In LABORATORIES across the United States and Europe, scientists this summer are performing genetic experiments that could not have been imagined two years ago. They have plucked single genes from human cells, copied them a trillion times in bacterial factories and analyzed them for clues to the nature of human genetic disease, using newly developed techniques.
The progress has been so rapid, and the implications for the study of inherited diseases so enormous, that vast expectations have been raised within the community of scientists aware of this research, virtually none of which has been reported to the public.
To Dr. Thomas Maniatis of the California Institute of Technology, the present work "represents a revolution in human genetics." Dr. Fred Blattner of the University of Wisconsin says, "Eventually [the experiments] will revolutionize our understanding of human genetic disease."
At the National Institute of Child Health and Development in Rockville, Dr. Philip Leder, a molecular biologist whose work with mouse genes helped set the stage for the latest research into the core of life, expects "that a number of inherited diseases will be understood at their basic genetic level within the next year or two. I can safely predict that by next year there will be dozens and dozens -- perhaps that's a conservative estimate -- of various cloned gene systems around."
One reason for the dramatic impact of the new research is that we still know so little about human genetic defects. The current experiments "should be put in the context of the staggering level of our present ignorance," says Dr. Richard Erbe of Boston's Massachusetts General Hospital, who counsels expectant parents on the risks of passing genetic diseases to their offspring.
But, like other scientists, he finally sees hope for answers to some very basic questions.
"The door has really been closed, but now it's open. We're over the brink," said Dr. Leder.
The human library of genes is so unexplored that no one even knows how many volumes it contains. It could be as few as 50,000 or as many as 3 or 4 million. Scientists do know that each cell contains the full library -- that is why it's theoretically possible to clone a twin from a single cell --but they have no idea how a cell's factory picks out the appropriate volumes to read so that it knows to make insulin if it's in the pancreas or blood parts if it's in the bone marrow.
The library is both vast and messy. Last year, scientists in the United States, Holland and France found that a gene was not bound in a single neat volume but had huge sections of some mysterious other book stuck inside, much as if a child thumbing through his first picture book found a Russian translation of Moby Dick in the middle. They found that the apparatus in the cell that reads genes can neatly edit out the foreign sections, called spacers or intervening sequences. The spacers must do something, but what? And further complications: The cell has hundreds of apparently identical copies of a few genes, but why?
The answers are in deoxyribonucleic acid, (DNA) the elegant double stranded spiral staircase structure whose discovery won a Nobel Prize 25 years ago for Francis Crick, James Watson and Maurice Wilkins.
Each step on the stairs is formed by a pair of nitrogen bases, one on each side, either adenine with thymine or guanine with cytosine. Read the order of the steps down either side, A-T-C-G, and you know DNA's four-letter alphabet. Each word contains only three letters, but a sentence can be hundreds of words long. The word, or codon, tells a cell to make a specific amino acid, and the amino acids link together in long chains to form proteins. Proteins make up bone, hair, blood and a thousand other tissues essential to life, and comprise the catalysts called enzymes essential to the body's chemical reactions. Reports Being Written
THAT MUCH has entered basic biology textbooks in the last decade or so. What is in no textbook, because scientists are still writing the reports for their professional journals, is this work:
A number of groups, working more or less simultaneously at Caltech, Columbia, Yale, Wisconsin, Amsterdam and London, this year isolated and purified the human genes which make the instructions for hemoglobin, the oxygen-carrying protein in the blood. These groups are using a technique developed at Harvard and Cambridge, England, to translate the sequence of letters and words making up the game. One gene, for what's called the beta chain of hemoglobin, is 146 words long.
University of Michigan pediatric scientists have found the two human genes which provide the blueprint for ribosomal RNA. Similar in shape to DNA but with only one spiralling strand, RNA forms the backbone of the hundreds of protein factories in each cell known as ribosomes. (A different kind of RNA, called messenger or m-RNA, is what goes into a cell's nucleus, hunts out and reads the DNA permanently shelved there and returns to the cytoplasm, rounding the nucleus, giving the ribosome on each trip an order to produce one protein molecule.)
To understand the feat of finding and mapping an individual gene, it helps to know something about the size of the DNA which makes up that gene. DNA is 20 angstroms, or two-millionths of a millimeter, wide. It is the main ingredient of the 46 chromosomes in each human cell carrying all of a person's hereditary instructions. Each microscopic cell has packed inside three to four yards worth of DNA. If this were laid end to end and enlarged to the thickness of kite string, the DNA in one cell would stretch from New York City to St. Louis, and about every two feet of kite string would comprise a new gene. Before very recently, the only kind of genetic defect detectable by looking directly at the chromosomes was something as comparatively huge as Down's syndrome, mongolism, in which each cell has an extra 21st chromosome, or a few hundred extra miles of string.
