HUMAN GENETICS HOW GENE SPLICING HELPS RESEARCHERS FIGHT INHERITEDDISEASE Whal Is NIGMS? The National Institute at General Medi— cal Sciences (NIGMS) is unique among the components of the National Insti— tutes of Health (NIH) in that its main mission is the advancement of the basic medical sciences. It supports selected research and research training programs in areas that underlie all medical investi- gation, such as cell and molecular biol— ogy and genetics. Ten at the scientists whom it funded have won Nobel Prizes since l970 for their pioneering work on the structure and tunction of genetic ma— terial, the action of hormones, and the structure at the cell. Knowledge resulting from this work contributes directly to the progress at research on specitic dis- eases in the other Institutes of NIH NIGMS also develops and supports in- terdisciplinary studies in biophysics, pharmacology, biorelated chemistry, physiology, and trauma and burn re- search. Many ot the researchers men4 tioned in this brochure worked with NIGMS support. “ll/THE NEW HUMAN GENETICS HOW GENE SPLICING HELPS RESEARCHERS FIGHT INHERITED DISEASE US. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health ' National Institute of ”G eeeee | Medical Sci eeeee v‘ Written by Maya Pines under contract 263-MD-33213t-2 Produced by the Office of Research Reports National Institute of General Medical Sciences 732(0’4727‘5’ PLABL, CONTENTS AF 66” [*7/15 / Introduction .......................................................... 5 The Beginning of Human Genetics ...................................... 8 Unlocking the Secrets of DNA .......................................... T4 The Development of Recombinant DNA Techniques ......................................... T8 A New Understanding ot Sickle-Cell Disease and Other Blood Disorders ................................................ 22 How to Use DNA Fragments to Detect a Disease ................................................... 25 How DNA Probes Find Their Match ...................................................... 28 Stalking the Most Elusive Genes ........................................................ 3T The Rapid Growth of Gene Mapping ....................................................... 36 Zeroing in on Cancer Genes ........................................................ 44 A Bank of Living Human Cells ......................................................... 47 GenBank: A National Database of Nucleic Acid Sequences ............................................... 52 The Promise of Genetic Therapy ...................................................... 53 Glossary ................. I ............................................ 56 INTRODUCTION AT firsT The young couple from Be- Thesda, Maryland, ThoughT They would never dare To have children of Their own. Though They were per— fechy healThy, They knew ThaT They boTh carried a gene for Thalassemia, a life-ThreaTening herediTary blood disease. Any child of Theirs would have a 25 percenT chance of inheriT- ing a double dose of This gene and acquiring The disease—a chance They were noT willing To Take. They changed Their minds in The laTe l970’s, when more sensiTive TesTs of red blood cells allowed sci- enTisTs To deTecT This geneTic disorder even in feTal blood. AT abouT The same Time, iT became possible To wiThdraw blood samples from The feTus Through 0 Technique called feToscopy. The young woman became preg- nanT in T979 and wenT To be TesTed aT The Yale UniversiTy Medical School's GeneTics CenTer in New Haven, ConnecTicuT, much of whose basic research is supporTed by The NaTional lnsTiTuTe of General Medi- cal Sciences (NlGMS). To her in- Tense relief, she learned ThaT The fe— Tus was unaffecTed by The disease. The baby is now a healThy and ac- Tive preschooler. However, The pro- cedure is noT wiThouT hazard and his parenTs were noT Too eager To risk iT again. By T982, The science of human geneTics had advanced so much ThaT iT was no longer necessary To Take blood from The feTus for pre— naTal deTecTion of Thalassemia. Ge- neTicisTs no longer needed To look for The abnormal producT of an ab— normal gene. lnsTead, They could look for The gene iTself by examining The ”masTer molecule of life”—The geneTic maTerial called deoxyribonu- cleic acid, or DNA—in The nuclei of cells. DNA could be exTracTed from any Type of feTal cell, and since fe- Tuses normally shed some of Their cells inTo The amnioTic fluid ThaT sur- rounds Them, These cells could be wiThdrawn wiTh a hypodermic needle Through The much safer Tech- nique of amniocenTesis. Early in her nexT pregnancy, The young woman wenT To The Johns Hopkins UniversiTy School of Medi- cine in BalTimore, Maryland, To see Dr. Haig H. Kazazian, an experT in The deTecTion of Thalassemia by means of recenle developed recom- binanT DNA (gene—splicing) Tech- niques. The DNA TesTs showed ThaT The feTus had inheriTed only her gene for Thalassemia—noT her hus- band’s—and would escape The dis— ease. And now The couple has an- oTher healThy baby boy. In The fuTure, feTal cells for DNA TesTs may be obTained even earlier in pregnancy Through a new, sTill- experimenTal meThod called cho- rionic villus sampling, which pro— vides resulTs 0T 9 To ll weeks of , pregnancy (compared To T8 T0 22 weeks for amniocenTesis). The science of geneTics offers The firsT real hope ThaT a large number of The 3,000 inheriTed diseases which afflicT human beings may be 5 (magnified for illustration) Chromosome Biochemical DNA analysis studies analysis Amniocentesis—the most wide- spread technique of prenatal di— agnosis. Cells shed by the de— veloping fetus are extracted from a sample of amniotic fluid withdrawn from the expectant mother’s uterus by means of a hypodermic needle. The cells are cultured and then tested for chromosomal defects, such as Down syndrome, and for certain biochemical defects. ln addition, scientists can now analyze the DNA of these cells directly, identifying specific genetic errors. prevented or controlled. At present this science is mainly diagnostic, al- lowing some people to make in- formed decisions about whether to have children or not, and warning others about their risks of develop- ing certain illnesses. But it holds the promise of many kinds of treatment. Some genetic diseases already can be treated effectively by chemi- cal means. For example, in l968 geneticists saved the life of an 8- month-old boy who was rushed to the Yale Hospital in a coma and near death. While doctors gave him emergency treatment, laboratory tests revealed that the child’s urine contained strikingly large quantities of methylmalonic acid, a chemical that is usually present to this extent only in cases of vitamin B-12 defi- ciency; yet his blood tests showed plenty of vitamin B-lQ. Geneticists were called in. They took a sliver of skin from the boy's forearm, minced it, and put it in a culture medium where the cells could grow and divide. Then they studied how these cells responded to various chemicals. This pointed to an unusual defect: The boy’s cells lacked a certain enzyme which nor- mally activates vitamin B-l2 and al— lows it to break down the methylma- lonic acid into simpler chemicals. Because of this genetic defect, his body needed 1,000 times the nor- mal amount of vitamin B-l2; no lesser amount would do. The doctors then iniected an enor- mous dose of vitamin B-l2 into the little boy’s veins. As if by miracle, he recovered. The quantity of methyl- malonic acid in his urine decreased dramatically. The basic defect re- mained, but from then on, with con- tinuous high doses of vitamin B-l2, he developed nearly normally. This was the first documented ex— ample of a recessive genetic dis- order (which appears only when genes for it are inherited from both parents) involving vitamin metabo- lism. Since its discovery, researchers have identified some 25 other inher- ited disorders that respond to high doses of vitamins. Many of these conditions affect the central nervous system and produce mental retarda- tion, seizures, gait abnormalities, or behavioral disorders resembling schizophrenia. Some of the disor- ders can now be diagnosed pre- natally, if the parents are aware of their risk. Treatment can begin even before birth, with high doses of vita- mins to the expectant mother for use by the developing baby. All too frequently, however, the primary biochemical defect in ge— netic diseases remains unknown, or else scientists do not yet have a way to deliver the missing chemical to the appropriate cells. Solutions to both these problems await the fur— ther development of human genetics and the application of recombinant DNA and other technologies. Ultrasound transducer Villi of chorion frondosum \— Plastic catheter Chorionic villus sampling—a new and still—experimental method of prenatal diagnosis which provides results as early as the 9th week of pregnancy. Fetal cells from the chorionic villi (protrusions of a membrane called the chorion which sur- rounds the fetus during its early development) are suctioned out through the uterine cervix and their DNA is analyzed. The pre- liminary results of this process can be obtained within a day. THE BEGINNING OF HUMAN GENETICS Genetic diseases used to be consid- ered quite rare. Today it is recog- nized that innumerable people suffer the consequences of disorders due wholly or in part to defective genes or chromosomes (the rod-shaped packages of genetic material inside the nucleus of a cell). Genetics is now progressing so rapidly, on so many fronts, that it is revolutionizing medical research. It is producing a new understanding of how cancer develops, for instance. it is detecting the differences be— tween various forms of heart dis- ease. It is helping researchers de— sign more effective and less harmful drugs. It is providing precise infor- mation on who is most vulnerable to what kind of illness, and who should particularly avoid certain environ- mental agents. Perhaps most impor- tantly, it is bringing new insights into the function of regulatory genes which affect all human growth and development, from birth to death. As recently as 1956, however, scientists were uncertain about the correct number of chromosomes in a human cell. Mammalian genetics still depended primarily on the slow— paced method of mating two ani- mals and studying their offspring. This approach worked quite well in mice. But since human beings have relatively few children, who take a long time to grow up and repro— duce, the study of human genetics was particularly difficult. It remained largely an observational science, Nucleus A human cell. The nucleus con- tains the genetic material; the chromosomes become visible only during certain stages of cell division. Chromosomes much as atomic physics was be- tween the time of the Greeks and the l9th century. Early physicists had deduced the existence of atoms from the properties of matter, but they had no proof of it. Similarly, geneticists deduced the existence of genes from the properties of organ- isms and their progeny, but could neither analyze nor manipulate the particles about which they built elaborate theories. Their experiments dealt with the entire animal, rather than with chromosomes or genes. The idea that human traits are under the control of distinct factors (later called genes), half coming from the father and half from the mother, goes back to the early l860's and the experiments of the Austrian monk Gregor Mendel with different types of pea plants. He showed that in some cases an in- herited trait will be expressed be- cause of the presence of a single ”dominant” factor, while in other cases two ”recessive" factors are required for a trait to be expressed. At about the same time, it be- came clear that, in animals, heredi- tary factors are transmitted through the egg and the sperm, and that each of these sex cells contains only half the normal number of chromo- somes, which scientists had just learned to see under the microscope by means of special dyes (”chromo” comes from the Greek word for ”color” and ”some” from the Greek word for ”body”). When a sperm Affected Normal Father Mother D d dd D d dd D d dd Affected Normal Affected Normal How Dominant Genetic Disorders are Inherited One affected parent has a sin- gle faulty gene (D) which domi— nates its normal counterpart (d). Each child has a 50 percent risk of inheriting the D—and the dis- order—from the affected parents enters an egg, these two haploid cells (cells with half the usual num— ber of chromosomes) fuse to form a new cell with the full number of chromosomes. One of the first things that re- searchers noticed about human chromosomes was a sex difference. In I905, they discovered that one particular chromosome, which they called ”Y," could be found only in male cells, together with an ”X" chromosome. By contrast, female cells have two copies of the X chro- mosome. Eventually it became clear that when sex cells are formed, each egg receives one X chromosome, while sperm carry either an X or a Y chromosome. Therefore the sex of offspring is determined by the sperm: If an egg is fertilized by a sperm that carries a Y chromosome, the fetus is male; if the sperm carries an X chromosome, the fetus is female. It took years of effort and many technological advances before chro- mosomes could be seen clearly enough to distinguish them from each other and count them accu- rately. Each species of