The Genetics of Conditional Mutations and Individual Developmental Programs in D. melanogaster
Siberian Department of Russian Academy of Sciences, Institute of Cytology and Genetics, Novosibirsk, Russia
Citation: Chadov BF, Chadova EV, Fedorova NB (2017) The Genetics of Conditional Mutations and Individual Developmental Programs in D. melanogaster. SCIOL Genet Sci 2017;1:3-21.
A new class of mutations referred to as conditional mutations was discovered in Drosophila melanogaster. These mutations cause death in a restrictive genotype but survival and reproduction are promoted in a permissive genotype. In addition to their conditional nature, mutations occurring in flies with a permissive genotype produce a set of specific features that drastically distinguish them from conventional mutations. The emergence of morphoses in mutants suggests that the genes are involved in controlling ontogeny. Correspondingly, these genes were named ontogenes.
The following features of these mutations were analysed using theoretical approaches: 1) Conditionality; 2) Lethality; 3) Reduced fertility; 4) The development of morphoses; 5) A parental effect on the inheritance of the mutant phenotype; 6) Effects of chromosomal aberrations on mutant phenotypes; 7) Long-range gene interactions; 8) The shift from mutation-induced lethality to early embryogenesis.
The considered facts are logically related, consistent, and sufficient to state that the ontogenes 1) Are DNA regions that; 2) Are active in germ cells; 3) Encode intranuclear RNAs; 4) Induce epinucleotide (epigenetic) changes in the genome; 5) Determine the order of developmental stages (program of development); 6) The corresponding program is read twice, namely; a) In the germ line cells (reload) and b) In somatic cells (implementation); 7) The first read is directed from the last developmental stage to the beginning of development and the second read occurs in the opposite direction from the initial stages to the later stages.
Conditional mutations, Ontogenes, Program of individual development, Drosophila
A living organism owes its existence to genes inherited from its parents, including the genes responsible for the individual developmental program, which indicates that "a due event occurs in the appropriate place and at the appropriate time" . Until recently, the genes controlling the ontogeny and the genes that are under their control were believed to be the same universal Mendelian genes. Developmental biology has not even attempted to consider any other types of genes .
Curiously enough, Mendelian genes, which form the background of genetics and were used to postulate the idea of an individual developmental program, are not the major carriers of the developmental program. A species-specific individual developmental program is singular and has no alternative, whereas genes have different variants known as alleles. Mutations in the corresponding genes should be lethal and eliminated by selection to retain the single-option nature of the developmental program. However, carriers of mutations in Mendelian genes not only survive but also are equally viable in certain cases. Thus, a consideration of the genetic provision of the individual developmental program poses the key question of "different-type genes" that are as yet unknown to genetics but absolutely necessary for the operation of a single-option genetic program of individual development.
Mutations referred to as conditional mutations [3,4] were discovered in the search for the genes responsible for intraspecific similarity [5,6]. The very first batch of mutations was identified in D. melanogaster as early as 2000 [7,8]. The manifestation of a conventional (Mendelian) mutation depends only on the mutant gene itself, for example, on the dose of the gene, whereas the remaining genes are completely unimportant for the phenotype. The manifestation of a conditional mutation strongly depends on other genes in the genome or on the overall genome. A description of a conventional mutation does not require a description of the genotype in which this mutation is manifested, but it is absolutely necessary when describing a conditional mutation. Researchers must know 1) The genotype in which the conditional mutation is manifested; 2) The genotype in which it fails to manifest. Some additional conditions are also important for the manifestation or non-manifestation of a conditional mutation . Mutations responding to external environmental factors are sometimes referred to as conditional, in particular temperature-sensitive lethal mutations [10,11] or lethal mutations that depend on the chemical composition of the nutrient substrate [10,12,13]. However, these factors are beyond the scope of our study. Here, we only focus on the conditional mutations that respond to changes in the genome.
The fact that the manifestation of conditional mutations strongly depends on other genes drastically distinguishes them from conventional mutations, thereby allowing researchers to search for these mutations as genes of an unknown type. Further studies of conditional mutations increased the list of differences. Conditional mutations in a permissive genotype display a set of specific features that drastically distinguish them from conventional mutations, namely, they 1) Are dominant; 2) Switch the genome from a stable to an unstable state; 3) Display a parental effect; 4) Elevate the basal metabolism [3,4]. A high rate of monstrosities (morphoses) and modifications in the offspring is a hallmark of the presence of a conditional mutation in an individual [3,4]. The defects in individual development (morphoses and modifications) emerging in the offspring of mutants have suggested that these mutations denote the previously unknown class of genes involved in the control of development [14,15]. These genes were named ontogenes [6,16-19] and are regarded as the candidates for these "other type of genes" that are required to construct the individual developmental program.
