American Naturalist 81(796): 30-37 (1947)
ON THE SYNTHESIS OF GENE-PRODUCTS IN EQUIMOLECULAR QUANTITIES1
DR. J. A. RAPOPORT
INSTITUTE OF CYTOLOGY OF THE ACADEMY OF SCIENCES OF THE USSR
|1A chapter from the dissertation ''Phenogenetic analysis of differentiation'' presented February 1941 at the Moscow University. During the war the author was in the Army.|
THE extensive experiments printed as preliminary communications in 1939, 1940, 1941, are based mainly on investigations of chemical substances known in biochemistry after the works of Duclaux (1879), Hata (1909), Jacoby (1920, 1926), Euler and Svanberg (1920, 1921), Olson (1921), Myrbäck (1926) and others, as enzyme inhibitors.
It is very important that the poisoning of enzymes in biochemical experiments proceeds as a stoichiometric process (Myrbäck).
Among the morphogenic substances, there are many which are not enzymes. However, according to the hypothesis lying at the foundation of this investigation, the majority of gene-products are of an enzymatic nature. The experimental material under consideration proves this hypothesis advanced in genetics by Hagedoorn (1911), Morgan (1916), Wright (1916) and Goldschmidt (1916). More than half of the specific morphoses found under the action of chemical substances in Drosophila melanogaster and other species of this genus, have been caused by enzyme inactivators.
For instance, mercury causes the phenocopy of the Minute-type, repeating all the finest morphologic peculiarities of this mutation. Silver calls forth the phenocopy of the types yellow or straw; antimony—the phenocopies of the bordeaux or brown types and some other manifestations; boron—the phenocopies with some peculiarities of eyeless. Fluorine causes the occurrence of small melanistic inclusions under the chitin of the larvae and imago; nitrous acid—the phenocopy of the thick and thickoid leg types; sodium perchlorate—sterility, caused by the absence of the external genitalia in both sexes; thiocyanate compounds—shortening of the body of the type chubby mutation; trichlorlactic acid—the phenocopy chrome; neutral red—intensive abdomen abnormal; p-aminobenzoic acid affects the structure of eyes, wings and legs. About thirty cases were found of the morphogenetic activity of enzyme inhibitors and a number of morphoses occurring under the influence of other substances as well. Very interesting among the latter is the action of tryptophane which not only acts on vermilion and cinnabar, as other authors have found, but in our experiments transformed the wild-type color of the eyes into an intense dark one. Only in the case of morphoses induced by the compounds of sodium perchlorate were the phenocopies found in 25 per cent of the individuals; in all the other experiments, the morphoses appeared in 100 per cent of the individuals developing in a medium containing definite concentrations of chemical compounds. All the modifications found were so highly specific that it was possible to investigate with their help also the quantitative relations regulating the synthesis of morphogenetic products during the embryonic period. It became possible to compare different morphogenetic products in vivo, when the pH of the external medium, the pH of body fluids, and other conditions are identical or do not vary to a great degree. These comparisons throw some light on the general peculiarities of the synthesis of morphogenetic substances in the organism.
MATERIAL AND METHOD
As test object served the Naltchik stock of D. melanogaster. The comparative study of morphoses was made on twenty other species of the genus Drosophila. Larvae of the first and second instars of exactly determined age were placed into small vials containing a definite quantity of the tested chemical compound. However, the sensitive periods of almost all the changes set forth below occupy more than one instar. The food medium consisted of molasses, maize flour, etc., standard for our laboratory and already described. Other recipes give slightly different results. The cultures developed at 25-27° C.
The great number of cations and anions giving a clear effect make it possible to expect definite quantitative dependencies when active concentrations of different ions are compared. Only the data about well soluble cornpounds will be used for this purpose because our interest is not in the quantity of the chemical introduced in the medium, but only in the quantity which is dissolved. Universally acting concentrations should not be expected since different chemical inactivators form different compounds with morphogenic products. For instance, some combine with carboxyl, some with carbonyl, others with amino-groups of the morphogenic products, etc.
A considerable, prevailing number of modifications caused by inorganic stuffs permits one to postulate that the morphogenic enzyme products have a protein nature even before a direct analysis of these substances. The poisoning effectiveness both among the anions and among the cations shows that the inhibited enzyme compounds have an amphoteric nature characteristic for proteins, and salt-like linkages are formed with acid or alkaline groups.
Some substances, nevertheless, apparently react with chemical groups having the same structure in different morphogenic products and they are, therefore, in the possibly nearest concentrations. A comparison is given of several anions, cations and of two reagents for the carbonyl group.
It is of interest to compare anions of sodium borate, of thiocyanate and of fluorine. Sodium borate acts in concentrations of 1:750, potassium thiocyanate—in concentrations of 1:750-1:1500 and sodium fluoride—1:3000.
|2The same modification is caused also by sodium hypochlorite.|
The comparison of modifications caused by perchloric acid2 and characterized by incomplete manifestation, shows that the lowest acting molar concentrations of sodium perchlorate almost coincide with active concentrations of sodium nitrite. The perchlorate has a higher molecular weight than the nitrite.
For cations, a comparison of mercury and silver is given, owing to the qualitative inactivation peculiarities of these ions. If the larvae are kept for twenty-four hours in an active concentration of one of these ions and are subsequently placed into normal media, modifications will, nevertheless, take place. Thus, in both cases partly insoluble complexes of cations and of corresponding formative products arise. It should be remembered that enzymes also regenerate feebly from salt-like compounds with silver.
Mercury chloride causes morphoses in concentrations of 1:2000-1:3000, and silver nitrate in concentrations of 1:800-1:1250.