So much work now is going on with human globin genes that Dr. Arthur W. Nienhuis of the National Heart, Lung and Blood Institute says flatly that the genes' entire structure will be known within two years at most. Since biology has a simple but profound rule that structure determines function, the geneticists expect to know soon much about several types of inherited anemia, a fatal or debilitating disease in which the body lacks enough properly constructed bits of hemoglobin to carry oxygen in and carbon dioxide out of the blood. New Tools in Use
BUT MORE IMPORTANT than these early gene discoveries are the new tools invented to find and translate them. Several microscopic workhorses have been harnessed to do much of the crude work. They include specially weakened strains, that can exist only in the lab, of the bacterium known as E. Coli and a virus called bacteriophage lambda, which grows in the bacterial cell. A set of chemicals called restriction enzymes is the scalpel for the work.
Each enzyme, when added to a cell's DNA, can snip it apart wherever it finds a certain sequence of four to six letters. The cut can be staggered, so that a single strand of DNA is sticking out at either end, making it possible to splice it into the new DNA. Different enzymes cut the DNA apart at different places.
The pieces are sorted out by size, and the piece that a scientist wants is inserted into a circle of DNA from the bacterium called a plasmid, then put back into the bacterium. When the bacterium reproduces itself every 20 minutes or so, it also reproduces the foreign DNA. That way, one fragment of DNA can be reproduced a trillion times a day.
The technique has been improved on considerably within the past couple of years by taking the entire DNA from a human, mouse or other animal cell and adding it to a virus first, then putting the whole thing into a bacterium. That makes DNA production by the bacteria much faster and for the first time provides copies of the entire hereditary library.
All of this multiplication is needed to improve the scientists' chances of finding the genetic needle in the haystack. Considering how small a single gene is compared to the total DNA in a cell, to get enough of a mouse gene for any kind of study at all would require 300,000 adult mice to get the 10 pounds of DNA which could be purified to roughly a milligram, or 3/100,000ths of an ounce, of the individual gene. And no one can do that even with enough mice.
The multiplication has only made more needles.To find them, scientists send in armies of messenger-RNA, each piece with a radioactive flag attached. The letter arrangement in each gene has complementing it a unique piece of m-RNA, which ordinarily the DNA produces to send out as a message to the ribosomal factory. By reversing the process and sending the m-RNA to the DNA, it's possible to track down an individual gene. Then the pieces of DNA lit up by the radioactivity can be separated out of all the other DNA and you have a pure gene reproduced many times. The globin genes were the first found this way because they were the easiest to get the m-RNA for, by pulling it out of red blood cells.The technique was developed at Stanford, Caltech and Wisconsin. Reading the Letters
HAVING A TRILLION a copies of a single gene would be a neat but somewhat useless exercise if it were not for a new technique which can rapidly read the gene's letters. Developed independently by Fred Sanger at Cambridge, England, and Allen Maxam and Walter Gilbert at Harvard, the technique works by taking a chemical that chews apart the DNA every time it finds a particular one of the four A, T, C or G letters. By lighting up those chewed-apart fragments with radioactive phosphorus, you can actually read that the letter A is in positions 1, 5, 8, 11 and so on. By repeating with the chemical corresponding to each letter, a piece of DNA several hundred letters long can be completely mapped in a few days.
With a gene from a normal person found and mapped, a scientist then can map the same gene from a person with a genetic disease and begin to learn what went wrong with the gene.
One such disease, sickle cell anemia, is known to be caused by the wrong letter -- a T instead of an A -- being in the 17th position of the 438 letters making up the beta-globin gene. That was found, not by recombinant DNA methods but by reasoning backwards from the fact that an amino acid defect was responsible for the disease, and knowing that the defective amino acid had to have received its instructions from a faulty length of DNA. Vernon Ingram of MIT found that in 1957. But with the new methods, an unborn baby could be diagnosed for sickle cell in a much safer procedure, taking some of the amniotic fluid surrounding it in the womb and looking at the gene, rather than invading its body to look for actual sickle cells in the blood.
Two other types of anemia, rare in this country but found frequently in Mediterranean and Southeast Asian countries, are known as alpha-thalassemia and delta-beta thalassemia. The first is fatal at birth and the second can be debilitating. DNA examination has found both result from the appropriate genes for alpha-globin and delta-globin being missing or shortened.
Using that discovery, doctors from Harvard, Yale and Haceteppe University in Turkey showed how the defect could be visualized on X-ray film and thus diagnosed in an unborn baby. A normal gene shows up on the film as a fuzzy black band but if the baby doesn't have the gene, the band will be missing.
The announcement of that application in The New England Journal of Medicine last month brought wide publicity for the research team and its head, Harvard's Dr. Stuart Orkin. Ironically, Orkin is considered in the scientific community as a minor researcher who happened to latch on quickly to a set of techniques and show their direct human applications. The New England Journal, which rushed his report into print, is being criticized privately by some scientists who say the weekly journal leapfrogged ahead of other journals carrying more important research findings but which won't be published for several more weeks or months.
