Chapter 35 Chapter 19 The Chemical Basis of Inheritance

In retrospect, all experimental methods prior to the rise of molecular biology were completely inappropriate for fully understanding genes.During the period from 1910 to 1950 it was increasingly recognized that the material basis of heredity was composed of highly complex molecules, and that the only way to make further progress was to learn more about the chemistry of genes.It is obviously inappropriate to regard the molecular basis of inheritance either as amorphous particles or as simple molecules.The study of genes is no longer a problem for traditional biologists; it has become a frontier, and at first no man's land, between biology, chemistry, and physics.In the 1940s, when the gene problem was seriously considered and studied by scholars from all walks of life, it was known that chemistry had taken the lead in solving the structure of genes, and a lot of work had been done (Calms, stent, and Watson, 1966).

By the mid-1880s, it was generally accepted that the nucleus was the base of heredity (see Chapter 16), or, more narrowly, the chromosomes, or more specifically, the chromatin, the true genetic material. The term "chromatin" was coined by Flemming in 1879 to refer to the dyeable substance in the nucleus.This immediately raises the question of the chemical nature of chromatin: is it a special substance that is different from other substances, or a protein that is different from cytoplasmic proteins?In fact, the answer to this question was made ten years ago (1869) by the Swiss physiologist and organic chemist Friedrich Miescher (1844-1895), who proved that chromatin is not a protein at all.

After graduating from medical school in 1868, Michel engaged in histochemical research following the advice of his uncle, the famous anatomist and histologist Sith.As Sith said, "Because I had repeatedly stated in my own histological studies that the ultimate questions about the development of tissues could only be solved by chemistry, Michel decided to use the experiments of the famous organic chemist Hoppe-Seyle received post-doctoral training in his laboratory, and arrived in Thünbingen, a small town in southern Germany, at Easter 1868." Hoppe-Seyle suggested that Michel study "the composition of lymphocytes" because of their medical importance.

Michel used pus as a material, which was abundant in hospitals before the invention of antibiotics.A cautious, industrious, and able young man, he developed entirely new methods of separation and was soon able to separate the pus cells from the other components of the pus.Then he tried to separate the cytoplasm of the pus cells from the nucleus, and analyze and determine the composition of the cytoplasm.At first all efforts failed.The end product obtained in one of his extraction procedures was a precipitate that did not have the properties of any known protein.Later, he washed the complete pus cells with highly diluted hydrochloric acid, and what he finally got was completely nuclei.So the unknown must come from the nucleus.Because the study of the components of the cytoplasm had reached a dead end, Michel decided to study the chemistry of the nucleus instead.

The reason I describe the sequence of events in some detail is that a myth was later created that Michel conducted his research in order to solve the mysteries of hereditary phenomena.Not at all!The reality was that an organic chemist wanted to add something to the chemistry of cells and tissues on the advice of his uncle.What is very impressive when reading Michel's article is the originality of his method.He was always using new techniques, especially new extraction and purification procedures, and because of his diligence and ingenuity he is a fully deserved, and I think it is correct, to say that he was the discoverer of DNA, even in Michel Whereas biochemists previously worked with whole tissues, Michel worked with isolated cells; or even parts of cells, such as the nucleus.When he analyzed the material obtained from the nucleus, he found that its outstanding feature was that it contained a large amount of phosphorus.Because this nuclear substance is different from known organic matter, Michel called it "nuclein".

Michel arrived in Tübingen in the spring of 1868 and completed his report on the discovery in the late autumn of 1869.But Honne-Seyler did not immediately publish the report, because the findings were so unexpected that he decided to examine them himself.The manuscript on nuclides was not published until the spring of 1871, when the results of his experiments, and those of some of his other students, were in perfect agreement with what Michel had stated in his report. After Michel returned to Basel, Switzerland in 1871, he discovered that salmon from the Rhine were a rich source of nuclides, because each sperm of salmon was a cell, and the head of the sperm was basically the nucleus.Michel now had a near-endless treasure trove of nuclides (salmon testes, he joked, provided tons of nuclides) and devoted his second year to studying it.He found that the nuclide was closely associated with a protein, which he named "Protamine".He also determined many chemical and physical properties of nuclides, including its experimental formula.

It is a great pity that after his first remarkable achievements, Michel's subsequent research career went downhill. It is all the more regrettable because he is an outstanding character.Perhaps this was due to the fact that he was the eldest of five brothers and possessed all the characteristics of an eldest son.The questions he asks tend to be conventional rather than pioneering (Sulloway).Although it quickly became clear that nuclides are what cytologists call chromatin, Michel never thought of it as a carrier of genetic information.He does not ask questions of genetics, but only questions of physiology or pure chemistry, such as "From where do organisms get so much phosphorus to synthesize a large amount of nuclides during sperm formation?"