Here, we describe Drosophila ontogeny data extracted from the information about conditional mutations. This topic is of special interest to researchers studying individual development, because they rely on formal genetic ideology rather than the ideology of developmental biology. According to Sidorov , it is not uncommon in the history of genetics when purely genetic (formal genetic) studies are much more advanced than more exact sciences, such as chemistry and biochemistry. The underlying reason is evident. The essence of all life is renewal [1,21]. If this hypothesis is true, then genetics, by focusing on the gene-trait problem and transmission of characters from the parent to its offspring, is the particular science that creates the framework for the knowledge about all life. Not surprisingly, new genetic knowledge once again is obtained via a hybridization procedure, which has already made an outstanding contribution to the establishment of genetics.
In this paper, we analyse the results of crosses between fruit flies (D. melanogaster) carrying conditional mutations. The crosses had been conducted and are described in detail in a set of papers published in 2000-2017, including two reviews . The goal of this analysis was to extract the information about individual development from the data on conditional mutations, which is the first attempt in this area. The new phenomenology discovered in conditional mutants  allowed us to expect the success of this type of study.
Materials and methods
Genetic analysis, i.e., an analysis of the composition of the progeny produced in crosses of mutant strains, was used in this work. A cross in an experiment repeats the reproduction of living organisms in its natural integrity, except for one difference: Strains carrying specific mutations are crossed instead of normal strains. The effects of some mutations are known to the researcher, whereas the effects of other mutations are unclear. The mutations examined in this work were original conditional mutations that had been previously generated by the authors and mutations obtained from international D. melanogaster stocks.
Mutations in the X chromosome were produced by γ-irradiation of wild D. melanogaster strain Canton S males and subsequent selection according to a specific protocol [4,7,8]. The conditionality of these mutations is confirmed because the (+) males are viable and fertile, whereas their y/+ daughters produced by yellow females die at the embryo stage. The mutations are maintained in laboratory stocks of two types: the females of one strain carry a mutation and a Muller-5 inversion and females of the other strain contain attached X chromosomes .
Mutations in chromosome 2 were produced by γ-irradiation of wild D. melanogaster strain Canton S males and subsequent selection using another protocol [4,24]. The conditionality of these mutations is confirmed because the "mutation/+" individuals are not viable, whereas the "mutation/In(2)CyO" individuals are viable. These mutations are maintained as "mutation/In(2)CyO" strains.
Mutations in chromosome 3 were obtained using the morphosis technique after γ-irradiation of wild D. melanogaster strain Canton S males . These mutations are recessive lethals and are maintained in laboratory stocks as "mutation/In(3LR) Dichaete" [3,4].
Fertility of the mutant male carriers of a conditional mutation in the X chromosome is determined as the ratio of the progenies to the number of laid eggs .
Thus, the conditional mutations 1) Have been generated using a typical mutagenic factor (γ-irradiation); 2) Have been isolated by hybridological procedures intended to detect the DNA damages in a chromosome of a specified pair; 3) The mutations are maintained in specially organized genetic cultures; 4) The mutations are inherited following the inheritance pattern for a defect in a particular chromosome; 5) The mutations reproduce their manifestation after a long-term cultivation [3-6]. Several tens of mutations in chromosome 2 have been localized on polytene chromosomes by deletion mapping. Four mutations have a visible manifestation and are inherited following the main rules for inheritance of visible mutations. All the above described facts unambiguously demonstrate that the basis of conditional mutations is damages in DNA sequences.
Although the conditional mutations according to all genetic criteria are the result of damages in the DNA sequence and have the same origin as the Mendelian mutations, they do not follow the rules of classical genetics, which are the law for the Mendelian mutations. This appears as 1) A distortion in the ratios of the progeny classes; 2) The absence of reciprocity of crosses; 3) The presence of parental effect in inheritance; 4) The effect of a chromosomal rearrangement in the genome on the composition of progeny; 5) Asymmetry in mutation manifestation; 6) Induction of genome instability; etc. [3-6]. Thus, it emerges that the classical Mendelian genetics and the genetics of conditional mutations are mutually exclusive.
Results and discussion
The section headings here are based on the conclusions generated from the experimental material described and discussed in the corresponding section. Taken together, these findings provide insights into novel aspects of individual development that are clarified by experiments with conditional mutations.
1. Ontogenes are the hereditary units responsible for the course of individual development (conditionality of manifestation, lethality, and morphoses)
The history of the search for conditional mutations began with the hypothesis that an invariant part of the genome is responsible for intraspecific similarity [5,6]. Mutations in the genes in this part of the genome can hypothetically emerge but have a lethal outcome, i.e., the mutations should be dominant lethals. According to this hypothesis, certain conditions (genomes) in which the dominant lethals continue to circulate in the population for a certain time period should exist for the evolution to occur . Therefore, we searched for conditional dominant lethal mutations. These mutations should be dominant lethals in one genotype and nonlethal in another genotype. Thus, we initially proposed that the mutations to be identified should combine dominant lethality and a conditional manifestation, features that are absolutely untypical of Mendelian mutations.
