GENETIC CONTROL OF CELLULAR BIOCHEMISTRY

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David M. Bonner, Ph.D.

Department of Microbiology, Yale University School of Medicine

T HE PRESENT PAPER deals with the cell, but we should not forget that the prob lems of the cell are at the same time the problems of the whole organism. I would like to consider briefly the gene and the cell, i.e., the relationship of genetic material to

metabolism. I would like to consider this

problem not only in terms of the cell, but in terms of man as well, since the field of biological research is not a field solely of academic interest. The problems of gene ac tion and of the genetic control of metabo lism are fields which are rapidly developing as fields of major importance to medicine, and, in fact, the impact of genetics may be perhaps felt most keenly in the years ahead in the field of pediatrics.

In discussing the genetic control of cellu lar metabolism, one might first ask: What is meant when we speak of the genetics of a cell or of man? Basically, we are concerned with the nature and action of the material

which is transmitted from cell to cell or

from organism to organism, and which de termines the traits of the succeeding gener ation. It has been clearly shown that the genetic material of the cell resides in its chromosomes, and that chemically, it is a polydeoxyribonucleotide, designated DNA for short.1'2

What makes one think that genetic mate rial controls cellular metabolism? In recent years, the cytology of human cells has en

joyed a great resurgence of interest. It is

now possible to grow human cells in tissue

culture, and by this means human chromo somes can be studied with great care and

precision. Recent cytologic investigations have proved of great value and have sug

gested many interesting problems. For in

stance, Ford and his associates in England

have recently found that mongolism is asso ciated with the duplication of an entire chromosome.@

This observation is of clear interest, but perhaps more importantly at the present time, it poses the problems which one faces when considering genes and metabolism.

Why, with the duplication of but 1 of 23

chromosomes should mongolism appear? What does this one chromosome do, and what kind of information does it provide to the organism essential for normal growth and development? A partial answer to this question can be seen by considering other hereditary defects in man.

A number of inborn errors in metabolism in man are known. A number of these are known to be inherited as single gene differ ences, alkaptonuria for instance. It is also known that alkaptonuria involves a defect in the metabolism of the amino acid, phen ylalanine. This inability to oxidize phenyl alanine in the normal way leads to the ex cretion of an intermediate product, a fact recognized some 30 years ago, and one of the earliest pieces of evidence pointing to a relationship between genes and biochemical reactions. Since at that time the steps in volved in the oxidation of phenylalanine in man were not known, a clear correlation between specific genes and the ability to perform specific biochemical reactions was lacking.

Phenylketonuria, likewise, is known to be a hereditary defect, and again, is one which is caused by abnormal metabolism of phen ylalanine. Metabolic defects of this sort, therefore, point to an ultimate genetic con trol of cellular metabolism.

In microorganisms, such as fungi or bac teria, this relationship can be shown very

Presented at the IX International Congress of Paediatrics, Montreal, Canada, July 20, 1959. ADDRESS: 333 Cedar Street, New Haven 11, Connecticut.

PmIArIucs, September 1960

459

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460 THE CELL

born. This disaccharide is first split to give glucose and gaiactose. Both of these hexoses are normally oxidized to furnish the neces

sary energy and carbon for growth. The

utilization of gaiactose requires a number of specific enzymaticaily catalyzed reac tions; it has been shown that children with galactosemia lack one enzyme which is vital for galactose utilization. Thus, individuals who are homozygous for the gaiactosemia gene cannot metabolize galactose, and the accumulation of this compound is believed to give rise to the many diverse aspects of the syndrome.

In this case, therefore, one can clearly see that a genetic difference results in an enzymic difference, in that persons with galactosemia are unable to form a specific enzyme vital for gaiactose utilization. Thus, galactosemia offers proof of the fact that genes control enzyme formation, and shows even more importantly that a specific gene appears to control the formation of but a single enzyme.