plant or ani- mal has a characteristic number of chromosomes; these also vary in size, length, and other properties. In I956, Drs. Joe-Hin Tiio, now at the National Institutes of Health (NIH), Carrier Normal Mother Father Xx xy xy xx Xy X X Normal Normal Affected Carrier Male Female Male Female How X-Linked Genetic Disorders are Inherited The mother, who has a defective gene on one of her two sex chromosomes, is protected against the defect because her normal sex chromosome (x) compensates for the defect on the other (XI. The father has normal male sex chromosomes (x and y). Each male child has a 50 per- cent risk of inheriting the faulty X and the disorder, and a 50 per- cent chance of inheriting the normal x chromosome. Each female child has a 50 per- cent risk of inheriting the faulty X and becoming a carrier like her mother, and a 50 percent chance of inheriting two normal x chromosomes. and Albert Levon of Sweden finally established the number of chromo— somes in human cells: 46, or 23 pairs, with each pair containing one chromosome from the mother and one from the father. Twenty-two of these pairs are called autosomes; the two chromosomes of the 23rd pair are the sex chromosomes. This led directly to cracking a major mystery: the cause of Down syndrome (mongolism). Geneticists soon found that this syndrome, which produces mental retardation and characteristic facial features in 1 out of 800 American children, is caused by a single extra chromo- some number 2] in each cell. The extra chromosome is believed to re- sult most often from the incomplete separation of the chromosomes dur— ing the formation of the egg—an error which is more likely to occur with increasing maternal age. It can be detected before birth by examin- ing the chromosomes in fetal cells collected from the amniotic fluid. Several other forms of mental re- tardation and dozens of different disorders have now been traced to gross errors in the number or shapes of the chromosomes in each cell. Going one step further and inves- tigating the genes on the chromo- somes proved for more difficult. it Carrier Carrier Father Mother G9 09 GO Gg Gg 99 Normal Carrier Carrier Affected How Recessive Genetic Disorders are Inherited Both parents, usually unaffected, carry a defective gene (9) but are protected by the presence of a normal gene ((3) which is generally sufficient for normal function. Each child has a 25 percent risk of inheriting a “double dose" of the 9 gene, which may cause a serious genetic defect,- a 25 per- cent chance of inheriting two normal genes; and a 50 percent chance of being a carrier like both parents. _a in: }l ail. i' 19 20 21 22 l!" H H 1. XY With this historic photo, Drs. Joe-Hin Tiio and Albert Levon showed that there are 46 indi- vidual chromosomes in a normal human cell (magnification: X 2,300). Halt of these chromo- somes come from the mother, half from the father, and they are usually counted in pairs, except for the sex chromosomes (X and Y chromosomes). In Down syndrome (formerly called mongolism), there is an extra chromosome number 2) (three rather than the normal two). The extra chromosome most frequently results from in- complete separation of the chromosomes during the forma- tion of an egg cell by the ovary. The condition thus represents a genetic accident rather than a strictly heritable disease. (The chromosomes in this photograph have been stained to reveal patterns of bands using more modern techniques than were available to Drs. Tiio and Levan in 1956.) Common genetic disorders. Over 3,000 disorders clue to a defect in a single gene or a chromosomal error have been identified. Some are evident at birth; others develop later. Geno“: "dun Bio-u Cause ollllnou Incidence Inherit-nu Down syndrome autosomal range of mental 1 in 800 sporadic chromosome retardation abnormality Klinefelter's sex chromosome defect in sexual 1 in 2,000 sporadic syndrome abnormality differentiation Cystic fibrosis ? complications of I in 2,000 autosomal excessively thick Caucasians recessive mucus secretion Huntington’s ? progressive 1 in 2,500 autosomal disease mental and dominant neurological degeneration Duchenne g muscular 1 in 7,000 X-linked muscular degeneration, dystrophy weakness Sickle-cell disease abnormal impaired 1 in 625 autosomal hemoglobin circulation, mostly black recessive anemia, pain attacks Hemophilia defect in blood uncontrolled 1 in 10,000 X-Iinked clotting factors bleeding Phenylketonuria enzyme deficiency mental deficiency 1 in 12,000 autosomal mostly . recessive Caucasians and Orientals Toy-Sachs disease absence of an buildup of fatty 1 in 3,000 autosomal enzyme deposfls in brain, Ashkenazic Jews recessive leading to early death Lesch-Nyhan enzyme mental 1 in 100,000 X-Iinked syndrome deficiency retardation, self- mutilation had been recognized since 1869 that a maior constituent of cell nuclei was a substance which is now known as nucleic acid. Researchers later found that it really consists of ‘ two different substances, one con- taining the sugar ribose (this is ribo- nucleic acid, or RNA) and the other containing the sugar deoxyribose (this is DNA). In the 1920’s it be- came clear that, unlike RNA, DNA was present only in the chromo- somes. In 1944, Dr. Oswald Avery and his associates at the Rockefeller Institute in New York City discovered that DNA was directly involved in transferring hereditary characteristics from one strain of bacteria to another. All living organisms, from bacteria to butterflies, from trees or fish to human beings, contain DNA that differs only in the number and ar- rangement of its components. But what is DNA? UNLOCKING 'I'HE SECRETS OF DNA DNA’s three-dimensional structure was finally deciphered in T953 by Drs. Francis H.C. Crick and James D. Watson, who were then working at the Medical Research Council laboratories in Cambridge, England. (Together with the English physicist Dr. Maurice Wilkins, they later won the Nobel Prize for this achieve— ment.) Their now-famous double helix model at the DNA molecule explains both how DNA is built and how it replicates (makes more of itself). According to this model, DNA is made up of two long and twisted strands. Each strand is composed of combinations of tour smaller chemi- cals called nucleotides. Each nu- cleotide consists of one sugar mol- ecule, one phosphate group, and one of tour nitrogenous bases—ad— enine (A), thymine (T), guanine (G), or cytosine (C). These nucleotides line up next to each other like the two sides at a zipper, with the phos- phate and sugar forming the outer ribbon, while the bases act like the interlocking teeth. Betore cell divi- sion, new DNA must be synthesized and every gene must be replicated. To do this, the two strands separate and each one acts as a template for the formation of a mirror image, thereby producing two copies of the original DNA—two daughter DNA molecules whose sequences are identical to those ot the parental double helix. When a cell divides, each daughter cell thus receives a SU (Jr-Phosphate Bagkbone The double helix: model of the DNA molecule which embodies the code of heredity, as con- ceived by Drs. Francis H.C. Crick and James D, Watson in l953. All living things reproduce themselves according to the ge— netic information in different se- quences of DNA subunits (bases). l4 complete copy of the original cell’s genetic information. The two sides of the zipper can fit together only in one way: A pairs with T, and G with C. Because of this specific base pairing, if the se- quence of one strand is known, that of its partner is also known. Each strand of the double helix thus spec- ifies its complement, in the some sense that a photographic negative ”complements" the positive image. The four letters of this genetic alphabet can be used to write an in- finite number of messages and in- structions through which the genes direct the synthesis of thousands of enzymes and other proteins inside the cell. The language in which such instructions are written—the genetic code—was deciphered in the l960's by Drs. Marshall W. Nirenberg of NIH, Severo Ochoa of New York University, and Har Gobind Khor— ana of the University of Wisconsin (work which also led to a Nobel Prize). The code consists of triplets of nucleotides (each triplet is called a codon) which are ”read" in se- quence along the DNA molecule. Genes—which were mere ab- stractions for earlier scientists—can thus be studied and analyzed in terms of their constituent chemicals. Each gene is a series of codons which gives the instructions for building a specific protein. Each co- don corresponds to one ”word”— either one of the 20 amino acids which are the building blocks of U 7 uracrl lthyminel C — cytosme A — adenine G — guanine Ala — alanine Arg 7 arglnlne Asn 7 asparogine Asp — aspartlc acid Cys 7 cysteine Gln — glutamine Glu — glU'Gth acid Gly 7 glycine His 7 histidine lle — isoleuctne Leu 7 leucine Lys — lysine Met 7 methionine [Start] Phe 7 phenylalanine Pro — proline Ser 7 serine Thr — Ihreonine Trp — twptophan Tyr — tyrosine Vol — vallne The genetic code. Each triplet of nucleotides codes for one amino acid, excepting three—the ”stops”—which signify the end of a protein chain. One amino acid, methionine, can also act as a signal to start protein pro— duction. Each gene that codes for a protein is a series of triplets {codonsl which gives the instruc- tions for building that protein and thus influences a specific trait. l5 llllv II. T“ ‘ III-l ‘IIIUII Cllllltllllltll'lllil I O Ollllll‘llllllll ‘i ‘[Ill 4!!” YA lullllllltu to proteins, or a signal to start or stop constructing an amino acid chain. These instructions are transmitted not by the DNA itself, but by a copy, made of RNA, which acts as an intermediary. The original DNA remains safely in the nucleus, some- what like the printing block in a printing press. The RNA copy is manufactured in the nucleus by tran— scribing iust one strand of DNA, which codes the instructions. In the late l970's, scientists were surprised to find that in higher ani- mals, including humans, the instruc- tions are often interrupted by inter- vening sequences of DNA (introns) whose functions are not fully under- stood, but which clearly do not code for the proteins specified by the genes. The actual coding regions of genes are called exons. When infor- mation from the genes is transcribed into new strands of RNA, the introns are spliced out of the RNA. The re- sulting molecule, called messenger RNA, then moves out of the nucleus into the cytoplasm, where it can di- rect the production of protein. DNA replication. To replicate before cell division, the DNA double helix separates and un- winds and each strand acts as a template for the formation of a mirror image according to the rules of base pairing: A with T, and G with C. This results in two daughter DNA molecules whose sequences are identical to those of the original DNA. , ' " ‘ ' _ I rComp‘lementa ‘ DNA transcription. The instruc _ DNA Strands W tions contained in DNA are _ transmitted to other parts of the cell by an intermediary—a copy manufactured out of a different _ nucleic acid, RNA—while the original DNA remains safely in the nucleus. The RNA copy is made in the nucleus by tran- scribing (producing a new com- ; plement to) iust one strand of DNA, which is sufficient to en» : code the instructions RNA Strand Beginning of i / protein ' _ Nearly completed— protein This end translated first - RNA translation to protein. The cleus. The ribosomes translate single strand of messenger RNA these instructionsinto growing , (mRNA) carries the DNA’s in— chains of amino acids that be— - structionstothe ribosomes, tiny come specific proteins. protein factories Outside the nu— ' 451—088'085w—2' "_ “I ,' __ , ___., t7, THE DEVELOPMENT OF RECOMBINAN'I' DNA TECHNIQUES In order to zero in on individual genes, scientists needed a method that would consistently cut the long, ultra-thin strands of DNA at specific places. For nearly a decade they had no way to do this and progress in genetics research was slow. Con- sidering that each human cell con- tains about 6 feet of DNA strands, coiled and packed into 46 tight bun— dles of chromosomes, and that this DNA is made up of 6 billion base pairs, the situation seemed almost hopeless. Even bacterial DNA was impossibly large, consisting of more than 4 million base pairs in the tini- est organisms, such as the intestinal bacterium Escherichia coli (abbrevi- ated E. coli). Therefore, researchers focused on even smaller and simpler systems, such as viruses. When they tried to break up larger DNA mole- cules into more manageable pieces, they ended up with a chaos of ran— dom fragments whose order in the original DNA could not be established. A happy finding in l970 radically changed this picture. While studying Hemophilus influenzae, a bacterium which causes meningitis, 0 Johns Hopkins researcher, Dr. Hamilton Smith, noticed that it produced an enzyme which broke down the DNA of bacteriophages (viruses that at— tack bacteria). This self—defense en- zyme did not harm the bacterium’s own DNA, but it cut the DNA of a different bacterium, such as E. coli, into little pieces. Furthermore, all the cuts occurred at one specific base— pair sequence, which was later identified. This was the first of a series of so— called “restriction enzymes," which have become the key to the manip- ulation and study of DNA. Soon af- terward, Dr. Daniel