Table 1 lists the first mutations produced in D. melanogaster [7,8,26]. These mutations reside in the X chromosome of (+) male. The male is viable and yields progeny when crossed to yellow females; however, yellow/+ daughters are not produced. The lethality of yellow/+ daughters that carry the mutation in a heterozygous state indicates that the mutation is a dominant lethal, and the conditional manifestation is confirmed by the observation that the male with mutation survives but mutant daughters die.
The conditionality of the mutant phenotype suggests a regulatory character of the gene, since the gene has both (+) and (–) activities in response to the genotype. The list of genotypic conditions influencing the mutation manifestations was supplemented further. Based on the regulatory trait, the identified genes likely belonged to the set of genes controlling the ontogeny. A lethal manifestation of the mutations was also consistent with our hypothesis, since lethality is an efficient way to maintain the constancy of a species-specific individual developmental program. The observation of developmental defects (morphoses) in individuals that carried conditional mutations was an unexpected confirmation that the conditional mutations occurred in genes controlling individual development.
The glossary of genetic terms defines morphosis as a non adaptive and usually unstable variation in individual morphogenesis associated with changes in the environment. The morphoses that simulate the shapes of known mutations are referred to as phenocopies . Here, the term morphosis is used for the nonhereditary morphological deformities caused by specific genetic features of the parent. Morphoses display characteristic features, allowing them to be reliably distinguished from phenocopies and modifications.
The morphoses in conditional mutants are represented by the appearance of aberrant flies with different degrees of alteration (Figure 1, Figure 2 and Figure 3). Most morphoses do not interfere with fly hatching from the pupa, survival, mating, and even production of progeny. Morphoses are observed when fruit flies of different strains are cultivated, but these morphoses are a rather rare event. However, morphoses frequently emerge in the progeny of obtained conditional mutants (Table 2) [3,14,26]; moreover, in some cases, the entire progeny display morphoses. The diversity of morphoses and extent of impairments in the fly appearance are amazing. However, these mutations do not lead to death. A high rate of the emergence of morphoses has allowed us to obtain a large collection of coloured images of various morphoses (approximately 1000) over a rather short time span. A set of 135 images classified according to the body parts of the adult Drosophila has been reported  and is available on the internet . Some of these images are shown in this study. A characteristic feature of the morphoses is their asymmetry. A morphosis develops on the left or right side of the body. The same morphoses are rarely present on both sides of the body. The important specific features of morphoses will be discussed further in the context of the hypothesis on the action of ontogenes.
Figure 1: The morphoses of the "plus tissue" type (surplus morphological structures). a) Groups of eye ommatidia (red spots) on the occiput; b) An additional eye on the right side; c) An additional thorax with an altered wing on the right side and a normal wing on the right side in a form of a structure-less bubble; d) An additional wing on the right side (directed forward) and an altered thorax on the right side; e) A tergite fragment with bristles on the abdomen; f) Doubling of the external male genitalia; g) Four wing-like appendages with bristles instead of a normal wing on the right side; h) Tarsus on the abdomen; i) An additional altered seventh leg. View Figure 1
Figure 2: The morphoses of the "minus tissue" type (lacking morphological structures). a) Loss of a wing (stump) and bristles on the left thorax; b) Loss of a prothoracic leg on the left side; c) Loss of the head capsule and a major part of the right eye; d) Loss of the left wing and circular bristle pattern on the left thorax; e) One pair of legs instead of three pairs in the normal fly and different shapes of the right and left legs in the remaining pair; f) Reduced tarsus of the left metathoracic leg; g) Loss of half of the thorax on the left side, including the wing, and a right wing with a Notch-type indentation; h) Circularly cut right wing; i) Loss of the left wing and a cone-like stretched left thorax. View Figure 2
Figure 3: "Two heads" morphoses. a) Reduced second head in place of the left eye, with the eye on the small head exhibiting a Bar phenotype similar to the eye on the main head; b) A process formed as a head with two wa-type eyes instead of the right eye; c) A head with appendages and two red eyes instead of the left eye; d) Two heads with appendages on one neck; e) A second head with wa-type eyes instead of the right eye; f) One head (from below) with two w-type eyes and the second head (from above) with one w-type eye. View Figure 3
2. Ontogenes are active in germline cells (parental effects of conditional mutations)
The traits generated by conditional mutations are inherited according to a parental effect [3,4,29]. Four examples of a parental effect are presented below as an illustration of all recorded cases of a parental effect recorded in conditional mutants .
