single gene, cannot be diagnosed in that way because we do not yet know which gene gives rise to the disease and so cannot make gene probes for the mutation. In such cases, researchers may draw on another approach based on genetic linkage.
Researchers have known for years that certain genes tend to stay together on a chromosome and so are inherited together. The genes are said to be 鈥渓inked鈥� as they are physically close together on a chromosome. If scientists cannot identify the product of a particular gene they wish to study, and if that gene cannot be located directly by a gene probe, the next best thing is to find a gene that is linked to it but can be located.
快猫短视频s can use such neighbouring genes as 鈥渕arkers鈥� to look for the presence or absence of the gene they wish to study. One problem is that few marker genes for genetic diseases have so far been found. In addition, the marker may not be close enough to the gene to be accurate. Recombination 鈥� where paired chromosomes physically swap parts of themselves 鈥� can break up the linkage between a marker and a disease-causing gene.
This relies on family studies to trace the inheritance of the marker and faulty gene through several generations.
Nonsense DNA
New marker genes
RESEARCHERS have since discovered non-coding regions of our DNA that contain base changes which produce either new cutting sites for restriction enzyme or remove existing sites. These have provided them with new markers. These harmless base changes follow the usual pattern of inheritance. The sequences are said to be polymorphic, that is, they occur in several different forms. Those that change the cutting sites of restriction enzymes are called restriction site polymorphisms.
The length of the fragments can also be altered by the presence of varying numbers of small, repeated sequences of non-coding 鈥渘onsense DNA鈥�, called hypervariable regions (HVR). HVRs occur throughout our DNA and are also inherited in the normal way. Because both HVRs and restriction site polymorphisms affect the sizes of DNA fragments they are collectively called restriction fragment length polymorphisms (RFLPs).
Diseases of the blood
Rogue haemoglobins
THE new techniques have spectacularly improved prenatal diagnosis of certain inherited diseases of haemoglobin. Haemoglobin is the protein that makes blood red and enables it to carry oxygen from the lungs to the tissues.
Some people have imperfect haemoglobin genes. These 鈥渇aulty鈥� genes can cause a range of diseases in which haemoglobin fails to do its job properly. In one of those diseases, called sickle cell anaemia, the haemoglobin forms long, rod- like filaments that change the shape of the person鈥檚 red blood cells when they give up their oxygen to tissues. This, in turn, causes the cells to be prematurely destroyed and to clog small blood vessels. The blockages cause severe illness which can be life threatening.
Knowing the sequence of the haemoglobin gene, and studying the patterns of inheritance of sickle cell anaemia in affected families, enabled researchers to work out the genetic mutation that gives rise to the disease. Haemoglobin is made up of two pairs of peptide chains called alpha and beta chains. The structure of those chains are controlled by corresponding alpha and beta genes.
Sickle cell anaemia is due to a single base change in sixth codon of the beta chain gene, where each codon specifies a particular amino acid in the protein product. Instead of GTG, the codon for valine, the triplet reads GAG which codes for glutamic acid.
That mutation of adenine for thymine 鈥� and the consequent substitution of glutamic acid for valine 鈥� changes the three-dimensional structure of haemoglobin. Once scientists knew the mutation it was possible to make a gene probe that would identify the mutation in a mixture containing the entire DNA of thousands of cells.
The discovery of RFLPs gives researchers the opportunity to study and, hopefully, diagnose genetic diseases in which the underlying cause is still unknown, such as cystic fibrosis.
Suppose doctors want to find out if a fetus has received a gene for cystic fibrosis from each of its parents. They know roughly where on the chromosome the mutation is but are unable to identify it. The idea is to find a RFLP close enough to the faulty gene that can act as a linkage marker for it.
Doctors examine the DNA of both parents and a previously affected child or other relative for a linkage between a RFLP and the gene carrying the cystic fibrosis mutation. If they find a linkage they then look for the presence of the linked RFLP in the DNA of the fetus. Should the fetus show the presence of the polymorphism on both chromosomes it must have received the mutant gene from both parents and thus would be expected to develop the disease.