In 1872 he publicly spoke of his desire to study "the biological problems of nuclide, its distribution, chemical association, its appearance and disappearance in the body, and its renewal." Influenced by Carl Ludwig, Julius Sachs, and Sith, Michel adopted a then-fashionable physicalist and very mechanistic approach to biological phenomena.This is best illustrated by his explanation of the fertilization process from the point of view of the contact theory. "It is assumed that the properties of the egg cell, compared with ordinary cells, are determined by the fact that it is a missing link in a series of factors controlling its complete structure, since in the egg cell all other essential cellular components are contained. But when When the egg matures, protamine (in the nucleus) breaks down to produce nitrogen (N)... This otherwise perfectly intact machine cannot function due to the lack of a screw. After the sperm reinserts this screw in the proper position, it resumes The original intact structure. It does not need anything else. When the chemical-physical static somewhere is touched or disturbed, the machine starts to work again. A certain law expands." There is not a single word mentioned here about the combination of the genetic material of the two parental gametes.How highly Michel values ​​purely mechanical aspects can also be seen in the questions he asks:

"In what direction and depth do spermatozoa of different species penetrate the protoplasm in eggs?" Michel seems to think that the study of nuclides is less important and turns to other research besides teaching work.During the 14 years from 1874 to 1887, he studied the life history and metabolism of salmon, the chemistry of sperm tails, the structure of the detailed morphology of sperm heads, the chemistry of egg yolks, the nutritional issues of Swiss federal government agencies, and the chemistry of human blood. The relationship between the change and altitude, etc.One gets the impression that the purpose of his research is determined by chance rather than by consideration of scientific importance.It was only later in life that he returned to DNA research and began to propose "correct"

question.But it was too late, because unfortunately he soon died of tuberculosis at the age of 50. Now that DNA is known to be the chemical basis of the genetic program, historians of science have paid much attention to the history of DNA research since Watson and Crick discovered its molecular structure in 1953.Five or six books and some long chapters in the general history of biochemistry have been published.I will only present some highlights here and focus on the biology-related aspects of DNA research. Michel worked with the isolated nucleus, that is, the nucleus after it has been separated from the cytoplasm.This allowed him to use various chemical reagents to test their reactions with nuclides.It is naturally logical to apply the knowledge thus gained to intact cells.Cytologist Zacharias (1881) first used this method to observe the reaction of cells to various reagents under the microscope.He found that the nucleus and chromosomes are resistant to pepsin and dilute hydrochloric acid, soluble in alkali, and swell in saline solution.All of these are characteristic of Michel's nuclides.Other cellular components, such as the spindle filaments, do not show nuclide reactivity, leading Fleming (1882) to conclude that "the chromatin may be identical to the nuclide, if not, from the work of Zacharias They are just one carrying the other. The term chromatin can be used until its chemical nature is known, and at the same time it means the easily stainable substance in the nucleus."

Later Hertwig, Strasburger, Kolliker, and Sachs also agreed to treat chromatin as identical to nuclides, at least that's what they actually did in their paper.This is not only the personal opinion of German cytologists, as Russian evolutionist Menzbir said it in 1891. "Therefore, there is no doubt that only the chromatin and the characteristics of the parents are transmitted to the children (and by extension, the traits of the species are passed from one generation to the next) are related." Zacharias' argument is also accepted by chemists, such as the German chemist Koser Kossel said in 1893: "What histologists call chromatin is essentially a nucleic acid compound with some albumin, and possibly pure nucleic acid to a certain extent." However, it was later claimed that the nuclides referred to by early scholars were extremely impure nucleoproteins, a mixture of DNA and a large amount of protein, and thus had nothing to do with the question of whether these early scholars should enjoy the honor of discovering that DNA is genetic material. The nuclides of Michel and Kossel are indeed not