2.1. Parental effect on the inheritance of lethality caused by conditional mutations in chromosome 2
Mutations in chromosome 2 are maintained as stocks of the "mutation (2)/In (2LR)CyO, Cy Bl L4" type. The cross of mutant males to yellow females only produces one class of progeny, Cy Bl L4, instead of the expected two classes, (+) and Cy Bl L4. The sons and daughters of "+/mutation (2)" class die (Table 3, left columns). The conditionality of mutations is confirmed by the observation that the combination of a mutation with inversion in the opposite chromosome 2 is viable, whereas the combination of the same mutation with a structurally normal chromosome 2 ("+/mutation (2)" class) is lethal.
The lethality of the "+/mutation (2)" class manifests as a typical parental effect: Lethality of mutation manifests in the cross (♀ y × ♂ mutation (2)/Cy Bl L4) but it does not manifest in the reciprocal cross (♀ mutation (2)/Cy Bl L4 × ♂ y). As expected, two classes of progeny are produced, namely, "Cy Bl L4/mutation (2)" and "+/mutation (2)" (Table 3, right columns).
2.2. Parental effect on the inheritance of lethality caused by conditional mutations in the X chromosome
Mutations in the X chromosome are maintained as laboratory stocks of two types: The females with attached X chromosomes and females with mutation/In (1) ?-5 [3,4]. The conditionality of mutations is confirmed because (+) males carrying the mutation are viable and fertile, but the yellow/mutation daughters (cross 2♀ y × ♂ +) that received the mutation from their father die (Table 1). As shown in Table 1, the progeny of mutant males exclusively consists of sons that received the Y chromosome from their fathers rather than the X chromosome, which was mutated in this case. A lethal effect of these mutations was observed based on the daughters’ mortality. However, this effect was broader and became apparent when the number of dying eggs in clutches was calculated. The rate of dead eggs was always greater than 50%. Therefore, daughters and some sons died, although sons did not receive a mutant X chromosome from their fathers. In other words, the observed lethality was inherited according to a parental (paternal) type rather according to the laws of Mendelian genetics, i.e., together with the factor that leads to the emergence of the trait (in our case, the X chromosome).
2.3. Parental effect on the inheritance of lethality caused by conditional mutations in chromosome 3
Each of the stocks with a conditional mutation in chromosome 3 carries the mutation in one of the autosomes and a pericentric inversion with a visible dominant marker, D (Dichaete), in the other. Only heterozygotes for a particular mutation survive in culture, whereas homozygotes for a conditional mutation that are also homozygous for inversion die. Six variants of crosses are possible between four mutations, and two types of crosses, direct and reciprocal, are possible for each variant (Table 4). Twelve crosses were conducted, and the progeny obtained from reciprocal crosses were compared to the progeny obtained from each of the six pairs.
Theoretically, the progeny in a pair of reciprocal crosses should consist of two approximately phenotypic classes, Dichaete+ and Dichaete, in approximately equal numbers. However, the compositions of the progenies in reciprocal crosses were drastically different. The F1 progeny from one cross (mutations 46 × 55) contains all classes at the expected ratios, whereas the other cross (mutations 55 × 46) did not produce progeny. The same dramatic difference was observed in another pair of reciprocal crosses (mutations 34 × 46 and 46 × 34).
The differences in the other reciprocal crosses were not as impressive but were statistically significant. In particular, 3.4-fold fewer (18.9%) Dichaete progeny obtained from the cross of mutations 27 and 46 compared with the reciprocal variant (64.4%). The same situation was observed for reciprocal pairs (27 × 34) and (27 × 55). Since the set of classes of progeny and the probability that the progeny classes will form are the same, the differences observed in the reciprocal crosses cannot be explained by a difference in the viability of the progeny. The differences in the number of progeny observed in the reciprocal crosses represent a typical parental effect.
2.4. Parental effect on the inheritance of morphoses
The conditional mutations in the laboratory stocks and resulting crosses are always performed in a heterozygous state. Half of the offspring of a heterozygote receives the mutation and the other half does not. Nonetheless, morphoses emerged in both the progeny that received the mutation and in the progeny lacking the mutation. In the cross of a yellow female with the mutant (+) male carrying a conditional mutation in the X chromosome, the yellow sons did not receive the mutant X chromosome, but some still developed morphoses (Figure 4). In the cross of C(1)DX, y w f females with the (+) males carrying a conditional mutation in the X chromosome, the C(1)DX, y w daughters did not receive the mutant X chromosome, but some daughters displayed morphoses (Figure 4f and Figure 4i). Thus, a progeny will develop a morphosis when its father carries a conditional mutation, but the progeny does not need to receive the mutation from the father. The parental inheritance pattern and subsequent paternal effect are evident.