Nathans, a col- Nick -X-X-X—G A-A-T-T—C-X-X-X- -X—X-X-C-T-T—A-A o-x—x-x- Nick 18 “Sticky ends” of DNA. The re- striction enzyme EcoRl, produced by the E. coli bacterium, recog- nizes the DNA sequence GAATTC and cuts it between the G and A. Since the two strands of the double helix are complementary, however, the restriction enzyme does not cut straight across both strands. In- stead, it produces a fragment with two exposed strips of sin- gle-stranded DNA at its ends. These exposed ends will ”stick” to other, similarly exposed single strands of DNA which have complementary sequences. league of Smith’s, coupled the ability of this and other restriction enzymes to cut DNA with a tech- nique for separating the resulting fragments on the basis of size. This enabled him to create the first ”re- striction map” of a DNA molecule. To date, over 200 restriction en- zymes have been isolated from bac- teria. Each one makes it possible to cut, or ”digest," DNA at the point where particular sequences of nu— cleotides occur (a different sequence for each enzyme), producing frag- ments of different lengths. These enzymes do not usually cut straight across the two strands of DNA, however. The breaks often occur in a staggered fashion, creating short, single-stranded tails on the ends of each fragment when the fragments separate. These tails are called "sticky ends” because they tend to stick to complementary fragments by base pairing. Two fragments pro- duced by the same enzyme can stick together in this way and then be permanently ioined by adding another enzyme, DNA ligase. Scien- tists can thus splice together, or “re- combine,” various pieces of DNA. The discovery of these recombi- nant DNA techniques produced ex- treme excitement among biologists. In 1973, Drs. Herbert Boyer and Stanley Cohen at the University of California, San Francisco, showed how these techniques might be used. They took advantage of the fact that inside many bacteria, in addition to a single large chromo— some, there are tiny circular DNA molecules, called plasmids, each consisting of only a few thousand base pairs. The two researchers cut such E. coli plasmids with a restric- tion enzyme called EcoRl, for which each plasmid had only one recogni— Circular Plasmids (several thousand base pairs each) Bacterium Main Circular Chromosome (4 million base pairs) Plasmids. In addition to a main chromosome, bacteria (as well as yeast and some plants) con- tain plasmids, small circular mol- ecules of DNA which replicate autonomously. Plasmids are easily moved from one cell to another or to the test tube. They can also be broken open with restriction enzymes. Scientists often insert foreign DNA into plasmids and then use them to transfer this DNA into other cells in recombinant DNA experiments. nmsgpiecfis , _ by reshiction tion site (the DNA sequence at which a specific restriction enzyme makes a cut); then they spliced in fragments of foreign DNA which had also been cut with EcoRI, and sealed the plasmids back together with ligase. The hybrid plasmids, each carrying one or more frag- ments of foreign DNA, were then transferred back into E. coli bacteria, where they carried out the instruc— tions of the inserted DNA and re- produced together with the bacte- ria’s own genetic material. This opened up the possibility of isolating and studying almost any human gene—a breathtaking vista for geneticists. Human DNA could be broken up with restriction en- zymes and the fragments inserted at random into bacterial plasmids. The recombinant plasmids could then be introduced into a population of bac— teria. One or more of these bacteria would be likely to contain a recom- binant plasmid carrying the specific human gene that was wanted. The bacteria could then produce enor- mous quantities of this gene, since bacteria reproduce every 20 to 30 minutes. At that rate, a single bac— terium could produce more than a billion copies of itself in 15 hours. The growth of gene-splicing tech— niques was greatly helped along by two related developments: In T975 and T976, research groups in England and the US. devised new means by which the nucleotide sequences of DNA seg— ments could be determined simply and rapidly. The approach taken by the American scientists was to use four different chemicals to break a DNA strand into a series of frag- ments whose lengths depend on the distance between the break and one radioactively labeled end of the strand. Because the chemicals cut at specific nucleotides or pairs of nu— cleotides, researchers are able to identify the last base in each frag— ment. By studying the pattern of fragment lengths they can determine the order of the bases in any DNA chain. The British method achieves similar results by using enzymes and modified nucleotides that are incor- porated into the chain and allow identification of the terminal base of each fragment. Both of these tech— niques have been in extensive use since their development and, as a result, the complete sequence of the DNA of several smaller viruses is now known, and the sequences of many human genes have been determined. Shortly after sequencing was de- veloped, the previously formidable task of putting together nucleotides to make specific DNA sequences in the laboratory was vastly simplified by new methods. Scientists can now produce sequences of T2 to 20 nu— cleotides in just a few days. By combining these techniques, researchers can now mass-produce a variety of important biological substances, including insulin, human growth hormone, and the interferons. 2i A NEW UNDERSTANDING OF SICKLE-CELI. DISEASE AND OTHER BLOOD One of the victories of the science of human genetics has been its in- creasingly precise analysis of what goes wrong in various kinds of in- herited blood disorders, particularly sickle-cell disease and the thalasse— mias (such as the illness for which the baby shown on page 4 was at risk). Occasionally—perhaps because of an error when the cell replicates, or because of some outside influ- ence such as a virus or radiation— the specific sequence of nucleotides in a DNA molecule is altered. Such changes are called mutations, and they can have either harmful, neu- tral, or, very rarely, beneficial ef- fects, It is thought that every human being inherits about six or seven deleterious recessive mutations that, when transmitted to offspring who happen to receive the same delete— rious gene from the other parent, can cause serious illness and even death. Over 2 million Americans, mostly blacks, are carriers of the sickle-cell gene, for instance. This is an exam- ple of a mutation which generally does no harm to carriers, who also have one normal gene which com- pensates for the deleterious effects of the defective one; it even pro- vides a selective advantage in cer- tain parts of the world, since it offers some protection against malaria. However, when two carriers of this gene marry, each of their children has a 25 percent chance of inherit- ing a double dose of the sickle-cell 22 gene and suffering from sickle-cell disease. This is a potentially lethal recessive disease which causes chronic anemia, iaundice, severe pain, and poor resistance to infection. Carriers of the sickle-cell gene can be identified by a simple blood test. The disease itself was originally traced to a defect in hemoglobin, the oxygen-carrying protein of blood, back in l949. With increasing precision, in T956 it was traced to the substitution of iust one amino acid (valine instead of glutamic acid) out of the nearly 150 amino acids that make up one subunit of the hemoglobin molecule. Recently it was tracked down to its fundamen- tal source: the mutation of a single nucleotide in the DNA codon for glutamic acid (GAG), changing it to the codon for valine (GTG). Because of this change in one nu- cleotide, the victim's hemoglobin is less soluble and under certain con- ditions it turns into a viscous, semi- Normal Hemoglobin - val — his - leu -thr - pro - glu - glu - Sickle-Cell Hemoglobin - val — his - leu - thr - pro - val - glu - Sequence of normal and sickle- cell hemoglobin differs by one amino acid. solid gel. As a result, the normally round and very flexible red blood cells may become so inflexible that they can no longer squeeze through the small blood vessels. Some of these cells are also distorted into the shape of a crescent or sickle. When the abnormal cells obstruct the small blood vessels, oxygen can no longer reach the surrounding tissue, caus— ing damage. Sometimes the abnor- mal cells are removed too rapidly by the spleen, causing anemia. Several new methods of treatment are now being considered, including gene therapy (see page 53). But children with this disease often die young, after many medical crises. ”I am the mother of five children, three of whom have sickle-cell dis- ease," says Ola Huntley, director of a sickle-cell self—help group in Los Angeles. ”At the time we married, there were no tests for carriers. My second, third, and fourth children are presently suffering; the fourth was born before the third was diag- nosed. lt’s rare that we can all be together at home, because they are constantly being hospitalized. For 25 years now, I’ve made those frequent trips to the hospital. There is only temporary relief. I see the pain in my children’s eyes. l see them face ma- ior surgery. My daughter has experi- 24 enced a stroke—a blood clot in the brain—and leg ulcers. My son has grand—mal seizures from blood clot— ting. In addition, they get ridicule from their peers. They have low self- esteem, and little motivation to live . . . ." The disease is extremely variable, however, and some people with sickle—cell disease lead fairly normal lives. As yet there is no way to pre- dict the severity of the disease with any reliability. With the aid of recombinant DNA techniques, sickle—cell disease can now be detected prenatally. This kind of screening became possible when researchers found a way to tell the difference between normal and abnormal forms of the beta- globin gene, which codes for one of the two types of protein chains that make up hemoglobin. At first they used an indirect ap- proach. If they could not recognize the defective gene itself, why not look for a ”marker"—any identifia- ble variation in DNA that was close enough to the beta-globin gene to be likely to be inherited together with it? That is what Drs. Yuet Wai Kan and Andrées lvl. Dozy of the University of California, San Fran- cisco, did in T978, and their method is worth describing in some detail. HOW TO USE DNA FRAGMEN'I'S 'I'O DETEC'I' A DISEASE As explained above, restriction en- zymes cut DNA at specific nucleo- tide sequences which they recog— nize. The length of each resulting DNA fragment depends on the dis- tance from one recognition site to the next. At a point about 7,000 nucleo- tides away from the beta—globin gene on chromosome ll, Drs. Kan and Dozy noted a natural variation in DNA which appeared to have no effect on health. This was the first DNA polymorphism, or inherited variant, ever discovered. The re- searchers found that the DNA of some people had a recognition site for the restriction enzyme Hpal at this point, while other DNA did not. If Hpal cut through the DNA at this point, one of the fragments it pro- duced was a fairly short piece con- taining the beta-globin gene—a fragment only 7,600 base pairs in length. However, if the DNA lacked this sequence and Hpal could not make a cut at that point, the beta- globin-containing fragment was longer—13,000 base pairs in length. The various fragments generated by such cuts can be separated by electrophoresis, a process in which an electric field makes the DNA fragments move through porous agarose gels. The smaller fragments move faster than the larger ones, so l I I I I WOO IIIIIIIIIIIIIIIIIIII IIII ||||1111l|lilllllllllll| lllilllill IIII IIII IIII ilillllllllillllllillllililIlllllllscc‘le Individual A 1,000 Base Pairs DNA a b c d e f g 800 1,200 2,000 500 3,500 2,400 1,600 IndividualB DNA a b c d e r h 500 1,200 2,000 500 3,500 4,000 Restriction fragment length poly- morphism (RFLP). When a spe— cific restriction enzyme cuts DNA, it may produce fragments of different sizes in the DNA of different people. For example, EcoRl will cut DNA wherever the base sequence GAATTC occurs (represented here by arrows). But while individual A has six of these recognition sites, yielding fragments a through 9, individ- ual B has only five recognition sites. Thus the length of frag» ment h from individual B's DNA (4,000 base pairs) equals the sum of the lengths of fragments fand g produced by EcoRl in in— dividual A’s DNA. Such natural variations, or polymorphisms, in restriction fragment lengths are inherited. 