absolutely pure DNA, but they are by no means mixed with a large amount of protein as some people said later.This can be clearly seen from the experimental formula proposed by Michel and Kossel: Michelle C29H49N9O22P3 Kossel C29H36N9O26P3 DNA (50% AT:50% GC) C29H35N11O18P3 (now believed to be correct) Michel's sample may contain some degree of water (hydration), but neither Michel's nor Kossel's experimental formulas appear to contain protein.If it contains protein, the value of C and N should be higher than that of P3 (this is what W. MeClure told me). At the end of the last century, E.B. Wilson pointed out in the second edition (1900) of his famous book "Cytology", "chromatin may be nuclides. . . . The nuclear material, especially chromatin, is The primacy of hereditary phenomena is strongly supported by the findings on maturation, fertilization, and cell division" (p. 332).However, he has some doubts "whether chromatin can really be regarded as the material basis of idioplasm or inheritance as Hertwig and Strasberger said" (p. 259). Shortly after the discovery of nuclides it was suggested (Sachs, 1882) that the nuclides of different species should also be chemically different.As early as 1871, Hoppe-Sevler pointed out that yeast contains nuclides, and in 1881 it was proved that higher plants also have nuclides. The 1880s was when the study of phylogeny was at its climax, and the nuclide research on lower invertebrates was an attempt to find a certain "original nuclide" that was much simpler than the salmon nuclide to prove the phylogeny.That wish came to naught when it was discovered that the nuclides in sea urchins were essentially the same as those in salmon. 19.1 Essence of Germplasm Soon after it was realized that chromatin was (mostly) composed of DNA and that chromatin was germ plasm, the question of whether the fundamental properties of chromosomes were morphological or chemical aroused debate.Biologists have almost unanimously opposed purely chemical explanations, saying that nuclides are chemically too simple substances to explain the extreme complexity of germplasm structure.Boveri (1904) uses metaphors to illustrate his point.If the nucleus is compared to a watch, "the shape of the nucleus involves all the mechanisms of the watch, but the chemistry of the nucleus can at most tell us what metal the gears of the watch are made of" (1904: 123).This was again a case of the blind man feeling the elephant, since the ultimate solution to the problem relied on the morphology of the polymer (which was not understood at the time in Boverly) to account for the unusual structure of the germplasm. Among the earlier scholars, de Vrij was the most correct because he had a solid foundation in both botany and physical chemistry.He emphasized that germplasm could never be a simple chemical substance: "The traits acquired in the course of history require a molecular structure so complex that present-day chemistry is utterly inexplicable" (1889: 31) Even before him Kolliker (1885:41) had said that "nuclei with exactly the same chemical composition may have different effects due to the molecular structure of their effective substances (heteroplasm)." What a prophetic insight! By the end of the 1880s cytologists were using their methods to make as much contribution as they could.They revealed as convincingly as possible that the chromatin fits all the requirements of genetic material and that the head of the sperm is actually the real genetic material.They were not particularly concerned with what these substances were chemically, nor did they pay attention to the size and structure of the molecules.This case is special because it should have been clear that unless the structure of DNA was known its role in heredity could never be explained.I found in the literature that this question has never been seriously raised, probably because there was no experimental method to provide the necessary data to answer this question at that time. At this time, the problem was taken over by chemists. For more than half a century, exploring the nature of DNA was entirely a matter of chemistry.The first requirement is to demonstrate that the nuclide is indeed a substance distinct from proteins and unrelated to other phosphorus-rich substances in living organisms such as lecithin.Michelle is still unclear about these issues.In order to confirm the unique characteristics of nuclides, it is necessary to establish a method of purifying (purifying) nuclides and ensuring that proteins are removed. Altmann (1889) successfully accomplished this task and named this protein-free nuclear substance nucleic acid. Nucleic acids and proteins are fundamentally different Chemists understand better than biologists.As late as 1900, Wilson thought that pure