The Orkin group was able to see a rough image of a single gene on paper but learn nothing else about it. As one scientist who's actually purified a gene in a test tube said, "They saw a fingerprint; we have the finger."
The grumbling among scientists over the Orkin incident illustrates ethical problems in the high-pressured competitiveness of a field widely regarded as one of the hottest in science today. Looking for the Switch
A MORE SUBTLE anemic disease, and much more common, is called Cooley's or beta-thalassemia. Unlike sickle cell anemia which produces a defective chain or alpha-thalassemia which produces no chain at all, the problem with beta-thalassemia is that the chains produced are perfect but there aren't enough of them. That means there aren't enough functioning hemoglobin units, as it takes two alpha chains and two beta chains linked together to form one hemoglobin.
Scientists believe that work now under way on beta-thalassemia not only will show the structural fault in the beta gene but also may provide clues to an area which no one knows anything about, the regulation mechanism that turns genes on and off.
One suspect for the position of the gene on-off switches are in the long pieces of foreign DNA, the spacers, which scientists last year found inside the gene itself. For example, the two genes making ribosomal RNA are a total of 25,000 DNA letters long, but the two huge spacer regions inside take up 70 percent of the total length.
As Michigan's David Jackson said, "Somewhere between 50 and 90 percent of the DNA in our cells is spacer. It doesn't code for proteins. What is it doing there?"
Answering the gene regulation question could tell us why genes sometimes work too slowly, producing a disease like beta-thalassemia, and at other times work at the frenetic pace of cancer cells.
A related mystery about gene regulation holds important pharmacological implications. With the knowledge now available about the rat gene for making insulin, researchers are confident they can isolate and put a human insulin gene into bacteria. But the real trick would be to turn the gene on, so that instead of resting silently side by side with the bacterium's DNA, it sends out the messenger-RNA to produce insulin.
This spring, Gilbert's group at Harvard announced limited success in getting bacteria to make a tiny bit of rat insulin precursor by putting the gene into a carefully chosen spot in the bacterium. But the next step, insulin itself, has proven elusive. And the insulin gene was not actually turned on itself but was simply put into a spot so that the bacterium mistook it for one of its own genes.
The genetic instructions for a number of other key chemicals now are being investigated. They include growth hormone, the pituitary gland secretion which causes dwarfism if cut off; interferon, which is apparently the body's natural virus-fighting substance, and the various types of immunoglobulin, the protein more commonly known as antibodies, a key part of the body's infection-fighting system. A human immunoglobulin gene has been located, and the growth hormone gene has been found in rats. Scientists believe it is a trivial step to isolating the pure human gene.
Susumo Tonegawa in Basel, Switzerland, recently found how the body can make a different specific antibody for each of the millions of foreign agents invading the body. That has long puzzled scientists because it was thought that specificity would require millions of genes. Tonegawa found in mouse immunoglobulin that enzymes -- the same catalysts that perform the spacer editing task -- can bring together entirely separate genes and fuse them into a new gene making a new type of antibody. A few genes shuffling in and out in new combinations is what produces the vast number of slightly different antibodies. Restrictions on Research
MUCH OF THE DNA research, both pharmacological and basic gene-mapping, has been stalled by the federal guidelines allowing work with adult human genes at only one laboratory in the country, Ft. Detrick, Md. It has such elaborate safeguards against bacteria escaping as airlocks, special protective clothing and showers for people exiting. A number of labs around the country have the next most secure designation -- with special ventilation systems costing tens of thousands of dollars -- and they can work with genes from human fetuses.
On July 28, the National Institutes of Health proposed an easing of the regulations so that labs now working with fetal genes could graduate to adult genes, enabling them to work with cells from adults with genetic diseases. NIH Director Donald Frederickson said the thousands of DNA experiments conducted since the techniques first were used in the early 1970s has shown that earlier fears of endowing bacteria with super powers -- the Frankenstein factor, some called it -- were unfounded.
One important piece of evidence for that belief is in the spacers within each gene. The scientists have found that bacteria don't have such spacers in their own genes, and they don't have the enzymes, which advanced organisms do have, that perform the neat editing of the spacers. Thus they can't use the inserted DNA to acquire new characteristics.
If the experiments go ahead -- and the regulations probably won't change until November or December at the earliest -- no overnight breakthroughs in treating some of the 2,300 known hereditary diseases can be expected.
Nor is anyone talking about actual genetic engineering, such as taking that misplaced letter in the beta-globin gene and substituting the right letter to cure sickle cell anemia. For one thing, it probably wouldn't work, since it would involve fixing the gene in not one but millions of cells in the bone marrow manufacturing hemoglobin. For another thing, no one knows enough about the human gene to even think of tinkering with it.
"These experiments may yield the genetic principles that are the key to the whole thing," said Massachusetts General's Dr. Erbe."No one has claimed this is going to allow the clinician to effectively treat a broad spectrum of genetic diseases in the near future. But indeed the promise is in filling some of the enormous void in knowing how these processes work."