How can a gene control the formation of an enzyme? As I mentioned, genetic mate rial is DNA, and yet this poiynucleotide can in some way control the formation of an enzyme, a polypeptide. At present we can investigate this problem more easily in microorganisms than in man. Thus, we

might consider what is now known concern

ing the relationship of a gene to an enzyme in microorganisms, for whatever relation ship we find there will probably hold true for man.

Tryptophan in Neurospora crassa and

Eschenichia coii is normally formed from anthranilic acid via the intermediate com

pound indole-3-giycerol phosphate. This latter compound is converted to tryptophan

by an exchange reaction with serine, which

is catalyzed by the enzyme tryptophan syn thetase. Tryptophan synthetase also cata lyzes two other reactionsâ€”the conversion of indole-3-glyceroi phosphate to indoie, and the condensation of indole and serine to tryptophan.Â° (Fig. 1) It is possible to isolate mutants of the organisms lacking trypto phan synthetase activity; such mutants are characterized by a growth requirement for elegantly by experiments in which certain

gene mutations lead to a loss of ability to

carry out specific biosynthetic @

In fact, the Nobel prize in medicine was awarded in 1958 to Professor George W. Beadle and Professor Edward L. Tatum for their clear demonstration a number of years ago that genes control biochemical reac tions. Thus, it can be clearly shown in the

fungus Neurospora or the bacterium Esch enichia coli that gene mutation can lead to

the loss of ability of the organism to syn thesize an essential amino acid, for example, tryptophan. In turn, this defect can be fur ther shown to represent a loss of ability to carry out but a single vital reaction in the normal biosynthetic sequence involved in tryptophan formation.

Thus, in man, as in microorganisms, we can state with considerable certainty that genes control the capacity to carry out spe cific biochemical reactions. In fact we can go still further, since it is also known that a specific gene must be present in a specific configuration for an organism to carry out a specific biochemical reaction.1'4

One might now logically ask: How can a gene control a biochemical reaction? What

is the action of genetic material which can

determine whether or not a given cellular reaction can occur? Biochemical reactions do not normally occur spontaneously at the pH and temperature of living cells. Thus, almost without exception, the biochemical reactions which a cell carries out require enzymatic catalysis. The specific reactions a cell carries out are, therefore, determined by its enzymic constitution. One might then logically predict that genes control bio

chemical reactions only indirectly by con trolling the ability of the cell to form spe

cific enzymes. This prediction has been veri fied in many instances.5'6

Galactosemia is a hereditary metabolic

defect in newborn infants. This defect is in herited as a single gene trait, and in recent

years has been the subject of intense bio

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SPECIAL ARTICLES

+ SERINE

> TRYPTOPH*@W

cH,H@ OH@HL

H-Câ€”C--COON

NH

N

R INE

I NI3OLE

I

V

TRYPTOPHAP4

SYNTHETASE

Ftc. 1. The reactions catalyzed by tryptophan synthetase.

tryptophan which cannot be replaced by indole. A large number of strains have been

isolated and studied, all of which require

tryptophan and cannot utilize indole for growth, and all of which lack detectable amounts of the enzyme tryptophan synthe tase; thus, we have here an example of the relation of a gene and an enzyme.

With such a group of mutants, one can ask a number of interesting questions. For instance, one can determine whether all of the genetic information relating to the for mation of this one enzyme is in fact con tained within a single region of the genetic material of the organism or is present in a number of different regions. In the case of tryptophan synthetase we can answer this question with assurance; we know that all of the major information relating to the for mation of this enzyme is contained within a restricted region of one chromosome of

Neurospora crassa.@'8

Having assured ourselves that all of the genetic information relating to the forma tion of this one enzyme appears to reside within a restricted chromosomal region, we can ask additional questions: Is this a re

gion of great complexity or is it a relatively simple region? Does this region consist of a single site which undergoes change and so gives rise to a uniform class of mutants, or are there many sites which when altered can affect the formation of this enzyme, each in a unique way? Or perhaps we can even phrase this question in semi-molecular terms: Is this a region which is composed of many nucleotide units, or does it appear to be a region consisting of relatively few such units? These questions can be answered ex perimentally by determining whether the mutant strains which lack the ability to form tryptophan synthetase are identical or not.