nucleic acids were transformed into albumin by a series of steps containing less and less phosphorus; "they change their composition in response to different physiological conditions". As far as the study of pure DNA is concerned, researchers theoretically have two options.They either decompose the DNA molecule and study its components; or study the entire molecule of DNA, which is the way of research after Staudinger created the theory of polymer chemistry in the 1920s.However, the latter approach was impracticable within the conceptual structure of organic chemistry, which was dominated at the turn of the nineteenth and twentieth centuries by ideas of colloidal chemistry. The two famous leaders of nucleic acid research in the ensuing fifty years were Kessel and Levine.Historians of biochemistry have described how the chemical nature of nucleic acid molecules was gradually elucidated (Fruton, 1972; Portugal and Cohen, 1977).By 1910 it was generally believed that DNA molecules contained four bases: two purines (guanine and adenine) and two pyrimidines (cytosine and thymine), a phosphate, and a sugar.But it took another 40 years to finally determine how these components were connected (1953). Kessel (1853-1927) began to study nuclides in Hoppe-Seyler's laboratory in 1879, and discovered a base, hypoxanthine, in the decomposition products of nuclides in that year.Later he proved that the xanthine came from another base (adenine) and successively discovered and confirmed the other three bases. In 1908 Lewin (1869-1940) began to study DNA and soon became a leader in this area. As early as 1893, Kessel pointed out that a pentose sugar was one of the components of yeast nucleic acid. In 1909, Lewin and Jacobs determined that the pentose sugar was ribose.Other researchers made individual nucleic acids ("thymonucleotides") from calf thymus and found a particular sugar in them.It was notoriously difficult to identify, but Levine and colleagues finally (1929) proved it to be 2-deoxyribose.For many years, it has been believed that ribose is the essence of plant nucleic acid, and deoxyribose is the essence of animal nucleic acid.Later, however, ribonucleic acid (RNA) was discovered in the cells of the pancreas and other animals.Deoxyribonucleic acid (DNA) is found in the nucleus of plant cells.But it wasn't until about the 1930s that it was fully understood that all animal and plant cells contain both DNA and RNA. Cytochemists have a very vague understanding of the function of nucleic acids in cells, most often mentioned as pH buffers or assisting in energy transfer. While much has been learned about the chemical makeup of DNA during the first 30 years of this century, little progress has been made in understanding the DNA molecule as a whole and its biological function.Throughout this period it was erroneously assumed that these four bases were present in equal amounts in nucleic acids and became the basis for the so-called tetranucleotide theory of the molecular structure of DNA.This theory regards nucleic acids as relatively small molecules, with a molecular weight of about 1500.It should be noted that in order to obtain the components of DNA, both Kessel and Levin used very violent analytical methods in organic chemistry.We now know that these methods disrupt molecules that are actually very large.However, the small molecular weight obtained by different methods at that time was in line with the popular concept of colloid chemistry at that time.It was not until the rise of polymer chemistry in the 1920s and 1930s that new advances were made. 19.2 The Nucleic Acid Theory of Genetic Phenomena The idea that DNA has the ability to control development gradually lost its weight when the idea that DNA was a rather small, simple molecule spread.Given the extreme complexity of developmental processes and pathways, how could such a simple small molecule possibly play an important role in the genetic phenomenon and control of the developmental process from the fertilized egg to the fully grown biological organism?In contrast, a protein macromolecule containing 20 different amino acids seems to offer an infinite number of permutations and combinations. Not only were chemical reasons led most biologists to abandon the idea that DNA was hereditary material after 1900, but they were also bewildered by the fact that during mitosis the chromosomal material was only heavily colored when chromatin condensed into chromosomes puzzled.Chromosomes appear to disintegrate into unstained granular matter during quiescence (no DNA-specific dyes were available at the time).Boverly mentioned as early as 1888 that chromatin disappears from the chromosome framework during quiescence and re-forms at the onset of mitosis.His view was later cited by more