Tomorrow鈥檚 genetics
A window on diabetes?
SO FAR, these new techniques have been used to study diseases caused by single-gene mutations. What use could they be for studying other diseases, such as coronary heart disease?
Doctors know that many genes play a part in the degeneration of the coronary arteries that supply the heart muscle with blood and can lead to heart attacks even in young people. The involvement of many genes, plus their interaction with the environment, presents a much more complex problem to solve: heart attacks do not run in families in the same way as do haemophilia or sickle cell anaemia. Even so, the new techniques may help to clarify what goes wrong in such diseases and eventually enable us to diagnose those people with a tendency to develop heart disease.
One approach would be to isolate genes that might be involved in the chemistry of the arterial wall and the way in which the body uses fats. For example, several genes alter the behaviour of the lipoproteins, which carry the fat that is deposited in the walls of blood vessels. As the vessels become narrower the risks of heart attacks increase.
Linkage studies will help researchers to identify the most important genes predisposing people to heart disease. 快猫短视频s can analyse families to see if particular genetic markers are associated with premature heart attacks. The same approach could be used for other diseases such as diabetes.
How our cells make proteins
THE DNA in chromosomes is tightlycoiled and folded. Stretched out, itresembles a long, spiral ladder. Each 鈥渟ide of the ladder鈥� is built of a chain of sugar- phosphate units. In DNA the sugar molecule is deoxyribose. The rungs consist of pairs of the four bases; adenine (A), thymine (T), guanine (G) and cytosine (C).
Because of the chemical properties of the bases, A pairs only with T, and C only with G. This means that the two complementary strands cannot fit together in any other way.
Proteins are built of chains 鈥� called peptides 鈥� of amino acid building blocks, each chemically linked to the next. Certain chemical properties of the amino acids force the chain to fold in particular places, producing a three- dimensional structure whose shape is critical if the protein is to perform its job properly.
The information that directs the order of amino acids in peptide chains is contained in the sequence of bases in DNA 鈥� the genetic code. A gene is a length of DNA that codes for one peptide.
The genetic code is read in triplets of bases in the DNA molecule. In other words, each amino acid is represented by a code word of three bases; for example, the amino acid valine has the code word: GTG. A triplet of bases that represents one amino acid is called a codon.
When a cell needs to make a particular protein, something has to 鈥渞ead鈥� the relevant genes and translate each codon. Then the appropriate amino acid must be found, and all the amino acids must be assembled together in the correct order to make the protein.
The first part of this process goes on in the cell nucleus, the home of the DNA. An enzyme called ribonucleic acid polymerase copies one of the strands of DNA to form a molecule called messenger RNA (mRNA). This process is called transcription. RNA is similar to DNA except that its sugar is ribose instead of deoxyribose and it contains the base uracil (U) instead of thymine.
The molecule of mRNA then travels from the nucleus to the cytoplasm of the cell. There, it acts as a template upon which the amino acids are joined together. This event is called translation, because the codons of mRNA are translated into the amino acids they encode.
Amino acids are brought to the appropriate place on the mRNA strand by molecules called transfer RNAs (tRNAs). Each tRNA has a triplet of bases, called an anticodon, which finds and joins up with its complementary codon on the mRNA strand. In this way amino acids are placed in the appropriate order as directed by the sequence of mRNA codons.
Gene therapy: hopes and fears
GENE THERAPY, to replace amissing or defective gene, may inthe future cure many inherited diseases. The most likely candidates for gene therapy are single-gene disorders such as the haemoglobin diseases, mentioned above, and disorders of metabolism arising from single-base mutations, because they are the most well-studied genetic diseases. For example it may prove feasible to correct defects in the precursors of red blood cells that are made in the bone marrow. Although plans are afoot to begin gene therapy for people who would otherwise die from genetic disease, there are still many difficult problems to tackle.