At first glance, these strains appear to be identical; mutations in all cases have led to a requirement for tryptophan and they have all resulted in a loss of detectable enzymic activity. But when these strains are exam med using still additional criteria, it can be clearly shown that the region must be mu tationally complex.6'8

For instance, these strains can be exam ined immunologically. Such strains can be compared for the presence or absence of a Q ANTHRANILIC ACID

NHL

COON

I

OH ON H

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462 THE CELL

protein which is immunologically related to the parental enzyme. Such an examination reveals that some strains possess a protein which is enzymatically inert, but which is still serologically related to the parental en

zyme. Other strains lack both enzymic and

serologic activity, while some strains possess a great deal of this serologically related pro tein and others possess relatively little of it. Thus, judged in immunologic terms, we find differences between these strains.6

One can also examine these strains by still additional criteria, such as genetic suppres sibility, temperature sensitivity, or ability to accumulate the compound indole. By so do ing, one is left with the impression that if a sufficient number of criteria were used, few identical mutants would be found.

Thus, by comparing these strains in a number of different ways, one knows that the region of the chromosome which con trols the formation of tryptophan synthetase is a complex region, composed of many mu tational sites.6'

@

What are these mutational sites? Do they represent discrete elements of the gene which can themselves undergo recombina tion, and must they be present in a specified order to permit the formation of normal en zyme? The problem of the discreteness of these mutational elements is difficult to an swer. Crosses between these strains yield rare wild-type progeny, but such wild-type progeny appear to arise by a copy-choice type of recombinational event, rather than by a reciprocal recombinational event. How ever, the fact that crosses between these strains do give rise to wild-type progeny permits the preparation of a mutational map of this genetic region.

Such a map can be prepared by deter mining the frequency with which wild-type progeny appear in interallelic crosses.8 This map suggests that the genetic information controlling the formation of this one enzyme appears to reside in a restricted region of a single chromosome of the organism and that the mutational elements of this region are linearly arranged (Fig. 2).

Thus, it now becomes of considerable in

MUTATIONAL SITE

Ftc. 2. Map of the mutational sites of the trypto

phan synthetase gene.

terest to determine whether the formation of

tryptophan synthetase requires that all of these elements, i.e., the sites which we nec

ognize as mutational sites, need be pres ent in a given sequence. In Neunospora this problem can be examined experimentally by determining whether enzyme formation will occur when all of the mutational ele

ments required for the formation of the en zyme in the parental strain are present in a

common cytoplasm, but distributed amongst different nuclei. One can thus determine

whether clones of mixed nuclear types

formed between different tryptophan-de pendent mutants are capable of growing in the absence of exogenous tryptophan, and if they do grow, whether or not they are capable of forming an active tryptophan

synthetase.

It has been found that certain tryptophan

synthetase mutants, when mixed together in a common cytoplasm, will grow in the ab

sence of tryptophan and in turn will form an active tryptophan synthetase. This phe nomenon has been called complementation. Not all of these mutants show this phenom enon of complementation, but rather appear to fall into three major groupsâ€”complemen tation groups. Thus, just as we mapped this region in terms of recombination, we can also map it in terms of complementation.8

Such a map can be constructed by put ting the strains with identical complemen tation patterns together in a group, and by overlap indicating strains which may not complement with each other but whose complementation patterns differ (Fig. 3).

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COMPLEMENTATI ON

RECIPROCAL RECOMBINATION

Fm. 3. Genetic fine structure of the tryptophan synthetase gene.

UNIT

functional areas. One question of obvious

interest, then, is whether or not the rela

tive positions of alleles in a complementa tion map are about the same as those found in a recombinational map. This problem has been examined, and such a correlation does appear to exist.8

Thus, the genetic region controlling the formation of this enzyme is found to con sist of many mutational sites, but judged in

terms of complementation, it appears to be

a region consisting of few functional sites. Each functional group appears, therefore, to contain many mutational sites. What are these complementational units? Do they represent discrete functional areas of the region? The explanation for complementa lion probably ultimately hinges on an un derstanding of the changes in enzyme for mation that are associated with gene change.