Two standard chemical reagents for the aldehyde group: hydrazine hydrochloride and semicarbazide hydrochloride act in exactly coinciding concentrations of 1:1000. Both these substances cause a pronounced melanic color, but semicarbazide causes also another morphological change.
It is astonishing that molar concentrations of ions affecting various morphogenic reactions either coincide or differ only by several times. The difference by two or three times may depend in some cases on the influence of other substances in the cells of the developing organism, an influence which cannot be excluded even when the principal reactions of inactivation are highly specific.
However, in many other fine physiological reactions, the range of the differences in similar comparisons is hundred and thousand times wider.
The most interesting and significant data may be produced in the investigation of highly soluble compounds when both anion and cation exhibit a pronounced morphogenic effect. An equal amount of dissociated active anions and cations appears in any concentration of these compounds.
For instance, the easily dissociating silver fluoride. Under the influence of sublethal concentrations of AgF in vials the walls of which were covered with paraffin, and in ordinary vials, 109 imago were obtained having yellow chitin and yellow bristles characteristic for silver, and small dark inclusions reminding of the mutation benign tumor characteristic for the fluorine compounds. Such changes did not appear in the controls.
It should be mentioned that silver chloride, nitrate, sulfite, acetate, citrate, nucleinate, etc., give only the yellow-straw effect. The sodium, potassium, barium, cerium, fluorides, fluoric acid, fluoroiodine and other cornpounds of fluorine, cause morphologically only melanistic inclusions in larvae and imagines.
Of interest are the results of the action of partly soluble compounds of cations and anions as, for instance, mercury thiocyanate, only slightly soluble in food media. In other compounds the thiocyanate ion apparently also forms a partly insoluble complex with the morphogenic product, since larvae, kept in a medium with ammonium thiocyanate for 24-26 hours, after being washed and placed in normal media manifest the modification at the pupal and imaginal phases of development.
Despite the low solubility of mercury thiocyanate, both modifications develop in sublethal concentrations, attributed to each ion separately. Thus, also the slightly dissociating cations and anions have closely coinciding thresholds of effective concentrations.
What are the quantitative relations between concentrations causing a morphological effect and lethal concentrations?
A comparatively insignificant increase of a concentration causing a phenocopy calls forth a lethal result. Sodium borate is toxic in solutions of 1:600, semicarbazide—1:700, potassium thiocyanate—1:600. Similar data have been obtained for silver and other investigated compounds. Concentrations, causing phenocopies, have already been given. Concentrations causing a clear visible effect are, therefore, followed by concentrations with a lethal action.
The influence of similar or equimolar concentrations of substances causing different phenocopies and, therefore, affecting various morphogenic products, may be interpreted as the consequence of synthetic processes, common for the cell as a whole.
Among various quantitatively dependent and even synchronic processes in the cell, the multiplication of genes and chromosomes is the most important or at least the most investigated. Characteristic for them are simultaneous and multiple augmentation and diminution of .large complexes. The conclusion may be, therefore, drawn that the investigated enzyme-like morphogenic substances appear during embryogenesis in simple molar quantities under the influence of chromosome structures.
In different tissues the chromosome mass of the intensely functioning cells augments. For instance, the nutritive ovarial cells of many animals. Even the extreme specialization of the biochemical function of the glandular cells is, nevertheless, correlatively connected with an increase of chromosome threads in every cell, either the chromosome number increasing or the manifold reduplicated elongated threads transforming into a giant chromosome. We suppose, therefore, that enzymatic products synthesized under the influence of the genes, possibly enter the cells in equal molar concentrations.
About the relative vicinity of the concentrations causing visible changes in morphogenesis and of the lethal concentrations, the following should be mentioned.
The allelomorph series of many or even of the majority of gene loci contain both lethals and visibles. Some of these lethals are caused by deficiencies, but some give reverse mutations and are, therefore, caused by chemical rearrangements. Lethals prove the primary role played by genes in normal development and the lethal effects of separate loci are very specific. It would be fallacious to identify the series of multiple allelomorphs with various quantitative gradations of phenocopies, and a comparison is merely given between a visible and lethal change. Each step of the multiple allele is a quantitatively singular chemical structure, and the gradation of phenocopies in the experiments under consideration is only the result of the inactivation of the unequal quantity of molecules in the morphogenetic substance of the wild type allele.
The proximity of the compared concentrations shows that the lethal action of the compounds used in the experiments is the result of the possibly strongest influence on this embryonic physiological reaction, whose lesser shifts are followed by phenocopies. A specific lethal action is present, which repeats the specific lethal action of the genes.
The similarity of morphologically significant and of lethal dosages of each substance proves that here the lethality is caused by more various toxic mechanisms than those characteristic for the poisoning of adult organisms.
Many specific phenocopies, corresponding to almost all principal mutation types, have been obtained by means of chemical enzyme inhibitors. Among the substances causing specific morphoses are the ions of heavy metals, inorganic and halogen substituted organic acids, hydrazine, aromatic ketons, ketenes, etc. Both the process of inactivation of morphogenic substances and that of enzyme inactivation depends on the quantity of enzyme inhibitors. The comparison of concentrations of several heavy metals, anions, organic enzyme poisons, shows that their effective dosages are either identical in molar concentration or closely coincide. The simultaneous occurrence of two morphoses under the influence of one easily dissociating compound, the first modification being caused by an anion and the second by a cation, may be regarded as a sufficient proof that the corresponding inactivated morphogenic products also form in the cell in equimolecular quantities.
Concentrations causing visible effects and those causing lethal ones appear to be adjacent. Thus, a lethal effect may in this case be regarded as the result of the most strong and noxious quantitative, change of the same reactions whose feebler shifts produce morphoses.