25 their sizes can be determined by ex— amining their positions in the gels. The researchers then used this procedure to analyze DNA from the cells of many American black fami— lies and established that, in 70 per- cent of these families, the presence of longer DNA fragments after ex- posure to Hpal was associated with sickle-cell disease. This meant that in families where this link was shown, the occurrence of such fragments after exposure to Hpal could be used as a predictor of disease. Shortly afterward, several re- searchers went a step further and found direct evidence of the sickle- cell gene. It was a matter of trying until they found the most appropri- ate restriction enzyme—in this case, Mstll, which cuts DNA right in the middle of the beta-globin gene as well as in many other places. When the beta-globin gene is normal, Larger 350 — Base Pairs 150 — l40——> Direction of movement of fragments through the gel Smaller 26 Electrophoresis. To separate DNA fragments according to their size and detect RFLP’s, sci- entists use electrophoresis, a process in which an electric field makes the DNA fragments mi- grate through a gel. Since the smaller fragments move through this gel faster than the larger ones, the length of any fragment can be determined by compar- ing its position in the gel to the position of DNA molecules of known sizes. Mstll cuts through it at the sequence CCTGAGG, producing two frag- ments of 1,150 and 200 base pairs in length. However, the sickle-cell mutation converts the DNA se- quence at this point to CCTGTGG, thereby abolishing the Mstll recogni- tion site,- this results in just one frag- ment of 1,350 base pairs after treat— ment with Mstll. To find out whether any given DNA contains the sickle-cell muta— tion, therefore, one need simply know what size fragments Mstll pro— duces at this point. But Mstll and other restriction enzymes cut human DNA in many other places as well, and picking out these fragments from many others of similar sizes is somewhat like finding a needle in a haystack. Geneticists get around this problem by using specially pre- pared, radioactively labeled DNA probes. Mstll Mstll Msi‘ll l Normal beta-globin gene 1 l — CCTGAGG CCTGAGG CCTGAGG 1,150 Base Pairs 200 Base Pairs Mstll Mstll l Beta-globin gene with sickle-cell mutation l — CCTGAGG CCTGAGG 1,350 Base Pairs Direct evidence of the sickle-cell if Mstll cannot make this cut be- gene. lt has become easy to de» cause the recognition site has tect the presence or absence of the sickle-cell gene through DNA analysis. If the restriction enzyme Mstll cuts a person’s DNA at the sequence CCTGAGG in the beta-globin gene, producing two fragments (1,150 and 200 base pairs in length), the gene is normal. But been abolished by a mutation that changed the sequence to CCTGTGG, there will be only one fragment, 1,350 base pairs in length—evidence that the per- son from whom this DNA was taken carries a gene for sickle- cell disease. HOW DNA PROBES FIND THEIR MATCH A probe is a short sequence of sin- gle—stranded DNA that is comple- mentary to the DNA sequence one seeks. Such probes take advantage of the fact that single strands of DNA automatically seek out com- plementary strands whose bases pair up with them, G with C and A with T. In order to be recognized by a probe, however, the DNA that is exposed to it must first be separated (“denatured”)*into single strands. The two strands of the DNA double helix are easily denatured by expos— ing them to near—boiling tempera— tures or to extremely alkaline conditions. Probes are often made out of DNA segments that have been cloned, or individually reproduced, inside E. coli bacteria. For example, the probe used to detect the sickle- cell mutation is a fragment of a cloned beta—globin gene. Before being used, it is treated to make it radioactive so that it can be de- tected in the midst of large amounts of other DNA. Any DNA that is suspected of having the sickle-cell mutation is first cut by Mstll. The resulting fragments are then separated by agarose gel electrophoresis and treated chemi- cally to denature them, producing single strands of DNA. Next these fragments are transferred (“blot- ted”) onto a nitrocellulose filter to which they become bound, still in the same pattern as they were in the agarose gel. (This technique is called Southern blotting.) At this 28 point, the filter is exposed to the ra- dioactively labeled probe, which will stick, or hybridize, only to the spe— cific DNA sequences that are com- plementary to it. These hybridized sequences will then give off a radioactive signal that can be visualized by exposing the filter to X—ray film, a procedure known as autoradiography. If a band corresponding to a DNA fragment of L350 base pairs shows up on the autoradiograph, it represents the sickle-cell gene. If two bands corresponding to the shorter fragments (l,l50 and 200 base pairs) show up, this represents a normal gene. If both the longer band and the two shorter ones ap- pear, the person has inherited one copy of the defective gene and one copy of the normal gene, and will be a symptomless carrier. The sickle-cell mutation can thus be diagnosed directly. Not many genetic diseases have their DNA errors right in the middle of a restriction enzyme’s recognition site, as is the case for the sickle-cell gene. Therefore, different strategies must be used to detect them. One strategy is to manufacture probes out of laboratory chemicals by synthesizing strings of nucleotides called oligonucleotides. Such oligo— nucleotides may be engineered to match normal genes perfectly and will hybridize to them. If the match is not perfect, however—if a single nu- cleotide does not match—the hybrid will be unstable and fall apart under DNA fragments _ g. T 15 echnique certain conditions. Mutant genes that are responsible for particular diseases and have a single ”point mutation" (a substituted, missing, or extra nucleotide) can now be de- tected in this way, assuming one knows exactly where to look. The most common form of thalas- semia among people who come from Mediterranean areas, for in— stance, has been traced to the sub- stitution of one nucleotide (A for G) in the beta-globin gene, which re- sults in a reduced production of hemoglobin and severe anemia. However, this substitution does not alter any known restriction enzyme site. Recently researchers have pre— pared synthetic DNA probes that are replicas of the normal and mu- tant nucleotide sequences. These probes are beginning to be used in prenatal diagnosis of thalassemia among families of Greek or ltalian ancestry, in whom the disease is most prevalent. Similar methods have made it possible to detect alpha l-antitryp- sin deficiency, an enzyme deficiency which predisposes those who har- bor it to severe and often fatal cir- rhosis of the liver in infancy, and to 30 obstructive emphysema in adult- hood. Parents whose children have died of cirrhosis often seek prenatal diagnosis before having other chil- dren. But until recently such diagno- sis required tests of fetal blood ob- tained through fetoscopy. ln T983, researchers succeeded in preparing a l9-nucleotide probe which was able to detect the cause of this deficiency—the substitution of one nucleotide in the alpha l—anti— trypsin gene—in cells taken from amniotic fluid. As a result, parents can now know early in pregnancy whether a fetus will be affected. Since the enzyme deficiency brings permanent damage to the lungs, children with this condition must stay away from industrial pol- lutants as well as cigarette smoke. ”If they do smoke, they will have emphysema by their late 30’s and die within a few years,” says Dr. Savio LC. Woo of the Baylor Col- lege of Medicine in Houston, Texas, one of the developers of this test. In the future, children who are found to have this deficiency may also be aided by iniections of the missing protein, much as diabetics are aided by insulin. STALKING THE MOST ELUSIVE GENES ln hundreds of other genetic dis- eases, the biochemical fault is still unknown and there is as yet no clue to where the error may lie among the 6 billion base pairs in human DNA. About 10 million Americans are carriers of cystic fibrosis, for in- stance—the most widespread reces— sively inherited disease among Caucasians in the US. If carriers marry, their offspring may inherit this life-threatening illness. Cystic fibrosis produces thick mucus in the pan- creas and lungs which prevents nor— mal digestion and breathing. It used to kill its victims before the age of 6; they starved despite an adequate diet or succumbed to severe infec- tions. Today, enzyme therapy and antibiotics help many of these chil- dren survive to adulthood, but the majority still die young, and they all require constant care, including reg- ular drainage of their lungs and other special measures to get rid of the mucus. Although the damage caused by this disease is obvious, its biochemi- cal basis is not. At present, carriers of the cystic fibrosis gene cannot be detected until they have produced a child with the disease. Nor can the disease be identified prenatally. However, a new approach is giving scientists a chance to locate some of the most elusive genes—and cys- tic fibrosis is a prime target for this kind of research. Scientists who studied genetic dis— eases in the past tried to identify the defective gene product, such as a faulty protein, and then worked back to the molecular basis of the disorder, such as the replacement of one amino acid by another. Studies of the DNA error that produced the amino acid replacement came later, if at all. “But we now have the ironic situation of being able to iump right to the bottom line without reading the rest of the page, that is, [to identify the gene] without needing to identify the primary gene product or the basic biochemical mechanism of the disease,” Dr. David E. Comings of the City of Hope National Medi- cal Center wrote in an American Journal of Human Genetics editorial in 1980. He called this approach the “New Genetics." The New Genetics makes it theo- retically possible to diagnose any disorder which can be linked to a DNA polymorphism in a family. It really began in 1978, when Drs. Kan and Dozy detected sickle—cell dis— ease by taking advantage of DNA variations that were inherited to— gether with the presence or absence of the sickle—cell gene within fami- lies. Researchers are now discover- ing so many other restriction frag- ment length polymorphisms (RFLP's) that, in any family, a given gene de- fect is likely to be linked to one. Per- haps the most dramatic achieve— ment in this area to date has been the discovery of a DNA polymor— phism linked to the dreaded Hun- tington's disease. Unlike other genetic disorders 31 which show up soon after birth, Huntington’s disease may not ap- pear until the victim is 35 or 40 years old. Then it produces progres- sive and irrevocable deterioration of the brain. Its first signs, such as per- sonality changes or abnormal move— ments, are often misdiagnosed. (The folksinger Woody Guthrie, who died of the disease, was originally thought to be an alcoholic, then schizophrenic.) Eventually facial expressions become distorted; head, hands, and shoulders seem to writhe in constant motion; speech blurs. As the disease advances, the victim becomes totally disabled and irrational. It is a slow process, lead- ing to death within TO to TS years. There is no effective treatment or cure. Huntington’s disease is caused by a dominant gene, so each child of an affected parent stands a 50 per- cent chance of inheriting the gene and therefore developing the dis- ease later in life. Generally the ill- ness is not recognized until the vic- tims have had children of their own; their offspring must then live in fear of suffering the same fate. Until T983 there was no way to AA AA AA AB Pedigree of an American family with many victims of Hunting— ton’s disease. The victims are fil-JZ firm 33%.er BD AB AAi AD IAA AC AC AA AD AA AA AA dren’s children were spared, but six others inherited the gene for the disease and 12 living mem- indicated in black. Circles repre— bers of this family have disease sent females and squares repre~ symptoms. Based on restriction sent males. The original victim (top circle, with slash indicating that she is deceased) transmit— ted the disease to all three of her children. Six of these chil- disease. 