Therefore we should consider briefly what happens to the enzyme as a result of gene mutation. The experimental data avail able at the present time strongly suggest that mutation in the region controlling the

formation of tryptophan synthetase results

in the formation of slightly altered en

zymes.8 This can be documented in a num ber of different ways. As was mentioned earlier, mutant strains are known which can form a protein which is serologically simi

lar to the tryptophan synthetase of the parental enzyme. These proteins appear to be proteins whose formation is controlled by the mutant locus, and which have under

gone an alteration such that they are no longer capable of reacting effectively with the normal substrate molecules. Addition ally, a mutation is known which results in

the formation of a tryptophan synthetase

molecule which is unduly sensitive to mac tivation by zinc. Still another mutation is

known which had led to the formation of a

protein which is serologically identical with the parental enzyme, but which cannot

carry out the normal reaction involving the

conversion of indole-glycerol phosphate to

tryptophan. It can, however, carry out a closely related reaction involving the con version of indole-glycerol phosphate to in

dole.

All such work stresses the correlation be tween genetic change and qualitative en

zyme alteration. What is the nature of this

alteration? An answer to this problem can be obtained from the classic studies of sickle cell anemia carried out by Linus Pauling and his collaborators. Sickle cell anemia in

man is known to be inherited as a single

gene difference. It is also known that sickle cell hemoglobin differs from normal hemo globin. It was shown a number of years ago by Pauling et al.9 that the electrical charge of sickle cell hemoglobin differs markedly from that of the hemoglobin formed by nor mal individuals. This difference in electrical charge has more recently been traced to a slight difference in the amino acid composi tion of the two hemoglobins.

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464 THE CELL

mal and sickle cell hemoglobin is a differ ence involving the substitution of a valine molecule for a glutamic acid molecule. Gene change, in this case, has led to an amino acid alteration.

At the present time, therefore, one would

obviously predict that mutation probably

gives slight differences in the amino acid composition of enzyme molecules. Substitu tion of one amino acid for another, or deie tion of one amino acid, could lead to the formation of inactive enzyme or to the for mation of a protein with physical character

istics that might not permit its recognition.

This line of reasoning obviously leads to the suggestion that the genetic region which controls the formation of tryptophan synthe

tase is a region which determines the amino

acid sequence of this enzyme. Mutational sites, therefore, might well govern the speci fication of a single amino acid. To follow this same line of reasoning a bit further, since all available data point to DNA as the genetic material, each mutational site could be one or a few nucieotide pairs. The prob lem of how DNA controls amino acid se quence still awaits elucidation. At present, one can only say that gene action in gov erning enzyme structure undoubtedly in

volves the triumverate, DNA, RNA and pro

tein. An understanding of the actual role of

each of these must await clarification of pro tein biosynthesis.

Having arrived at the general conclusion that mutational sites lead to the specifica tion of single amino acids, we might again

ask: What is the function of the genetic ele ment which is delineated in the compie mentationmap? I believethatthesemajor functional areas, which are found character istic of the genetic regions controlling en zyme formation, probably reflect the struc tural nature of enzymes themselves. In the past we have been tempted to consider an enzyme molecule as one large macromoie cule, or to state it in slightly different terms,

as a long string of amino acids. During this

past year, a good deal of work has pointed

to the fact that enzymes probably represent

a conglomerate of a number of independent

polypeptide chains.11 Experimental verifi cation of this point of view has been ob tamed by studies of the ribonuciease of mammals,1' and from the studies of trypto phan synthetase of microorganisms.1'

Thus these regions might indicate that tryptophan synthetase is composed of three

essentially independent poiypeptide mole

cuies. The functional region A is perhaps required for the formation of a polypeptide A; region B for the formation of a polypep tide molecule B; and region C for the for mation of a poiypeptide molecule C. These three chains may have to be united in some

manner for the formation of an active tryp tophan synthetase.