Figure 4: Morphoses observed in the offspring of conditional mutants. I. Parental effect of a paternal type. The conditional mutation was located in the X chromosome of a normal male crossed to yellow females. The yellow sons did not receive the mutant chromosome from their father but still developed morphoses. In two cases, a male was crossed to the ?(1)DX, y w f females. The y w f daughters (f and i) did not receive the mutant X chromosome from their father, but exhibited morphoses. The morphoses included a) The absence of the left metathoracic leg; b) A shorter right wing; c) An altered tergite pattern on the left side; d) Altered shape of the right wing; e) Lack of a tarsus in the right metathoracic leg and altered shape of this leg; f) Altered wing shape and structure; g) Reduction of the left thorax and left wing; h) Replacement of the left wing with two appendages; i) Reduction of the left wing; j) Myeloma of the right arista in the lower male; k) Shorter and deformed tibia of the metathoracic legs in males; l) Impaired wing veining. View Figure 4
One way to maintain conditional mutations in the X chromosome is to maintain them in females heterozygous for the inverted chromosome In(1)Muller-5, B wa. In the stock Muller-5/mutation, the In(1)Muller-5, B wa/In(1)Muller-5, B wa (B wa phenotype) females and In(1)Muller-5, B wa (B wa phenotype) males do not carry the conditional mutation; however, some displayed morphoses (Figure 5). This situation also occurs when a progeny is not necessarily required to carry the chromosome with a conditional mutation to develop a morphosis. The presence of the mutation in the mother is sufficient. A parental type of inheritance occurred in this case as well, but in a maternal variant. The B wa daughters and the B wa sons with morphoses developed from eggs that did not contain any conditional mutation in the X chromosome. Other evidence suggesting a parental effect of conditional mutations has been also reported .
Figure 5: Morphoses observed in the offspring of conditional mutants. II. Parental effect of a maternal type. The conditional mutation was located in the X chromosome (+) of a +/In(1)Muller-5, B wa female. The In(1)Muller-5, B wa/In(1)Muller-5, and B wa daughters and In(1)Muller-5, B wa sons with a B wa phenotype (bar-shaped apricot eyes) did not receive the X chromosome with the conditional mutation from their mother, but developed the following morphoses: a) Wings of different lengths with bubbles; b) A narrowed wing, impaired veining, and a bubble; c) Opaque wings of various shapes; d) A right wing filled with lymph and an impaired veining pattern; e) Asymmetric wings; f) Tissue overgrowth instead of left eye ommatidia; g) A smaller right wing with an irregular shape and a bubble; h) Loss of the right wing and coloured tissue in the thoracic base; i) A reduced wing blade on the left side; j) Absence of macrochaetes and microchaetes in the right side of the thorax; k) A deformed femur of the right mesothoracic leg; l) A sickle-shaped right wing. View Figure 5
2.5. A parental effect on the inheritance of a trait indicates that the gene responsible for this trait is active in germline cells
In general, conditional mutations are a unique case of the diversity in the manifestation of the parental effect. Variants (2.1) and (2.3) exemplify the nonreciprocal nature of reciprocal crosses. Variants (2.2) and (2.4) are examples of when a trait is inherited without the corresponding transmission of the mutation. Variant (2.4) is an example of maternal and paternal cases of the parental effect, and variant (2.2) is an example of a paternal case. In addition, variant (2.2) is an example of a character (lethality) manifesting in early embryogenesis, and variant (2.4) is an example of a character (morphosis) developing during the overall ontogeny from the early to late stages. The parental effect on the inheritance of the characters generated by conditional mutations definitely represents an inheritance pattern rather than an exception.
The regularity of the parental effect of conditional mutations and their diverse manifestations suggests that the cause underlying the effects, such as the parental effect, is a unique specific feature of ontogenes, namely, their activity in germline cells. This activity should lead to the consequences described below.
First, conditions for the implementation of differences in the manifestation of a gene in the mother and father actually exist. In particular; a) A nonreciprocal pattern of reciprocal crosses and b) Two types of the parental effect (maternal and paternal) should emerge.
Second, the activity of a gene should lead to an unusual situation before the germline cell undergoes reduction division. The meiocyte should contain a gene (ontogene) that has lost its activity and its product (potential character). Since the ontogene and the product are not physically connected, two variants of gametes should be formed: 1) "product (character) + ontogene"; 2) "product (character) without the ontogene". The presence of gametes of the latter type induces the most striking form of parental effect, namely, the inheritance of a character without the inheritance of the corresponding gene.
The above considerations regarding the causes of parental inheritance of the characters determined by ontogenes poses a logical question on why the Mendelian genes are inherited according to the Mendelian pattern, as, similar to ontogenes, they are simply DNA regions. We must focus on one of the features characteristic of Mendelian genes that, as a rule, is overlooked, namely, their activity in the zygote, to answer this question. Both the maternal and paternal alleles reside together in the zygote; thus, a basis for the differences in their activities does not exist. This features is why classical Mendelian genetics appears "asexual": The direction of crosses does not influence the qualitative or quantitative composition of the progeny. The absence of differences between the progeny of the reciprocal crosses has long been perceived as a standard of inheritance and posed no questions. Questions appear when this reciprocal pattern "suddenly" disappears, such as in the case of conditional mutations.