32 fragment length polymorphism studies, one other family mem- ber is expected to develop the identify carriers of the gene before their symptoms developed. Nor was there any clue to what caused the disease or where to seek remedies. Since scientists had no idea where the Huntington's disease gene was located, they could not even look for a restriction enzyme site that was close to it, as Drs. Kan and Dozy had done for sickle—cell disease a few years earlier. At Massachusetts General Hospi- tal in Boston, molecular biologist Dr. James F. Gusella decided to cut up the DNA of Huntington’s disease victims with restriction enzymes and compare the resulting fragments with fragments of similarly treated DNA from unaffected members of the some families. He wanted to see whether the lengths of these frag- ments varied in any way that could be linked to the appearance of Huntington’s disease. This required, first of all, finding a very large family with many living members who were willing to be tested and were old enough either to show signs of Huntington’s dis- ease or to be likely to have escaped it. (Past the age of 60, the risk of de- veloping Huntington’s drops to l percent.) Through Dr. P. Michael Conneally, a professor of medical genetics at Indiana University in In- dianapolis who maintains a Hunting- ton's disease roster, such a family was found. Next Dr. Gusella began to look for patterns in the millions of frag- ments which he obtained after cut- ting up DNA taken from the cells of both Huntington’s disease victims and their healthy relatives. He in- tended to expose these fragments to a whole ”library" of hundreds of ra- dioactively labeled probes, one by one. Any variations in the resulting autoradiographs would then be checked for possible linkage to Huntington’s disease; the statistical analysis involved would be done by Dr. Conneally with the aid of a spe- cial computer program. The very first batch of T2 probes which Dr. Gusella used produced a promising lead. One of the probes, a DNA sequence arbitrarily called G8 and whose function was un- known, stuck to fragments of differ- ent lengths in DNA from various members of the family; these frag- ments could form any one of four patterns, designated with the letters A, B, C, and D; and it appeared that the A pattern was inherited along with Huntington’s disease. In any study of linkage, scientists need a large number of cases in which a disease correlates with some identifiable marker; the greater the number of such cases, the higher the odds that the two fac- tors are linked. The American family was large by American standards, but had only 25 living members of appropriate ages. While this was enough to provide a hint of a link between the DNA fragment lengths and the disease, it was not enough to prove it. Fortunately, an NIH team had 33 been studying an extraordinary group of interrelated families who live along the shores of Lake Mara- caibo in Venezuela and have the largest concentration of Hunting— ton's disease in the world—nearly 100 living victims and 1,100 persons at risk for the disease. For 3 years in a row, this team took blood and skin samples from people who clearly had the disease, as well as from their healthy relatives. One complete set of blood samples was carried to Dr. Gusella. Another set, together with the skin samples, was taken to the NIGMS Human Ge— netic Mutant Cell Repository in Camden, New Jersey, where they were established as cell cultures, frozen, and stored. These cells are now available to researchers. At Massachusetts General, Dr. Gusella extracted DNA from the Venezuelan families’ white blood cells (red blood cells have no nuclei, and therefore no DNA) and set to work. He cut up the DNA with a re— striction enzyme called Hindlll, as he had done with the DNA from the American family, then exposed the fragments to the G8 probe. Next Dr. Conneally analyzed Dr. Gusella’s data on the families’ DNA fragments. Although he found that in this family a different pattern of frag- ment lengths—the C pattern—was inherited together with Huntington's disease, the link between this pat- tern and the disease held up very well. As the information poured in, he became exuberant. ”The chances were 600 million to 1 that we had the marker," he said. ”And when we studied some more of the Vene- zuelan samples, the chances in- creased to over a billion to one— about as certain as you can get.” If G8 continues to be a suitable N°rm°l w DNA Marker for Huntington’s disease I I gene. .—.—_. Restriction Normal Gene Enzyme Cut DNA of Person with W Huntington’s Gene 1 Huntington’s Gene 34 .___, l probe, it may soon be possible to diagnose who in a particular family carries the Huntington’s gene and who is free of it—assuming that there are enough key family mem- bers to determine which pattern of DNA fragments travels with the Huntington’s disease gene in that family. The l00,000 Americans who are at risk for Huntington's may thus be able to learn their fate, if they wish to do so. The burden of Hunting- ton’s disease could then be drasti- cally reduced within a few genera- tions, it couples who know that one of them has the gene refrain from bearing children. Prenatal diagnosis would be another potential option for such couples. At the same time, prospective parents who might hesi- tate to have children for fear of transmitting the disease would be freed to do so if they knew that they did not carry the Huntington's gene. Another happy feature of Dr. Gu- sella’s and Dr. Conneally’s finding was that Dr. Susan L. Naylor of the Roswell Park Memorial Institute in Buffalo, New York, who had been working with the G8 sequence for unrelated reasons, had already lo- cated this sequence on human chro— mosome number 4. This indicates that the Huntington’s gene is also on chromosome 4. Knowing this, scientists can begin to zero in on the Huntington’s gene itself. Ultimately, the gene can be isolated and se- quenced. Once this happens, diag- nosis should be much easier and applicable to anyone, even without analyzing many family members. Even more importantly, scientists may then be able to decipher the biochemical defect that is involved in Huntington’s disease and perhaps find ways to counteract it. 35 'I'I'IE RAPID GROWTH In 191 l, scientists noticed that only men had a certain kind of color blindness. The trait seemed to be passed down from color-blind fa— thers to their color-blind grandsons, through daughters who had normal vision. The scientists deduced that the gene for color blindness must be on the X chromosome. They rea- soned that a female (who has two X chromosomes) must be protected against color blindness by inheriting one X chromosome carrying 0 nor— mal gene and another carrying a defective gene, and that the normal gene compensates for the defective one; however, a male (who has one X chromosome from his mother and one Y chromosome from his father) lacks this protection. Therefore each son of a woman who carries a de- fective gene on an X chromosome has a 50 percent chance of inherit- ing the defect. This was the first recorded in- stance of gene mapping, the at- tempt to locate genes on specific chromosomes—a field which is now undergoing explosive growth. Gene mapping is proving extremely impor- tant both in the diagnosis of genetic diseases and in research on the controlling regions that turn certain genes on and off. Although it was relatively easy to assign certain genes to the X chro— mosome, scientists had to wait half a century before they could assign genes to particular human autoso- mal chromosomes, and even longer to map the position of any gene on 36 a chromosome. A first breakthrough came in the 1960’s, when researchers learned how to fuse human cells with mouse cells in a test tube. This created hy- brid cells in which human chromo- somes, or chromosomal fragments, could be isolated and recognized. The human cells used for such hy- brids are mixed with cells derived from mouse tumors and treated with a variety of agents that promote the formation of bridges between them; new fused or hybrid cells with a large number of both human and mouse chromosomes soon appear. Generally all the mouse chromo- somes in these hybrid cells are re— tained, while the human chromo- somes are gradually lost as the cells multiply. With luck, the hybrid cells end up with only one or a few hu— man chromosomes remaining in each cell, or better yet, a fragment of one human chromosome. Any human protein produced by such cells is evidence that the gene for this protein is located on the particu- lar human chromosome or fragment remaining in the hybrid cell. With the aid of this procedure, more than 100 human genes were localized to spe- cific chromosomes. An even newer technique is in situ (the Latin term for ”in place") hy- bridization, in which scientists stop the division of human cells at a stage when each chromosome is highly condensed and clearly visible under a light microscope. Then they bind or hybridize the chromosomes Hybrid Ce" Clone 8 fqgi Iv. . 1‘, 9“, w 'i‘ m \h. 1/. 18...: UWSO §‘( «If... t. Hw%s nnl __, ) , ‘ omhcmw , i I)"; ndmmo , 2 o m 9%an :1: ,1 ‘1 _ omwnmu m..m fat, _ c mmm®3 .9. 95f L-.. mprQ ’x".\\ mbnwn (in. mmfifl 1“ '41:“. 5 hasoa Cm mb 38 in these cells to a radioactively la— beled copy of the piece of DNA that they are trying to map. When scien- tists find out, through autoradiogra- phy, which chromosome this piece of DNA sticks to, they can deduce the location of the DNA. Techniques such as in situ hybridi- zation and cell fusion are useful for mapping genes whose products are known. But in many cases, as with the genes responsible for Hunting- ton’s disease or cystic fibrosis, scien- tists know absolutely nothing about the nature of the gene's products. If they wish to map such genes, they must rely on the fact that any identi— fiable genetic variation, such as an RFLP, which is inherited along with a disease must be relatively close to the gene for this disease, on the same chromosome. Other tech- niques for mapping genes by direct chromosome analysis are currently being refined. Once a gene is known to be on a particular chromosome and rela- tively close to a specific marker, sci- entists can begin to ”walk” along the chromosome, using different DNA probes to come closer and closer to the gene. The hope is ulti- mately to isolate the gene, to under— stand what it makes, and to deci- pher how its malfunction can cause disease. Gene mapping relies heavily on the finding that certain chemical stains produce characteristic pat- terns of light and dark bands on chromosomes. After chemical stain— ing, each human chromosome or fragment of one has its own identifi- able pattern when viewed under the microscope, and the individual bands serve as landmarks for the positions of specific genes. In l976, Dr. Jorge J. Yunis of the University of Minnesota developed high-resolution chromosome band— ing techniques which make it possi- ble to see many more bands than before——up to 5,000 bands on a set of 23 human chromosomes. At this high degree of resolution, each band may hold as few as to genes. By now over 3,500 human traits have been associated with specific genes, according to Dr. Victor A. McKusick of The Johns Hopkins Uni- versity School of Medicine, who publishes a continually updated cat- alog of dominant, recessive, and X- linked traits. While a few of these traits are normal variations (such as the ABO blood types), 90 percent of the genes listed in his catalog are associated with diseases. Over 600 of these genes have been assigned to specific chromo- somes. For about half of them, the general area of the chromosome on which they are located is also known. In many cases their location has been narrowed down to just a few bands on the chromosome or to individual bands. Nearly all of this mapping has occurred in the past decade. Eventually the entire set of genes in human cells may be mapped. Drs. David Botstein of the Massa— 39 chusetts Institute of Technology, Raymond L. White and Mark Skol- nick of the University of Utah, and Ronald Davis of Stanford University suggest using a variety of restriction enzymes to cut up DNA (especially that of people who belong to large families whose traits over several generations are well known), using DNA probes to study the resulting fragments, and systematically look- ing for restriction fragment length polymorphisms on each chromo- some. They estimate that if scientists find just 150 polymorphisms spaced throughout the genetic material at reasonably regular intervals, ”then all genes will be linked to one or another of the regions containing RFLP’s and can thereby be mapped.” It might then become possible to understand better each person’s ge- netic composition, or genotype—— and also to unravel the mechanisms of many complex diseases, such as diabetes or breast cancer, which may be different in separate fami- lies. Diobetes may actually consist of several distinct disorders, each caused by one or more separate genes. These disorders only look alike because we do not understand them well enough. Over the past few years, researchers have discov- ered that more than 20 separate mutations can produce thalassemia. In order to prevent such illnesses or treat them most effectively, it is sometimes important to know which mechanism is involved in a given 42 patient or family. llPart of our goal is to sort out who is genetically predisposed to what disease," says Dr. Skolnick. If people know that they are particu- larly at risk for colon cancer, for in— stance, they can be examined more frequently and make sure that any benign growths which are found are removed, to prevent their becoming malignant. Gene mapping is also helping re- searchers investigate some of the controlling elements in DNA that in— fluence the expression of adjacent genes. These small sites, only about 100 base pairs long, act as l/pro— moters," ”terminators," ”enhanc- ers," ”attenuators," or other kinds of regulators of genes. They hold the key to many of the changes that occur during growth and develop- ment, and they play a vital role in certain diseases. For example, several related hemoglobin genes—those for fetal hemoglobin as well as those that are expressed in adulthood—are located next to each other on the same chromosome, but at various times some are switched on and others are switched off. In people with a double dose of the sickle—cell gene, the switch from fetal to adult hemoglobin usually leads to symp- toms of the disease. Scientists would like to find out what controls this turning on or off in a specific region of DNA. Once they know where a gene is located, they can begin to look for its controlling elements, which often are not too for away. It is generally believed that each human cell (except the egg and sperm cells) contains all the genes that are available anywhere in the body. Only some of these genes are turned on at any particular time in a given cell, however. Learning how to turn on specific genes in any human cell might make it possible to acti— vate certain functions so as to cor- rect genetic detects. Learning how to turn off other genes might prevent some diseases, such as cancer. 