Such speculation is of interest at the pres ent time but unfortunately requires experi mental verification. To contrive a general

picture of the nature and action of the ge netic area controlling the formation of a specific enzyme, at present, we would be tempted to say that there is a single region of the genetic material of an organism which controls the formation of a specific enzyme. This region consists of many mutational sites and of a relatively smaller number of functional sites. The mutational sites may well determine the specification of single amino acids, while the functional sites may well represent the independent polypeptide

chains that compose the enzyme molecules.

Experimental evidence points strongly in this direction, but proof of these statements

is far away. In any event we can say that

genes control the reactions carried out by a

cell, and this control is a reflection of the

genetic control of enzyme formation. The final elucidation of the action of genetic ma terial is still a subject which will prove ex perimentally rewarding in the years ahead. In conclusion, we might pause for a mo ment and ask: Of what ultimate importance

is a knowledge of the nature and action of genetic material to the problems of pedi atrics? Obviously, no one of us can answer such a question with assurance. However,

I think it is safe to say that information of the general sort that I have just discussed

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McEkoy, W. D., and Glass, B. Balti more, Johns Hopkins Press, 1957, pp. 3-22.

3. Ford, C. E., et a!. : Chromosomes in a pa

tient showing both mongolism and the

Klinefelter syndrome. Lancet, 1:709, 1959.

4. Bonner, D. : Biochemical mutations in

Neurospora. Cold Spring Harbor Sym. posium on Quant. Biol., 11:14, 1946.

5. Idem: Gene enzyme relationships in Neuro spora. Cold Spring Harbor Symposium on Quant. Biol., 16:143, 1951.

6. Idem: Gene action, in Genetics and Can

cer, 13th Annual Symposium on Funda mental Cancer Research, M.D. Ander son Memorial Hospital and Tumor In stitute. Austin, Texas, University of

Texas Press, 1959, pp. 207-225. 7. Kalckar, H. M.: Some considerations re

garding biochemical genetics in man. Perspectives Biol. & Med., 1:3, 1957. 8. Bonner, D.: The genetic unit. Cold Spring

Harbor Symposium on Quant. Biol., 21: 163, 1956.

9. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a mo lecular disease. Science, 110:543, 1948. 10. Ingram, V. M.: Gene mutations in human hemoglobin. The chemical difference be tween normal and sickle cell hemo globins. Nature, 180:326, 1957. 11. Singer, S., and Itano, H. A.: On the asym

metric dissociation of human hemo globins. Proc. Nat. Acad. Sc., 45:174, 1959.

12. Richards, F. M.: On the enzymic activity

of Subtilysin-modified ribonuclease. Proc. Nat. Acad. Sc., 44:162, 1958.

13. Crawford, 1. P., and Yanofsky, C.: On the

separation of the tryptophan synthetase of Esehenichia coli into two protein com ponents. Proc. Nat. Acad. Sc., 44:1161, 1958.

lems. We all know that an understanding of the nature of a defect is a major step to ward its control. Thus, in the case of galac tosemia, the very fact that we now know that this defect stems from the inability to carry out a single biochemical reaction, clearly points to methods which may be used to circumvent this metabolic block. In the case of other congenital defects, the very fact that a metabolic defect is known to be inherited as a single gene difference leads to the conclusion that such a defect probably represents an alteration in but a single biochemical reaction. This fact again points clearly to methods which may be used to circumvent such metabolic blocks. For instance, it may be feasible to use bac terial enzymes to circumvent blocks of this nature. It is likely in the years ahead that enzymes can be prepared which will have retained their catalytic activity, and yet have lost their antigenic characteristics. Or

it may even be ultimately feasible in man,

as it is now in microorganisms, to add ge netic material by transformation or viral transduction. Thus an understanding of the basic relationships between genes, bio chemical reactions and enzymes is not only a field of great basic interest, but it is a field which in turn may well prove fruitful in the problems of pediatrics.

REFERENCES

1. Beadle, G. W.: Biochemical Genetics. Chem. Rev., 37:15, 1945.

2. Idem: Role of the nucleus in heredity, in

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1960;26;459

Pediatrics

David M. Bonner