Another specific feature also distinguishes the genes and ontogenes and affects the inheritance pattern. During the course of mitosis, Mendelian genes do not "leave" with their derivative, ensuring that the rule "where the gene is, the trait is" is not violated. Ontogenes must undergo meiosis and reduction division, leading to the inheritance of a trait without the inheritance of the gene determining this trait.
Thus, accepting the activity of ontogenes in germline cells, we obtain an overall range of parental effects observable during the inheritance of conditional mutations. These effects allow us to infer that the ontogenes are active in the germline cells and to state with sufficient certainty that the ontogeny of an organism commences from the very moment when the parental germline cells start to develop into gametes.
This hypothesis has been proposed in the relevant literature. A change in the methylation level of gametes [30,31] and somatic changes in the progeny induced by an impact on the germline cells in a parent (epigenetic impacts) also confirms this hypothesis [32,33]. The obtained data verify this hypothesis. Notably, this hypothesis allows researchers to name the genetic factors that display this early activity. However, these factors do not refer to all genes constituting the genome or Mendelian genes, which have long been the focus of genetics, but rather a separate and specific category of genes, which we refer to as ontogenes.
3. Ontogenes encode nuclear RNA (interaction between conditional mutations and chromosomal aberrations)
According to the dogma of F. Crick, a gene is implemented into a trait by following a DNA-mRNA-protein script. The known regulatory pathway assisted by RNA suggests that this script is not the only way to exploit the information contained in DNA. The interaction between conditional mutations and chromosomal aberrations, which are discussed below, definitively reveal that the dogma does not apply to ontogenes. The ability of chromosomal aberrations to influence the manifestation of conditional mutations became evident immediately after the first set of conditional mutations in the Drosophila X chromosome was identified . The protocol for producing and detecting conditional mutations in Drosophila chromosome 2 was elaborated based on this property of rearrangements [4,24], as well as one of the methods used to maintain conditional mutations in the X chromosome in fly stocks [3,4]. Results from two groups of experiments reveal the effect of a rearrangement on the elimination of a particular class of progeny in the offspring of a conditional mutant are described below as an illustration.
3.1. Effects of chromosomal aberrations in the X chromosome of yellow female on the prohibition on daughters in the progeny of mutant males
Conditional mutations in the X chromosome of males in the cross with yellow females cause the absence of daughters in the progeny. The phenomenon of prohibition on daughters in progeny is depicted in Table 1. We examined how the presence of rearrangement in the X chromosome of yellow females would influence this phenomenon. For this purpose, several rearrangements in the X chromosome were produced in the yellow strain. These rearrangements included two inversions and two translocations with one of the breaks localized to the X chromosome (Table 5). The rearrangements were maintained in the genome of the yellow strain; therefore, these stocks only differed from the initial yellow strain by the presence of a rearrangement in the X chromosome . The presence of each rearrangement removed the prohibition on daughters to different degrees (Table 5).
3.2. Effects of chromosomal aberrations in chromosomes 2 and 3 of yellow females on the prohibition on daughters in the progeny of mutant males
This experiment differs from the previous experiment because we assessed the role of the rearrangements in the other chromosome pairs of yellow females (Table 6). These rearrangements comprised the Cy and Pm inversions in chromosome 2 and D in chromosome 3. The stocks carrying rearrangements were produced to ensure that the differences between these stocks were only associated with the presence or absence of rearrangements [3,4,19,34].
As shown in Table 6, the rearrangements in other chromosome pairs also affected the intensity of the effect of prohibition on daughters in progeny. This intensity varied, depending on the particular rearrangement and mutation, but the general pattern of response was quite clear: Rearrangements removed the prohibition on daughters in progeny. The study of conditional mutations also identified another effect of these mutations. Chromosomal aberrations in the genome of a mate to which a strain is crossed not only remove the prohibition on the appearance of a particular class of progeny but also introduce a prohibition .
The ability of a chromosomal aberration that changes the spatial arrangement of chromosome regions in the nucleus to influence the manifestation of genes, including the conventional genes residing in other chromosome pairs, is a unique phenomenon that is not typical of the conventional Mendelian genes (with rare exclusions). Notably, the most widely used method to maintain mutations in genetic collections are their maintenance in the chromosome pair with a rearrangement, which in no way changes manifestations of genes (the traditional Mendelian genes). Thus, the cause underlying the interaction of rearrangements with conditional mutations is associated with the specific features of these mutations [3,4].