and more people, and in 1909 Strasburger believed that chromatin "may be the nutrient for the carrier of the genetic unit... Chromatin itself cannot be the genetic material, because it is then separated from the chromosome and in the Its content in the nucleus also varies with developmental stages" (Strasburger, 1909: 108). Goldschmidt pointed out in particular in 1920, "If the nuclides in chromosomes are traditionally considered to be genetic material, then there is absolutely no chemical concept that can explain its diverse effects." Bateson (1916) In the same vein, he asserts: "The assumption that chromatin particles (indistinguishable from each other and identical in structure by virtually any inspection method) can endow organisms with all the characteristics of life on the basis of their materiality completely goes beyond even The most convincing range of materialism." Even with the discovery of the highly specific and sensitive Feulgen stain (Feulgen stain, see below) in 1924, nuclei in some preparations (e.g. sea urchin oocytes) appeared to be devoid of chromatin.By 1925 even Wilson had abandoned the idea that nuclides were hereditary material: "As far as the staining reaction is concerned, what persisted was not the basophilic component (nucleic acid) but the so-called achromatin or eosinophilic Substances. Nucleic acid components are sometimes absent at different stages of cell activity." Reasons for thinking that nucleic acid is not genetic material include some archaic notions about chemical interactions, in addition to the destructive nature of organic chemistry methods in general and the lack of adequate methods for measuring DNA content at various stages of mitosis.The botanist Strasburger (1910: 359) vehemently opposed to treating "real fertilization as a purely chemical process, and thus against any chemical theory of heredity... As far as I am concerned, the essence of fertilization lies in the Factors combine with each other." He can be forgiven for saying this in 1910, when naive and muddled notions of chemical processes still prevailed, and the concept of complex, three-dimensional polymers had not yet been born. The new concept of aggregated macromolecules is very attractive because it seems to satisfy many mechanistic biologists' old assumption that all biological matter is "ultimately composed of crystals".Once Koltsov (1928; 1939) appeared Koltsov (1928; 1939) in German chemist Standinger's new theory of polymers, he speculated on the crystallographic properties of chromosomal material. Sixteen years later the famous physicist Schrodinser (1944) presented his theory of non-periodic vibrating crystals, publicly declaring that he had been influenced by an article whose lead author was Timofeeff-Ressovsky, once Koltsov's assistant. Because polymeric macromolecules are easily degraded into their constituent parts, their extraction requires more delicate and gentle methods than those used by Kossel and Levin.When such methods are used, especially those of the Swedish school of Hammarsten, the products obtained are "snow-white, with a peculiar consistency like gunwool", quite different from the degradation products obtained by vigorous extraction methods. Studying such large molecules requires entirely new approaches.When Caspersson and others employed these methods (ultracentrifugation, filtration, light absorption, etc.) in the 1930s and 1940s, to everyone's surprise the molecular weight of the DNA molecule was 500,000 to 1,000,000 higher than previously measured (1500) two orders of magnitude larger.They are actually larger than protein molecules.These new findings completely rule out an important objection to the theory of DNA as the carrier of genetic information.The next step, and the more difficult one, was to find a way to cleanly separate the DNA and proteins and demonstrate biologically that the DNA components were directly related to hereditary transmission.This task was accomplished in 1944. Avery and colleagues provided such evidence when they studied the transforming factor of pneumococci.It has long been known that there are several types of pneumococcus, which differ in their virulence.British bacteriologist F. Griffith (1877-1941) found in 1928 that when he injected live R (rough) type avirulent pneumococcus and heat-killed S (smooth) type virulent pneumococcus into mice at the same time, many mice died soon, and their The blood contains live S-type bacteria.This finding suggested that the live, avirulent R-type bacteria had acquired something from the dead, virulent S-type bacteria that transformed the avirulent R-type into the virulent S-type pneumococci.It was later thought that some of the genetic information was transferred by "transforming factors".After years of