43 ZEROING IN ON CANCER GENES One of the most spectacular results of the new human genetics has been the discovery that certain genes, called oncogenes, play a key role in the development of cancer. These findings are revolutionizing the study of cancer and may lead to entirely new methods of diagnosing, treating, or preventing many kinds of malignancies. More than 20 human oncogenes have been identified as of mid-T984, and laboratories around the world are competing with each other to find more. Individual oncogenes ap- pear to be necessary, but not suffi- cient, to trigger cancer, a process which is believed to require several steps. In some cases, chromosomal breaks, deletions, translocations, or insertions of foreign DNA place a potential oncogene near a regula- tory element that activates it; in other cases, single point mutations in the coding sequences of certain genes may turn them into onco- genes. In addition, it is now believed that two or more oncogenes of sep- arate classes need to cooperate with each other to make normal cells become malignant. Most of the oncogenes were found after scientists discovered that certain viruses constantly move in and out of cells, picking up bits of DNA as they go. ”Out of the mil- lions of times this happens, on occa— sional virus picks up genes that make a recipient cell go crazy,” ex- plains Dr. John Cairns, professor of microbiology at the Harvard School 44 of Public Health. ”If you can find this virus, it hands you the cancer gene on a plate.” Thus a virus that infects chickens, called the Rous sarcoma virus, con— tains a powerful oncogene—the src (for sarcoma) gene—which stems from a closely related, normal gene of chickens. All mammals, fish, and even fruit flies have normal genes that are almost identical to src, which shows that these genes have persisted over millions of years of evolution. They are believed to code for some key proteins involved in cell growth and regulation, becom- ing activated as oncogenes by as- sociation with a retrovirus (an RNA- containing virus) such as the Rous sarcoma virus. An entirely separate line of re- search which has also uncovered some oncogenes started with the question of how various chemicals and other non-viral mutagens cause tumors. Many kinds of chemicals, as well as ultraviolet light and radia- tion, can cause chromosomal breaks, translocations, or deletions, or produce point mutations in DNA. In most cases these changes are not significant or are taken care of by the cells’ self—repair mechanisms. But sometimes they result in placing a gene that has the potential to be- came an oncogene next to a pow- erful activating signal which pro- vokes it to malignant activity. It had long been known that var- ious leukemias (cancers of the blood) and lymphomas (cancers of Normal Chromosome 8 Normal Chromosomal translocation. As cells replicate and divide, chro» masomal errors may occur in which pieces of chromosomes break off, attaching to other chromosomes. One reciprocal translocation, in which two chro~ mosomes exchange pieces, has been linked to a rare form of cancer called Burkitt’s lym- Chromosome 14 Tronslocation Chromosome 8q — phoma: A piece of chromosome 8 containing a gene called myc breaks off and moves to chro- mosome 14, while a piece of chromosome 14 moves to chro— mosome 8. As a result, the myc gene seems to lose some of its regulatory sequences and be- comes an activated oncogene. Translocation Chromosome l4q + 45 the lymphoid tissues that play a key role in the body’s defense against infection) are correlated with specific chromosomal translocations. Now researchers have found that in Bur- kitt’s lymphoma a gene called myc is moved from its normal location on chromosome 8 to chromosome M, where it lands near a gene for im- munoglobulin. This juxtaposition ap- pears to increase transcription of the myc gene. Through the chromo- somal rearrangement process, the myc gene seems to lose some of its own regulatory sequences, acquires more active sequences which are normally involved in the production of antibodies, and becomes an acti- vated oncogene. Recently these two lines of re- search have converged, as some of the oncogenes that were found in chemically induced tumors turned out to be the same as the onco- 46 genes previously found in retrovi- ruses. The myc gene, for instance, is associated with four distinct strains of viruses known to cause cancer in chickens. Thus a common set of genes may hold the key to many kinds of can- cer. Apparently these genes can be- come activated in various ways: by association with a retrovirus, by iux- taposition with activating signals from other genes, by point muta- tions, or by other means. Each acti— vation is a step in the conversion of a normal cell into a tumor cell, but several such steps may be neces- sary to produce cancer. Once the protein products of these cancer genes are identified, it may be possible to interfere with the action of either the genes or their products and thus halt or reverse the progress of cancer. A BANK OF LIVING HUMAN CELLS In order to study the role of muta- tions or other genetic errors in dis— ease, scientists need to have access to living cells which contain these genetic changes. Since T972, NIGMS has maintained a special facility for this purpose, the Human Genetic Mutant Cell Repository, generally known as the Cell Bank, at the Institute for Medical Research in Camden, New Jersey. It is the largest cell bank of this type in the world. Here, stored in liquid nitrogen at 3T6 degrees below zero Fahrenheit, are over 6,000 cell lines representing more than 300 genetic diseases. There are cells with every known type of chromosomal abnormality, including deletions, additions, inver— sions, translocations, and ”fragile sites” where breaks are likely to oc- cur. There are cells with identified biochemical defects, such as those from people with Toy-Sachs disease, in which a fatty material accumu- lates in the brain because of an en- zyme deficiency; and cells in which the biochemical defect is still a mys- tery, as in cystic fibrosis. There are cells from individuals and cells from large families; cells taken from am- niotic fluid; cells from victims of var— ious hereditary diseases, as well as cells from healthy carriers of these diseases; and also cells from normal volunteers, used for comparison. All cell cultures in the bank are available at modest cost to anyone doing research on genetic diseases. A sliver of skin is removed from a young woman’s forearm. After incubation in a nutrient solution, some of the cells in this tissue will grow and divide, forming a line of cultured cells. 47 Each year the Cell Bank ships out some 3,000 cell cultures to research- ers in the U.S. and abroad. Until re— cently, the demand has been mostly for fibroblasts—connective tissue cells obtained from samples of hu- man skin—which are particularly useful for studies of cell biochemistry and chromosomes. Many diseases which seem, in a patient, to be lo- calized to a particular organ are ac- tually caused by biochemical defects that show up even in cells taken from the skin. Now the demand is shifting toward lymphoblasts, the im— mortalized progeny of white blood cells that have been exposed to the Epstein-Barr virus (a virus associated with Burkitt's lymphoma and with in- fectious mononucleosis) and can grow forever. Lymphoblasts are proving more practical for studies of gene linkage, as in Huntington's disease. Establishing such cell lines from skin or blood takes considerable ex— pertise, as well as time. The cell cul- tures must be sterile, guaranteed to have specific abnormalities, and able to grow. Fibroblasts can reproduce only a limited number of times—usually not more than 40 to 50—in tissue cul— ture, and well before the end of this lifespan they slow down, taking much longer to divide. Lymphoblasts are immortal, but after being kept in continuous culture for a long time they often undergo chromosomal changes which may interfere with research. Researchers therefore 48 need cells that are still in their prime. ”Before the repository, if you wanted to work on a genetic disor- der like galactosemia, which is ex- pressed in fibroblasts, you usually got a cell line from a friend,” says Dr. Fred Bergmann, director of the Genetics Program at NIGMS. ”But the cells might be contaminated, or half dead! 50 the repositow does not usually begin with a cell line al- ready in tissue culture, but with a primary source, such as a piece of skin from a patient.” A small circle of skin, 3 millimeters in diameter, is punched out—usually from the donor’s forearm—under a local anesthetic. At the Cell Bank, this sample is cut into tiny pieces and ”seeded" in a plastic flask, where it is covered with culture me- dium and left to incubate at body temperature for a few weeks. The cells divide, and when the surface of the flask is covered with cells, the population is split into other flasks. This process of tissue culture‘is re- peated several times until there are enough cells for up to l00 glass ampules of 1 million cells each. But meanwhile the cells go through doz- ens of tests to make sure they are not contaminated by microorgan- isms such as bacteria, yeast, fungi, or mycoplasma. Finally—3 to 6 months after the skin sample’s ar- rival—the cells are frozen inside their ampules and stored for future use. Research by Drs. Joseph Gold- stein and Michael Brown of the University of Texas Health Science Vials containing cultured cetls heat-sealed before being P liquid nitrogen at ~316 °F (— 196 °C) in the NIGMS Bonk. 49 Center at Dallas provides a good example of how such cells are used. Working with fibroblasts, They dis- covered various defects in a specific Type of receptor on the surface of some cells (receptors are proteins which recognize specific chemicals from outside The cell and send ap- propriate chemical signals into The cell’s interior). These defects are de- termined by various forms of a dom— inant gene and greatly increase The risk of hearT disease and early death. Among people who have suffered heart attacks before The age of 60, 1 out of 20 has been found To carry a gene for familial hypercholesTerolemia, a disorder in which Too much cholesterol is pro- duced. Such genes occur in roughly 1 in 500 persons in The general pop- ulation; They are Thus among The most prevalent genes which, in sin- gle dose, lead To a specific and sometimes fatal disease. Very infre— quently, two persons carrying These mutant genes marry. Each of Their offspring Then stands a 25 percent chance of inheriting a double dose of The gene for hypercholesTerole— mia. Such extremely rare individuals have a high likelihood of dying from hearT disease before The age of 25. All mammalian cells need a small supply of Cholesterol, Normal cells either Take up some cholesterol from The blood or produce it Themselves, in response To an intricate feedback mechanism. Special receptors on The cell surface bind To LDL (low-density lipoproteins, which carry cholesterol 50 in The blood) and bring cholesterol into The cell; This acts as a signal To The cell to reduce its own production of cholesterol. The system works pretty much like a Thermostat to en- sure that cells always have enough cholesterol for life and growth wiTh- ouT producing Too much of it. But Drs. Goldstein and Brown discov- ered that cells from people with familial hypercholesTerolemia do not have a sufficient number of working LDL receptors on Their surfaces, breaking The normal chain of con- Trol. These cells Then go right on producing cholesterol, which spills out of The cells, accumulates in The victims’ blood, and clogs Their arteries. ”We have identified seven differ- ent mutant alleles—seven alternative forms of The gene for LDL receptors —which can produce familial hyper- cholesTerolemia,” says Dr. Brown. ”Some patients can'T make any LDL receptors at all. Others can make These receptors but can’T Transport Them To The cell surface, so The re- ceptors don’T function. Or else The receptors get to The surface but are relatively less efficient." Now that The researchers have isolated and sequenced most of The LDL receptor gene, They expect to develop techniques To diagnose these variant forms of hypercholes- Terolemia. Meanwhile, the insights produced by this sort of research have led To progress in treating the disorder. Instead of using drugs To reduce The amount of cholesterol ih the blood of such patients (and thus forcing their cells to produce even more cholesterol to make up for the loss), scientists are now developing drugs that appear to stimulate the production of normal LDL receptors. Genetic research pinpointing the most appropriate treatment for each type of hypercholesterolemia could lead to a dramatic reduction in the rate of circulatory disorders and heart attacks. The Cell Bank now contains 22 different lines of fibroblasts from people with familial hypercholester- olemia, including the cells on which Drs. Goldstein and Brown based their discoveries. The bank also has lymphoblasts grown from the white blood cells of members of some very large and special families. These cells are used primarily for linkage studies, in ef- forts to find reliable markers for var- ious disorders. For instance, there are cell lines from 49 members of an Amish family with a high incidence of depressive and manic—depressive disorders, in whom the psychiatric status of each person is known. And there is the collection of cells from 341 interrelated members of the Venezuelan Huntington’s disease community, which scientists can use to study a variety of genetic disor- ders in addition to Huntington’s disease. The Cell Bank’s collection is con— sidered so valuable that elaborate steps have been taken to prevent its loss in some disastrous fire or other local accident. A complete duplicate of this collection is stored at the Cooper Hospital Medical Center in Camden, New Jersey, and both sets are protected by various automatic sensors and security systems. 