Figure 6 shows the hypothesis that explains different responses of a rearrangement to conventional (Mendelian) genes and ontogenes. We postulate that the Mendelian genes follow the DNA-mRNA-protein script and ontogenes follow the DNA-nuclear RNA script. The first script implies that an mRNA leaves the nucleus for the cytoplasm and is transported to the ribosome and the corresponding regulatory protein then returns to the nucleus. This pathway is so long that a change in the distance between two genes in the nucleus, resulting from a rearrangement, has no consequences. This situation occurs for Mendelian genes encoding protein molecules. Ontogenes respond to a rearrangement because they utilize RNA molecules residing within the nucleus in their pathway. The nuclear distances between the site of synthesis and the ultimate location are critical for the function of these molecules .
Figure 6: Protein or short RNA? Selection of the interface product explaining the interaction between chromosomal aberrations and conditional mutations. From Fedorova, et al. . The large circle represents the cell and the small circle represents the nucleus. A and B represent genes (or ontogenes) in a pair of homologous chromosomes, and the grey ellipse represents a ribosome in the cytoplasm. Blue arrows show the routes followed by the corresponding interface product to implement the interaction between genes A and B. In the norm (left top), the distance between A and B is rather short and a chromosomal rearrangement (right top) alters this distance. If the interface product is represented by a protein (crossed out), this change in the distance will not be perceived, since it is negligible compared with the distance to a ribosome and back. However, the change in the distance will be noticeable if a short RNA is the interface product in question, since the trajectory of its movement resides within the nucleus and the distance between A and B is comparable. View Figure 6
The hypothesis that nuclear RNA functions in germline cells logically complies with the currently available information about meiosis. The first set of cytological data are related to lamp brush chromosomes [35-37], which display a high level of RNA synthesis, and the second set of molecular data confirms the existence of regulation via RNA interference [38,39]. This hypothesis explains not only the effects of rearrangements on ontogenes but also the absence of these effects on conventional Mendelian genes, making it particularly convincing.
4. A short RNA that forms duplexes is the most likely type of nuclear RNA formed on ontogenes
The overwhelming majority of manifestations of conditional mutations appear when a mutation is present in one dose [3,4]; thus, the products of ontogenes are likely double-stranded short RNAs [38,39]. A disturbance in the DNA sequence in one homologous chromosome interferes with the formation of the RNA duplex, eventually leading to a mutant phenotype in the carrier of a conditional mutation.
5. The DNA molecule of germline cells is changed by the RNA of ontogenes ("long-range gene interaction" phenomenon)
Classical genetics distinguishes several types of gene interactions, namely, complementation, polymery, modification, and epistasis. A characteristic shared by all types is that the interaction event occurs in the presence of the interacting genes. Conditional mutations exhibit another type of gene interaction, an interaction "at a distance". The corresponding genes reside in the parental genomes, whereas the interaction is implemented in the progeny. For the interaction to occur, the genes themselves and their derivatives, RNA and proteins, do not need to be transferred .
The chromosomal aberrations Cy, Pm, and D in the female genome and the conditional mutations in the X chromosome of males demonstrate the type of interaction described above (Table 6). Conditional mutations induce the prohibition on daughters in progeny, whereas the rearrangements remove this effect. Conditional mutations reside in the paternal genome and rearrangements reside in the maternal genome. The interaction appears in their progeny as the production of previously absent imago daughters. Although a chromosomal aberration initiates the emergence of daughters in the progeny, its presence in the resulting daughters is not obligatory. The emerged daughters can be both Cy and Cy+, both Pm and Pm+, and both D and D+ (Table 6).
The question arises: Which particular products must be present in the zygote along with the paternal and maternal gametes for the interaction event to occur? Based on the description provided above, these products are clearly not genes (DNA). What are other likely candidates, RNA or protein? As shown in the next experiment, the examined phenomenon does not require RNA or proteins.
Table 7 lists the data on the cross of yellow females with mutant males. Unlike the previous experiment (Table 6), mutant males are the carriers of the Cy inversion (left, control and right, experiment). The presence of both the Cy inversion and the mutation has no restorative effect on the production of daughters. Thus, a situation occurs when the RNA and proteins of two mutations (conditional mutation and chromosomal aberration) in the same cell does not provide an interaction event. In other words, colocalization of two derivatives of interacting genes is not the cause of the gene interaction.
Table 7: Absence of an effect of Cy on the phenomenon of prohibition on daughters in progeny if this inversion is present in the genome of the (+) mutant male. View Table 7
The interaction of an aberration and conditional mutation requires that they should be present in different parents. A certain change caused by the aberration should occur in the maternal genome and the change caused by a conditional mutation should occur in the paternal genome. We presume that these changes are of an epigenetic nature, i.e., they consist of a certain alteration of chromatin that does not affect the nucleotide code. These particular changes are transferred to the zygote by the gamete. These changes affect the entire DNA of the chromosomes transferred by a gamete, rather than a specific mutant region. Therefore, the presence of the mutant DNA region is not required for this interaction. From our perspective, these changes should be more precisely referred to as epinucleotide changes rather than epigenetic changes. Indeed, they are not related to the nucleotide sequence in DNA, and yet are related to DNA, the genetic material.