experiments, Avery, Macleod and McCarthy (1944) successfully demonstrated that the transforming factor in a cell-free aqueous solution was DNA. A series of very sensitive tests (immune response, etc.) proved that it was indeed pure DNA and not a protein related to DNS as some of Avery's opponents claimed.This DNA solution showed no reaction to any method of detection of protein.In addition, Avery and colleagues demonstrated that no chemical mutagens were involved in the experiments because the characteristic genetic changes were known in advance.The independence of nuclear material was further confirmed by its self-propagation (self-replication) in transformed cells and subsequent linkage experiments.Finally, the conversion factor is completely and irreversibly inactivated when treated with a highly specific enzyme, deoxyribonuclease.It has a molecular weight of about 50, and its UV absorption shows characteristics unique to nucleic acids. Avery and his team were still very cautious (perhaps too cautious!) in interpreting their findings, but the evidence was so strong that they didn't have to prove their point; the situation was the exact opposite, and it was the turn of the opposition Now to refute Avery's argument. The shock of Avery's discoveries was like an electric shock.I can attest to this from my own personal experience. I was vacationing in Cold Spring Harbor one summer in the latter half of the 1940s.My friends and I are convinced that this is the definitive proof that DNA is genetic material.The famous immunologist Burnet wrote to his wife after visiting Avery's laboratory in 1943: "Avery has just made a very exciting discovery which, quite frankly, is by no means inferior to isolated a pure gene in the form of deoxyribonucleic acid" (Olby, 1974: 205).In 1946 alone it was the topic of six important academic conferences.Of course, not everyone has changed their attitude. Muller (1947) expressed great skepticism, and Goldschmidt was still skeptical as late as 1955. In his famous book "Theoretical Genetics" ( 1955) wrote: "Our conclusion is that .Resistance isn't limited to aging geneticists, however.Certain biochemists, such as A. E． Mirsky, was even more skeptical. The question raised by the skeptics was whether the transforming factor was pure DNA or a small amount of protein mixed with the DNA, a possibility raised by Mirsky and some other skeptics.It is worth noting that most of these people are members of the "Phage group", including Delbrück and Luria, who do not know much about biochemistry.Although they too were well aware of Avery's discovery, they were still too attached to the tetranucleotide theory to believe that DNA could have the complexity necessary for genetic material.Their skepticism had considerable influence because the phage group dominated the field of molecular biology at the time.They finally changed their tune when two of their own group, Hershey and Chase, experimented with radioactively labeled bacterial viruses (aka phages) and came to a positive conclusion. Details of the Hershey-Chase experiment can be found in a genetics textbook.Although the analytical proof of this experiment was actually less precise than Avery's analysis, the phage group regarded it as the final conclusive evidence (Wyatt, 1974). The publication of Avery's results sparked, as Chagaff later put it, a true "avalanche" of nucleic acid research.Chargaff himself spoke of dropping everything else he was doing to work on nucleic acids (Chargaff, 1970).Only a few were qualified to do so at the time.Geneticists in particular, however enthusiastic they may be about Avery's new discoveries, don't have the requisite skills.The lack of research practice by some of them does not justify their ignorance, or at least the young geneticists, of the significance of Avery's discovery. Two researchers who made important contributions in the ensuing years were Chagaff and Andre Boivin.Chagaf proved that in any type of organism the ratio of adenine A to thymine T and the ratio of guanine G to cytosine C is always close to 1 (this ratio happens to be 1 and its molecular significance is obviously not Chagaf's first discovered), the ratio of A+T to G+C varies with different biological species.For example, in his early studies he found that the ratio was 1.85 in yeast and 0.42 in tuberculosis. Chargaff's discovery completely disproved Levine's tetranucleotide hypothesis, according to which all bases should be equal in content.By this time, the ways to propose a new DNA molecular theory have been opened up, and the time is ripe.It was later discovered that the base pairing (purine and pyrimidine) revealed by Chagaf was one of the most important clues to the subsequent solution of the double helix structure.It should be remembered that