5i GENBANK: A NATIONAL DATABASE or NUCLEIC ACID SEQUENCES There are no cell cultures and not even a scrap of DNA or RNA in storage at GenBank®, the genetic sequence data bank created by NllelS in l982—iust computers and lists of nucleic acid sequences from organisms as varied as ba- boons, bacteria, yeast, or E. coli, plus over 500 sequences of human and mouse DNA and RNA. Now that stretches of nucleic acids of various kinds are being se- quenced by researchers all over the world with increasing speed, a cen- tral library of sequences has be- come essential to prevent duplica- tion of effort and to enable scientists to compare what they are looking at with all other known sequences. it would be impossible for researchers to do their own work and also keep up with the flow of information in this field. GenBank is an international reposi- tory of all published nucleic acid se— quences greater than 50 nucleotides in length. The sequences are cata- logued, checked for accuracy, and annotated for sites of biological interest at the Los Alamos National Laboratory in Los Alamos, New Mexico. By the end of l983, GenBank contained more than 52 4,000 separate sequences compris- ing over 2.8 million base pairs of DNA. Users can call up on their own computers or request a printout of any sequence in which they are in- terested and find out many of the significant features within this se- quence—for example, which regions code for proteins. The database al— lows researchers to search for simi- larities between a new sequence and all existing sequences, and to do sophisticated analyses that might reveal other important regions. It has proved particularly useful to sci- entists who are working on onco- genes, as well as to those who are looking for the DNA sequences that control gene replication or expression. GenBank is supported by NIGMS as well as by several other Institutes or Divisions of NIH and some branches of government. It is ad- ministered by the Computer Systems Division of Bolt Beranek and New- man lnc. in Cambridge, Massachu- setts, and shares information and collaborates with the Nucleotide Se- quence Data Library of the Euro- pean Molecular Biology Laboratory in Heidelberg, West Germany. 'I'HE PROMISE OF GENETIC THERAPY Many genetic diseases are lethal or extremely debilitating, and their treatment still leaves much to be de- sired. A few of these diseases can be treated by providing substances that are deficient in the body, or by removing other chemicals that accu- mulate to dangerous levels. In the disorders of vitamin metabolism mentioned earlier, for instance, large doses of specific vitamins can save lives. Some hormonal defects can also be remedied. Thus, inherited growth hormone deficiency is treated with growth hormone; this can either be taken from the pitui- tary glands of cadavers or—now that the gene for this hormone has been identified and cloned—mass- produced by bacteria. Recently, scientists have found the gene for factor Vlll, the rare and ex- pensive blood-clotting factor that hemophiliacs need so they will not bleed to death (hemophilia is an X- linked bleeding disorder). Before this discovery, factor VIII had to be ex- tracted in minute quantities from many donors' blood and thus car- ried a high risk of transmitting hepa- titis, as well as some risk of passing on acquired immune deficiency syn- drome (AIDS). Furthermore, many hemophiliacs could not afford to pay $l0,000 a year or more for fac- tor Vlll therapy. But now that the gene has been isolated, it can be inserted into bacterial cells and fac- tor Vlll can be produced in large quantities, at low cost, without any risk of these complications. Another highly treatable genetic defect is phenylketonuria (PKU). Children with this condition suffer from a metabolic disorder which prevents them from breaking down the amino acid phenylalanine, a normal constituent of most foods. As a result, phenylpyruvate, a toxic chemical, builds up in their blood, causing severe mental retardation. Fortunately, the damage can be re- duced or prevented by a stringently restricted diet started right after birth, and screening of newborns for PKU is now mandatory in many states. Such treatments help, but they do not cure the diseases involved. Moreover, most genetic disorders cannot be treated at all by this sort of environmental manipulation. Therefore researchers are now working on entirely new ways to correct genetic defects—.by acting directly on the DNA in people's cells. As mentioned earlier, one ap- proach might be to switch on certain genes which would otherwise be in- active, so that they can take over the job of defective genes. This has already been tried on some victims of thalassemia and sickle-cell dis- ease. However, the overall useful- ness of this therapy remains to be determined. Another possible approach is gene therapy—the introduction of normal genes into the chromosomes of cells that contain defective genes in the hope that the manipulated 53 cells will ultimately replace the de- fective ones, thereby curing the pa- tient. The technical problems that must be solved before gene therapy can be used in medical practice are overwhelming, but according to Dr. W. French Anderson of NIH, ”it is a procedure with enormous potential." ”Gene therapy. . . is conceptually no different from any therapy in medicine that attempts to improve the health of a sick patient,” ex- plains Dr. Arno G. Motulsky, direc— tor of the Center for Inherited Diseases at the University of Wash— ington, Seattle. ”The only difference is that DNA, rather than other bio— logicals, drugs, or surgery, is used as the therapeutic modality. This point is important because some critics claim that gene replacement represents a revolutionary departure in medical treatment. In fact, gene therapy for diseased tissues is no different from any other therapy. No change in the genes of the repro— ductive organs is attempted." Only the first prerequisite for such therapy—the ability to clone 0 nor- mal counterpart of a defective gene—is available so far. Three ma— ior hurdles remain: learning how to deliver the normal gene to the proper target cell and having it stay there; getting the normal gene to produce what it should and regulat- ing it properly; and ensuring that the new gene does no harm. Many new techniques of gene de- livery are being developed. For ex- ample, some researchers insert 54 genes into viruses and then let the viruses infect cultured cells. Others inject DNA directly into the nuclei of cells. ”In animals, we can sometimes deliver genes to target cells,” sum- marizes Dr. Anderson. ”Once in place, a few of these genes can be made to express themselves in the desired manner. ”For humans, we cannot carry out the first two steps in the transfer process [delivery and regulation] and therefore know nothing about the safety.” Nevertheless, the recent progress in work with animals implies that the time is approaching when gene therapy on somatic (non-reproduc- tive) cells could be done in humans. Such therapy would affect only the patient, not the patient’s offspring, and would in some ways resemble an organ transplant. It might allow some single-gene disorders such as Lesch-Nyhan syndrome, which causes severe mental retardation and self-mutilation, to be treated for the first time. Meanwhile such maior illnesses as heart disease, cancer, and schizo- phrenia, each of which probably in- volves many different genes, seem far too complex for any kind of gene therapy. At present the manipulation of several genes at one time is not possible. However, some multigene diseases may be prevented or miti- gated by means of drugs, or by manipulating lifestyle or other envi- ronmental variables. As scientists discover the genetic risk factors for these diseases, people who find out that they are at particular risk may be able to reduce their chances at illness by changing specific aspects of their environment, thereby keep— ing the diseases at bay. In these various ways, many med- ical mysteries are beginning to yield to the genetic approach. The dis- coveries sketched above represent only a traction of current research on chromosomes, genes, recombi- nant DNA, gene mapping, onco- genes, genetic diseases, and treat- ment. As this research advances, it is enabling doctors to help millions of people prevent or control inher- ited diseases. At the same time, this research is laying the groundwork for a new medical science for the 21 st century. 55 GLOSSARY agarose gel—a porous, semi-solid ma- terial used for many research pur- poses, among them electrophoresis. alleles—alternative forms of a gene, oc- cupying a specific site on a chromo- some, which determine alternative characteristics in inheritance. amino acid—a building block of protein. Each protein consists of a specific se— quence of amino acids. amniocentesis—a method of prenatal diagnosis that involves withdrawal of a small amount of fluid from the am- niotic sac that surrounds the fetus,- the fluid contains cells shed by the fetus which can be analyzed. amniotic fluid—the fluid that cushions the fetus inside the amniotic sac. autoradiograph—image produced on an X-ray film by a radioactively la- beled substance. autosome—any of the non-sex chromo- somes; in the case of normal humans, there are 22 pairs. base—one of the five molecules that make up the informational content of DNA and RNA. In DNA, bases pair across the two chains of the double helix: adenine with thymine, and guanine with cytosine. RNA is single— stranded and contains uracil instead of thymine. base pairs——pairs of complementary nu— cleotides forming the DNA double helix. carbohydrate—a class of compounds such as starch or cellulose that is bro— ken down to sugars such as glucose and fructose. 56 carrier of genetic disease—a person who possesses a defective recessive gene together with its normal allele. Although the defective gene’s product is not detectable, the gene can be transmitted to progeny who will have the genetic disease if another copy of the same recessive gene is inherited from the other parent. cell—the basic subunit of any living or— ganism; the simplest unit that can exist as an independent living system. cell culture—the propagation of cells or tissues outside an organism, using special nutrients conducive to their growth; also known as tissue culture. cell-surface receptor—a protein in the cell surface which selectively recog- nizes a specific chemical from outside the cell. This chemical must fit the re- ceptor like a key in a lock. cholesterol—a fatty substance found in all animal tissue. chorionic villus sampling—a still-experi- mental method of prenatal diagnosis that involves withdrawing a sample of tissue from protrusions of a membrane called the chorion, which surrounds the developing fetus. The sampling is done directly through the uterine cer- vix and allows the detection of many fetal defects as early as the 9th week of pregnancy. chromosome—a rod—like structure found in the cell nucleus and containing the genes. Chromosomes are composed of DNA and proteins. They can be seen in the light microscope during certain stages of cell division. chromosome bands—patterns of light and dark bands produced by chemi— cal staining of the chromosomes. Each chromosome or fragment of one has its own identifiable pattern of bonds seen under the microscope, and the individual bands serve as landmarks for the positions of specific genes. cloning—asexually producing multiple copies of genetically identical cells or organisms descended from a common ancestor. Compare with gene cloning. codon—a triplet of nucleotides in the DNA or RNA molecule that codes for l of the 20 amino acids in proteins or for a signal to start or stop protein production. Each gene that codes for a protein is a series of codons which gives the instructions for building that protein. cytoplasm—all the substance inside a cell, excluding the nucleus. differentiation—the series of biochemical and structural changes that groups of cells undergo in order to form a spe- cialized tissue. DNA (deoxyribonucleic acidl—the sub- stance of heredity; a large molecule which carries the genetic information necessary for the replication of cells and for the production of proteins. DNA is composed of the sugar deox- yribose, phosphate, and the bases adenine, thymine, guanine, and cytosine. DNA denaturation—the separation of DNA into its two strands of nucleo— tides, for example by exposing it to near-boiling temperatures or to ex— tremely alkaline conditions. DNA probe—a specific sequence of sin— gle-stranded DNA used to seek out a complementary sequence in other sin- gle strands. The probe is usually made radioactive so that it can be detected. DNA sequencing—determining the nu- cleotide sequences of DNA. dominant—refers to a characteristic that is apparent even when the gene for it is inherited from only one parent. dominant gene—a gene that is ex— pressed even when its allele on the paired chromosome is different. electrophoresis—a method of separating substances, such as DNA fragments, by using an electric field to make them move through a medium at rates that correspond to their electric charge and size. enzyme—a protein which speeds up or catalyzes a specific chemical reaction. Epstein-Barr virus—a virus associated with Burkitt’s lymphoma and infectious mononucleosis. Escherichia coli (E. coli)—a common in- testinal bacterium which geneticists have used for many studies. exons—coding sequences of genes which are retained (after excising in- trons) when mature messenger RNA is made. factor Vlll—a clotting factor which is lacking in the blood of people who suffer from hemophilia, exposing them to the danger of uncontrolled bleeding. fetoscopy—a technique for withdrawing blood samples from a fetus with the aid of a device that permits direct vis- ualization of the fetus and placenta. 