First, there are still many obstacles to placing a gene in the chromosomes of each one of millions of host cells. Secondly, getting the gene into a cell, or even integrated into the host cell鈥檚 DNA, does not mean that the gene will do its job. The regions of DNA that flank a gene are known to contain base sequences that are crucial in deciding whether the gene will be 鈥渞ead鈥� and in regulating the rate of its transcription. Finally, the additional gene must not interfere with any other processes in the normal life of host cells.
At present, animal experiments are providing some insight into solving these problems but it will be years before they are satisfactorily answered. However, researchers are putting a lot of effort in persuading viruses to solve the first problem for them.
Certain viruses, called retroviruses, insert their own genes into the chromosomes of the human cells they naturally infect. Although retroviruses cause serious diseases, including cancer, it is possible to disable them with the new techniques of molecular biology. Their disease-causing genes can be removed and a therapeutic gene 鈥渟titched鈥� on to the gene that enables the virus to enter cells. Some experiments in animals show that the technique is feasible.
The idea of introducing foreign genetic material into a human being can be frightening. If gene therapy became possible would it mean that we could choose the eye colour, musical ability or sex of our children? Would that be desirable or ethically acceptable? Even if gene therapy were used as a last resort to treat only life-threatening diseases, could doctors guarantee that it would be safe and not lead to unforeseen diseases such as cancer?
In June 1988 The Lancet, one of the world鈥檚 most important medical journals, published a joint statement concerning gene therapy in human beings, by the medical research councils of a number of European countries including Britain. The statement made it clear that gene therapy should be used only to correct genetic defects and that 鈥渁ttempts to enhance general human characteristics should not be contemplated鈥�.
It said that gene therapy should be carried out on body cells (somatic cells) only and never on germ cells (sperm and egg). This means that any gene therapy would benefit only the individual treated and not his or her children. 鈥淔urther technical improvements in the expression of transferred genes in somatic cells will be necessary before successful gene therapy can be achieved even in animal models; in the meantime trials in man are not justified鈥�, the report said. It recommended that countries form groups to consider proposals for gene therapy and to oversee trials.
Linkage analysis
CONSIDER TWO parents who are both carriers (heterozygotes) for adeleterious gene A. Each parent has a chromosome that carries gene A and another that carries its normal counterpart N. If the mutation that produces A cannot be identified directly, linkage analysis may help.
In this example, one of the parental chromosomes carries a polymorphic restriction enzyme site P, which is close enough to the loci A and N so that they will not be separated in successive generations. Here, the chromosome containing the polymorphism is designated + and that which does not -.
From the 鈥� chromosome, a restriction enzyme cuts out a piece of DNA 10 kilobases (kb) long containing locus N for which there is a gene probe. (1 kb = 1000 bases.) The + chromosome, however, contains a single base change that produces a new site for the restriction enzyme at P. Hence the fragments containing locus M from the + chromosome are now only 7 kb long.
Mapping the parents鈥� DNA with probe M produces two bands, each representing either the + or 鈥� chromosomes. But which chromosome carries gene A and which the normal gene N?
That answer comes from a family study. A previously born child has received gene A from both parents and its gene map reveals the ++ chromosome arrangement; that is, only a single 7 kb band from mapping with probe M. For prenatal diagnosis in the next pregnancy, doctors can similarly map the fetal DNA with probe M. In this example, the gene map of the fetus also shows the ++ arrangement and so it must also have received the defective gene A from both parents.
Further Reading
The New Genetics and Clinical Practice, by David Weatherall (OUP, 拢9.95), is an account for the nonspecialist of what the new techniques of molecular biology offer medicine now and may offer in the future.
Human Gene Therapy, by Eve Nichols (Harvard University Press, 拢7.95), gives an accessible account of state-of-the-art gene therapy and how it may develop.
Unnatural Selection?: Coming to Terms with the New Genetics, by Edward Yoxen (Heinemann, 拢14), is a readable introduction to the biology of prenatal diagnosis and gene therapy and the social and political issues they raise.