there are two classes of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).After showing that they are not restricted to animals and plants respectively, the question of what their role is in the cell and how they are distributed in the cell is raised.It has been known since Michel's time that DNA is unique to the nucleus, and there have been early indications that RNA is typically nucleic acid in the cytoplasm, but is it true that diffuse DNA is also present in the cytoplasm and that some RNA is also present in In the nucleus there is still controversy.What is needed are new technologies that can be used in intact cells and can distinguish DNA from RNA.In other words, further progress depends on technological breakthroughs. In 1923, cytochemist Feulgen (R. Feulgen, 1884-1955) created a new staining method (aldehyde reaction), which was later called Feulgen staining, which was specific to DNA.Only by using this staining method can it be finally confirmed that DNA exists only in the nucleus (except for special DNA in some organelles).It took more time to discover a specific RNA staining reaction (Brachet, 1940, 1941; Caspersson, 1941).The presence of RNA in the nucleolus and cytoplasm was clearly confirmed by this reaction. The previous generation of cytological studies allowed us to make quantitative and qualitative predictions about the DNA in the nucleus: (1) Since chromatin is equally distributed after multiplication (replication) in each cell division, all cells arising through mitosis should have the same amount of DNA. (2) Due to meiosis, the content of gamete DNA should be half the amount of DNA in the cells of a diploid organism. (3) Based on the fact that mutations are quite rare, DNA should be a very stable compound. (4) Because two very different pieces of DNA come together at fertilization, they must have the ability to work in harmony. (5) Given the enormous number of genetic variations observed at all levels, from local gene pools to the highest taxa, DNA must be able to display an extremely large number of possible configurations. Boivin and his colleagues, the Vendrely brothers (1948), developed a new method for determining the precise amount of DNA in each cell, and soon confirmed two quantitative predictions.They demonstrated that diploid cells have twice as much DNA as haploid cells.It was also later found that polyploid cells had exactly the multiple of the DNA content expected for haploid.All of these findings confirmed that DNA is bound to chromosomes.Further research showed that DNA and RNA behave very differently in cells with different metabolic activities.For example, even in strictly fasted, starved mice, the amount of DNA in the nucleus remained constant, while in some of these individual mice the amount of RNA dropped rapidly. "The invariance of DNA is a natural consequence of the special function now assigned to it, that function as a repository of hereditary traits for a species" (Mandel et al. 1948: 2020-2021). 19.3 Discovery of the double helix The understanding of DNA has come a long way in these years of research, and the conclusions drawn from it are often prophetic.DNA's metabolic inertia, for example, also seems to confirm the common assumption among gene theorists that genes are "templates" Corollary: "The logical conclusion is that the gene (in the metabolism of the cell) does not 'do' anything, it just provides a blueprint for synthesis (metabolism)" (Mazia, 1952: 115). The absolute stability of DNA in quantity is fully consistent with this assumption. To answer the question of how genes can serve as templates, more must be learned about the structure of the DNA molecule.This has been realized by many scholars, some since Levin who have predicted that DNA must have a longitudinal linear structure, consisting of a backbone of deoxyribose sugars and phosphates (to which bases are attached in some way).The question to be investigated is how these three molecules are linked to each other.Only by clarifying this question can we determine how DNA performs its hereditary function.Three labs in particular are all working on this problem, and when they do, they all have an equal chance of success.One of these is the laboratory of Linus Pauling at the California Institute of Technology.Pauling had elucidated the a-helix structure of proteins and made a major contribution to understanding the forces that hold molecules together. The other is the laboratory of Maurice Wilkins at the Royal College of London.Wilkins and his colleagues specialize in X-ray crystallography, and Rosalind Franklin of this group has taken some excellent pictures of X-ray diffraction (refraction) images of DNA.Her research and other discoveries have led to the following questions: Is the backbone of the DNA molecule straight or twisted into a helix?Only one spiral or two and three?How are purine-pyrimidine bases attached to the backbone?Is the alkali attached to the outside of the frame like the bristles of a bottle brush?If there are two or three helices, will these bases be inside the backbone, and how are these bases connected to each other?