57 fibroblast—a connective tissue cell, found in skin and other tissue. fragile sites—chromosomal areas that are particularly vulnerable to breakage. gene—a unit of heredity; a segment of the DNA molecule containing the code for a specific function. gene cloning—isolating a gene and making many copies of it by inserting it into cells and allowing it to multiply. gene expression—the manifestation of the genetic material of an organism as specific traits. gene library—a collection of DNA frag- ments from a cell type or organism which have been introduced into vi- ruses or plasmids and which, taken together, represent the total DNA of that cell type or organism. gene mapping—determining the relative locations of different genes on chromosomes. gene splicing—joining pieces of DNA from different sources by using recom- binant DNA technology. gene therapy—the introduction of 0 nor— mal, functioning gene into a cell in which that gene is defective. genetic code—the language in which DNA's instructions are written. It con- sists of triplets of nucleotides (codons), with each triplet corresponding to one amino acid in a protein structure or to a signal to start or stop protein production. genetic engineering—altering genetic material to study genetic processes and potentially to correct genetic de- 58 fects. See recombinant DNA technology. genetics—the scientific study of hered— ity——of how particular qualities or traits are transmitted from parents to offspring. genome—the total genetic endowment packaged in the chromosomes. The normal human genome consists of 46 chromosomes. genotype—the full set of genes carried by an individual, including alleles that are not expressed. germ cell—a sex cell (sperm or egg). It differs from other cells in that it con- tains only half the usual number of chromosomes. Male and female germ cells fuse during fertilization. haploid cell—a cell with half the usual number of chromosomes, such as a sperm or egg cell. hemoglobin—the oxygen-carrying pro- tein found in red blood cells. hormone—a “messenger” molecule of the body that helps coordinate the ac- tions of various tissues; it is made in one part of the body and transported, via the bloodstream, to other parts, where it has a specific effect on cells. Huntington's disease—a disease that generally appears in adulthood, pro- ducing progressive mental and physi- cal deterioration; it is caused by a dominant gene. hybrid cells—fused cells, usually of dif- ferent organisms, which contain chro— mosomes from each organism. hybridization—the placement of comple- mentary single strands of nucleic acids together so that they will stick and form a double strand. The Technique of hybridization is used in coniunction with probes to detect the presence or absence of specific complementary nucleic acid sequences. intron—a DNA sequence that interrupts the sequences coding for a gene product (exons). After information from the genes is transcribed into new strands of RNA, the introns are cut out of the RNA. The function of introns is still being explored. linkage—the relationship between two genes, or between an identifiable trait and a genetic disorder. Genes that are located relatively close to each other on the some chromosome are said to be linked and generally are in- herited together. lipoproteins—compounds consisting of lipids (fatty substances such as Cho— lesterol) and proteins. lymphoblast—an immature lymphocyte that is immortalized when grown in cell culture. lymphocyte—a white blood cell which is part of the immune system. marker—a detectable genetic variant, such as one of the ABO blood types. Some markers are found only among the victims of certain diseases and can be used to determine the presence of these diseases. medical genetics—the study of the causes, symptoms, treatment, and prevention of genetic disorders. messenger RNA—the ribonucleic acid molecule that transmits the genetic in— formation from the nucleus to the cy- toplasm, where it directs protein synthesis. molecular genetics—the study of genetic mechanisms at the level of the mole— cules DNA and RNA and their components. mutation—a change in the number, ar- rangement, or molecular sequence of a gene. mycoplasma—tiny organisms, smaller than bacteria but larger than viruses. nucleic acids—DNA and RNA, the mol- ecules that carry genetic information. nucleotide—a building block of DNA or RNA. It includes one base, one phos- phate molecule, and one sugar mole- cule (deoxyribose in DNA, ribose in RNA). nucleus—~the structure in the cell contain— ing the genetic material. oligonucleotide—short string of nucleotides. oncogenes—genes that may play a key role in the development of cancer. phenotype—the entire expressed physi- cal, biochemical, and physiological constitution of an individual, resulting from the interaction of the genetic en- dowment with the environment. plasmid—a small, self-replicating mole- cule of DNA that is separate from the main chromosome in bacteria, yeast, and some plants. Because plasmids are easily moved from one cell to an- other or to the test tube, scientists often insert foreign DNA into them and use them to transfer this DNA into other cells in recombinant DNA experiments. point mutation—a change in a single base pair in an organism’s DNA. 59 polymorphism—an inherited variation, such as the ABO blood groups. probe—see DNA probe. protein—a molecule composed of amino acids arranged in a special or— der determined by the genetic code; proteins are required for life processes. receptor—see cell-surface receptor. recessive—refers to a characteristic that is apparent only when genes for it are inherited from both parents. recessive gene—a gene whose product is detectable only when its allele on the paired chromosome is the some. recognition site—see restriction enzyme recognition site. recombinant DNA—the hybrid DNA produced in the laboratory by ioining pieces of DNA from different sources. recombinant DNA technology—tech— niques for cutting apart and splicing together pieces of DNA from different sources. replication—formation of an exact copy. DNA replication occurs when each strand of DNA acts as a template for a new, complementary strand formed according to base-pairing rules. restriction enzyme—an enzyme that rec- ognizes a specific base sequence (usually four to six base pairs in length) in a double-stranded DNA molecule and cuts both strands of the DNA molecule at every place where this sequence appears. restriction enzyme recognition site—the DNA site where a specific restriction enzyme cuts the DNA molecule. 60 restriction fragment length polymorphism (RFLP)—the presence of two or more variants in the size of DNA fragments from a specific region of DNA that has been exposed to a particular re- striction enzyme. These fragments dif- fer in length because of an inherited variation in a restriction enzyme rec- ognition site. restriction fragments—fragments of DNA produced by cuts made with restric- tion enzymes. retrovirus—an RNA—containing virus that replicates by means of an enzyme (re- verse transcriptase) which, upon infec- tion of a host cell, makes a strand of DNA that complements the infecting virus strand; the double-stranded DNA produced in this way then be- comes part of the host cell's chromo- somal DNA and reproduces along with it, eventually also producing an RNA strand identical to the original virus. RNA (ribonucleic acid)—a single- stranded nucleic acid which contains the sugar ribose. There are many forms of RNA, including messenger RNA, transfer RNA, and ribosomal RNA (all involved in protein synthesis), as well as several small RNA’s whose functions are unclear. sex cell—a reproductive or germ cell (egg or sperm). sex chromosome—one of the chromo— somes (X or Y) involved in sex deter- mination. Normal human females have two X chromosomes in each cell, while normal males have one X and one Y. sickle-cell disease—a potentially lethal recessive blood disorder caused by the mutation of a single nucleotide in the gene for beta-globin, one of the protein chains that make up adult hemoglobin. somatic cell—a non-reproductive cell. One of the cells composing all the parts of the body (e.g., tissues, or- gans) other than the germ cells. Southern blotting—a procedure for transferring DNA fragments from an agarose gel to a filter paper without changing their relative positions. thalassemias—recessively inherited blood disorders caused by various mutations which reduce the synthesis of one of the protein chains that make up hemoglobin. The victims of severe thalassemia require frequent blood transfusions and often die in their teens or early twenties. tissue culture—see cell culture. transcription—the transfer of information from various parts of the DNA mole- cule to new strands of messenger RNA, which then carry this information from the nucleus to the cytoplasm. translation—the process of turning in— structions from messenger RNA into protein in the cytoplasm. translocation—an error occurring during chromosomal replication, whereby a fragment of one chromosome be- comes attoched to another chromosome. X chromosome—a sex chromosome. Normal human females have two X chromosomes in each cell, while nor- mal males have one X and one Y chromosome in each cell. X—linked—refers to any gene found on the X chromosome or traits deter- mined by such genes. Refers also to the specific mode of inheritance of such genes. Y chromosome—a sex chromosome. Normal human males carry one X chromosome and one Y chromosome in each cell. 61 PHOTOGRAPH CREDITS Crawford, J.T., Division of Research Services, NIH, page 4. Tiio, J.—H., National Institute of Arthritis, Diabetes, and Digestive and Kidney Dis— eases, NIH, page I2. Yunis, J.J., University of Minnesota, Min- neapolis, MN, page I2. Murayama, M., National Institute of Ar— thritis, Diabetes, and Digestive and Kid— ney Diseases, NIH, page 23. Yunis, J.J., Human Pathology 12:494, I98I. W.B. Saunders Company, Phila— delphia, page 38. Institute for MedicaI Research, Camden, NJ, pages 47, 49‘ 62 ILLUSTRATION CREDITS Watson, JD., Tooze, J., and Kurtz, D.T., Recombinant DNA: A Short Course. W.H. Freeman and Company, New York, 1983, pages 16, 17, 19, 26, 29. Splicing Lite, President’s Commission for the Study of Ethical Problems in Medi— cine and Biomedical and Behavioral Re— search, 1982, pages 18, 20. Gusella, J.F. and Housman, D., in Ger— shon, E8, Matthysse, S., Breaketield, X.O., and Ciaranello, RD. (eds.), Ge— netic Research Strategies for Psychobiol- ogy and Psychiatry. The Boxwood Press, Pacific Grove, CA, 1981, page 25. Fiddes, J.C., California Biotechnology Inc., Palo Alto, CA, page 26. Gusella, J.F., et al., Nature 3062235, 1983. Macmillan Journals Limited, Lon— don, page 32. Ruddle, PH. and Kucherlapati, RS, Sci- entific American 231:1, 1974. W.H. Free- man and Company, New York, page 37. McKusick, V.A., The Johns Hopkins Hos— pital, Baltimore, MD, pages 40, 41. Marx, J.L., Science 2182985, 1982. Amer— ican Association for the Advancement of Science, Washington, DC, page 45. U.S. GOVERNMENT PRINTING OFFICE : 1985 O - 451—088 63 DISCRIMINATION PROHIBITED: Under pro- visions of applicable public laws enacted by Congress since I964, no person in the United States shall, on the grounds of race, color, national origin, handicap, or age, be ex- cluded from participation in, be denied the benefits of, or be subiected to discrimination under any program or activity (or, on the ba- sis of sex, with respect to any education pro— gram or activity) receiving Federal financial assistance In addition, Executive Order TIMI prohibits discrimination on the basis of age by contractors and subcontractors in the performance of Federal contracts, and Execu- tive Order II246 states that no federally funded contractor may discriminate against any employee or applicant for employment because of race, color, religion, sex, or na- tional origin. Therefore, the programs of the National Institute of General Medical Sci- ences must be operated in compliance with these laws and Executive Orders. US. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institute at Health National Institute of General Medical Sciences NIH Publication No. 84-662 September I984