鲍林和威尔金斯小组所提出的上述问题以及其它一些问题在剑桥大学的沃森-克里克小组开始研究DNA时还都没有解决。 这三个小组的前进步伐、错误猜想以及所遭受的许多挫折的细节用不着再来介绍，因为这些已讲得太多，也有很多精采的介绍（Olby，1974；Judson，1979）。特别值得提到的是沃森比其它研究者更清醒地认识到DNA分子在生物学中的决定性重要意义。正是这种认识，激发他百折不挠地将他的研究推向前进并取得成就，尽管他在技术上是难于完成这一任务的。威尔金斯则一直到1950年还对“核酸在细胞中究竟是干什么的”感到很奇怪。 沃森（1928年出生）曾在美国印第安纳大学S． E． Luria指导下从事博士生研究。 他在该大学和冷泉港了解到DNA的重要性，当他的某些研究计划由于技术上的原因无法进一步开展时便决定去英国从事DNA研究。在剑桥大学的卡文迪什实验室他碰上了一个性格癖好相同的人、克里克（1916年生）。克里克和沃森具有同样的才华，并且在实验技术上很内行（这是沃森所不及的），但是至少在开始时他对DNA的重要性的认识并不如沃森那样清楚。他们两人对某些方面的知识都很缺乏，然而通过与许多人交谈请教、访问有关的实验室、并无休止地采用各式各样的模型进行试验，他们终于在1953年2月和3月得到了正确的答案。DNA的各种组成分子的拚剪模型大大有助于他们弄清楚DNA分子的三维（立体）结构。 最关键的“信息毕特”（bit of information）是恰伽夫（1950）所发现的嘌呤和嘧啶（AT和CG）的1：1比值。虽然这比值已经发现了两年，但是这三个研究小组多少都一直没有重视。当沃森和克里克最后认识到这数值关系的重要意义之后，他们只花了三个星期摆弄他们的拚剪模型就得出了DNA分子的正确结构。 最后的结果（现在每个中学生都知道）是，DNA是一个双螺旋，两条带就像盘旋楼梯的梯级由一系列的碱基对相联。四种可能的碱基对（AT，TA，CG，GC）的顺序，正如后来很快就发现的，提供遗传信息。这信息作为装配多肰和蛋白质的蓝图从而控制细胞分化。沃森和克里克的双螺旋如此圆满地解释了一切有关事实因而几乎立即被所有的人接受，包括那两个与之激烈竞争的实验室，鲍林的和威尔金斯的实验室。这就排除了一切关于DNA是否真正是遗传物质的最后怀疑。 茹在1883年曾认为传递遗传学的基本过程是细胞核分裂成“两个完全相同的半个”，这样措词是将重点放错了。最重要的实际上是遗传物质的倍增，然后将之分离到两个子细胞中去。因此细胞分裂中最关键的事态是DNA的精确复制。在发现双螺旋之前怎样才能做到这一点完全是一个谜。沃森和克里克一眼就看清了这一点，正像他们在原文中（相当忸怩地）所说的那样（1953a：737）：“我们注意到我们所提出的特殊（碱基）配对立刻暗示了遗传物质的一种可能的复制机制。”在随后的一篇文章中他们扼要指出，螺旋解开连同嘌呤和嘧啶碱之间的键断裂产生了两个模板作为DNA的复制机制。 了解双螺旋及其功能不仅对遗传学而且对胚胎学，生理学，进化论，甚至哲学（Delbrhck，1971）都有深刻影响。遗传型和表现型的问题现在可以用明确的语言说明，对获得性状遗传学说这是一道催命符。虽然早在1880年代和90年代就一再有人怀疑遗传物质可能和躯体的结构物质有所不同，而且即使1908年创用了“遗传型”和“表现型” 这两个词，直到1944年才充分认识它们在根本上是多么不同。从1953年以后才知道遗传型的DNA本身并不进入发育途径而只不过是一套指令。1950年代分子生物学的突破和信息科学的诞生在时间上正好巧合，信息科学中的一些关键词，如程序、编码。也在分子遗传学中使用。 编码的“遗传程序”一代又一代的经过修饰并且编入历史信息，成为了一个强有力而又为人们熟悉的概念。这一概念的历史演变还没有缕述成文。Hering（1870）和Semon（1904）的“记忆单位”（mneme）概念，虽然起初是用来支持获得性状遗传的，肯定属于这一范畴。更接近的是His（1901）将种质的活动比作讯息（message）的产生，种质活动的结果当然远比简单讯息复杂。遗传程序作为不动的运转者（Unmoved mover，Delbruck，1971）的概念是如此新颖在1940年代以前还没有人理解它。 在全部生物学历史上还几乎没有比发现双螺旋更具有决定意义的突破。我同意Beadle（1969：2）的评断：“我曾经说过多次我认为研究出DNA的细致结构是20世纪中生物学的一大成就，其重要性可以和19世纪达尔文及孟德尔的成就相媲美。我这样说是因为沃森-克里克结构立刻说明了它在每一细胞世代中是怎样复制自己的，它在发育和功能中是怎样被运用并发挥作用的，它是怎样经历作为生物进化基础的突变性变化的。” 对双螺旋的了解开拓了一个广阔的、激动人心的研究新领域而且可以毫不夸张的说由于这一发现的结果分子生物学在随后的15年中完全左右了生物学。对遗传现象真正本质的长期研究已告结束。没有解决的问题越来越多的是生理学问题，涉及基因的功能以及它在个体发生和神经生理学方面的作用。然而传递遗传学的情节已经完结。 传递遗传学的一切发现（在第十七章已作总结）在主要方面并没有被分子生物学的发现加以修正。值得提起的是基因细微结构的分析（Benzer发现亚单位）是由经典遗传学方法而不是生物化学方法取得的。有时能听到这样的议论，说什么由于分子生物学的新的研究路线和方法，传递遗传学已“还原”成分子遗传学。这种说法完全没有事实根据（Hull，1974）。早在1880年代就有生物学家认为基因是化学分子，大部分着名孟德尔主义者都同意这一假定。但是在1944年以前这只是一种假说。是分子生物学无可置疑的成就提供了传递遗传学有关现象的化学解释。DNA的结构（双螺旋）（1）解释了基因的线性顺序的实质，（2）表明了基因精确复制的机制，（3）按化学观点说明了突变的实质，（4）指明为什么突变、重组、功能在分子水平上是可以区分的现象。 分子生物学对我们认识基因功能的影响更大，从而开辟了一个完全新的研究领域。 将基因分为几类，如结构基因、调节基因，重复DNA等，仍然还处于初期阶段。核小体（nycleosomes）以及真核生物染色体中各种蛋白质的作用还只是粗略地有所了解。内含子、转位子（转座子）以及假想的“不活动”（silent）DNA的作用还是谜。几乎每个月都有新现象被发现同时也提出了新的疑难问题。我们知道的确实很少这一情况也许表明所有这些现象都和基因功能的调节有关。分子遗传学仍然很像一个未讲完的故事。 19．4现代观念中的遗传学 生物学中很少有其它的分支像遗传学这样对人类的思想和人类事务具有如此深刻的影响。这是一个很大的论题难以在几页篇幅内充分讨论，我所能做的只是指出遗传学思想的某些应用。 早就知道某些人类疾病可能是由于遗传原因，因为它们往往发生在家族之中。在维多利亚女王的男性后裔中非常流行的血友病也许是最出名的例子。18世纪Maupertuis和Reaumur就曾叙述过多指现象。到了现代已经知道人类有几百种遗传病，在很多病例中已经确定突变基因位于哪一个染色体上（McKusick，1973）。 人类遗传学有三个方面值得特别注意。第一个是某些人类遗传病表示代谢失调。英国医生Garrod早在1902年就指出尿黑酸病是由于某一代谢途径被阻遏引起，这阻遏又是因为某种特异性酶的先天缺陷所致（另见Garrod，1909）。虽然Garrod的学说第一次发表时没有引起重视，但经Beadle和Tatum重新发现后，对生理遗传学的发展起了重要作用。 人类遗传学的第二个重要方面是它促使遗传学家去研究那些具有某种非正规遗传方式的表现型情况。目前已经相当清楚与精神分裂病直接有关的基因或基因组具有低“外显率”，这就是说一个人尽管具有所必需的遗传素质但可能并不表现。具有低外显率的基因在果蝇中很普遍（TimofCCff－Ressovsky及Goldschmidt皆曾指出），但是由于明显的原因，遗传学者都不去研究它。有一些其它基因的表达强度是可变的（例如糖尿病基因），研究这样的基因同样可以提高对遗传方式的认识。 也许遗传学思想对现代人影响最深远的是几乎人类的一切性状都可能有部分的遗传学基础这种认识的提高。这种看法不仅限于体质而且也包括智力或行为特征。遗传素质对人类非体质性性状（特别是智力）的影响是目前争议最多的生物学和社会学问题。 最后，第三个方面是遗传学在动植物育种上非常重要。奶、蛋生产是动物遗传学家所取得的辉煌成就的两个例子。抗病作物的育种和杂交玉米以及短茎作物的培育是另外的例子。尽管所谓的绿色革命并不像预期的那样成功，然而它却提高了（有时甚至是激动人心的成倍增加）许多作物的产量。原始人在成千上万年过程中努力于提高作物产量所办不到的事而现代遗传学却能在十年左右的时间就能办到。 任何一个阅读遗传学现代教科书的人都会被书中的大量事实和解释弄得茫无所措。 对一个非专门家来说。即使最基础的教科书所包含的内容也不仅仅只是“你所要知道的遗传学知识”，实际上是大大超过了你所要知道的。由于现代遗传学多少已经分成三个或四个基本上独立的分支：传递遗传学（或经典遗传学），进化遗传学（或种群遗传学），分子遗传学，生理遗传学（或发育遗传学），所以情况更加严重。 这种情况对一个想要用很少的文字来总结从1865年到1980年所进行的研究和所发表的文章中所提炼出的重要概念的思想史家来说的确是难于克服的困难。下面是我本人的尝试，不可否认这只是暂时的，以后还需要修正。 （1）最值得重视和（直到19世纪40年代）完全没有料想到的发现是遗传物质（现在知道是由DNA构成）本身并不参与新个体的躯位塑造而只是作为一个蓝图，作为一组指令，称为“遗传程序”。 （2）密码（借助于它将程序译入个体生物）在生物界是完全相同的，从最低等的微生物到最高等的动、植物。 （3）一切有性繁殖的二倍体生物的遗传程序（基因组）是成双的，由来自父本的一组指令和另一组来自母本所组成。这两个程序在正常情况下是严格同源的，共同作为一个单位起作用。 （4）程序由DNA分子构成，在真核生物中和某些蛋白质（如组蛋白）相联；这些蛋白质的详细功能还不清楚但显然协助调节不同细胞中不同基因座位的活性。 （5）由基因组的DNA到细胞质的蛋白质的代谢途径（转录与转译）是严格的单行道。 躯体蛋白质不能诱发DNA中的任何变化。因此获得性状遗传在化学上是不可能的。 （6）遗传物质（DNA）从一代到下一代是完全固定不变的（硬式），除了非常罕见（百万分之一）的“突变”（即复制失误）以外。 （7）有性繁殖生物中的个体在遗传上是独特的，因为几个不同的等位基因在某个种群或物种中可能在成百上千个座位上表现。 （8）这种遗传性变异的大量储存为自然选择提供了无限的素材。