The interaction occurs when two genomes (maternal and paternal) carrying epinucleotide changes meet in the zygote. The interaction (comparison) has two outcomes, namely, the progression of development or cessation of development (death). Thus, the chain of events related to ontogeny in this case comprises two new hypothetical events - 1) The formation of the epinucleotide code of a gamete and 2) A comparison of the epinucleotide codes of gametes in the zygote.
6. The epinucleotide change in the DNA molecule is part of the individual developmental program (the phenomenon of a shift in the lethal effects of mutations)
6.1. The phenomenon of a shift in the lethal phase
The main manifestation of the mutations generated in ontogenes is lethality. The male carriers of a conditional mutation in the X chromosome produce few progeny (Table 1), since the overwhelming majority of the progeny die at the egg stage. None of the 21 studied mutations induced lethality at the subsequent developmental stages (larva and pupa).
A special experiment was conducted (Table 8) in which the lethality rate was recorded at each of the three stages, namely embryonic (egg), larval, and pupal stages, using maximally synchronized egg clutches. Ninety percent of the lethal cases in the progeny of 18 studied mutants were recorded at the embryonic stage (white and brown eggs). As the rate of lethal cases at the embryonic stage was 90% (72 + 18) and was 3.6% (3.0 + 0.6) in the larval and pupal stages combined, embryonic lethality was 25-fold more frequent (90/3.6) than lethality in the two subsequent stages and 225-fold more frequent after considering the duration of the embryonic stage (1 day versus 9 days; 90/3.6 × 9). The high lethality of the progeny at the very first developmental stage, the embryonic stage, is evident.
None of the 18 studied mutations caused regular death at a larval or a pupal stage (Table 8), despite the expectation of 16 mutations (0.9 × 18). Thus, the shift in the lethal effect of conditional mutations to the stage of early embryo is evident. The absence of lethality at the larval and pupal stages is obviously not random.
All (or almost all) genes constituting the genome are involved in individual development. They are intended to function at all developmental stages and are involved in the developmental processes and their progression. Thus, irradiation is reasonably believed to disrupt genes in a random manner. In this case, the mortality rate of zygotes at a certain stage should correspond to the duration of this stage, which is observed in conventional recessive lethals . However, in our experiment, the mortality rate does not correspond to the duration of the developmental stage in the conditional lethals. We believe that this phenomenon stems from the way in which the individual developmental program is organized. The operation scheme of the program is shown in Figure 7.
Figure 7: Two scenarios of the individual developmental program constructed by ontogenes. The first scenario is implemented during gametogenesis and the second is implemented during somatogenesis. Black circles denote the ontogenes constituting the program, lines denote the functional links between ontogenes, arrows denote the directions of successive gene activation, and horizontal lines 0-5 denote successive stages in the program. The switch-on sequence in gametogenesis is ordered from 5 to 0 (red arrow goes downward) and in somatogenesis from 0 to 5 (red arrow goes upward). The red circle denotes the mutant ontogene and the red colour denotes the disturbance of the developmental program below the mutation. View Figure 7
6.2. Two reads of the individual developmental program (model)
The individual developmental program unfolds according to two successive scenarios (Figure 7). The first scenario is implemented in the germline cells and the other is implemented in the soma after fertilization. The program consists of successive stages (see Figure 5) with certain ontogenes functioning in each of the stages. In the first scenario, a successive activation of ontogenes forms the pattern of the program from level 5 to level 0. The programs constructed during the gametogenesis of parents and obtained with gametes are implemented during somatogenesis. The implementation of the program during somatogenesis proceeds in an opposite direction from level 0 to level 5. A mutation in an ontogene will interrupt the level below the caused defect. A mutation in any ontogene at levels 5 to 1 produces a functional defect of ontogene 0 and kills the zygote at the early embryonic stage (the initial stage of the first scenario).
As described above, conditional mutations are a dominant type of mutation, RNAs act as duplexes (see Section 4), and epinucleotide changes should manifest in a dominant manner. Thus, an impairment of the program in one gamete or mutual mismatch of two programs should lead to a cessation of development. However, not every defect in an ontogene causes a functional failure in the first stage of the program (0-1) and the subsequent cessation of development. For example, an ontogene that is inactivated during gametogenesis can also mutate. In this case, the mutation will not appear as a lethal mutation, but rather as a morphosis, a local developmental defect (Figure 7, right panel).
Ontogenes are encoded with the assistance of an epigenetic code, which is established during gametogenesis, is mutually matched when pronuclei are encountered in the zygote, and is implemented during the somatic portion of ontogeny. The most important part of this ontogenetic segment is the involvement of the Mendelian genes that produce proteins.
The moment when the zygote is formed in this scheme is not the initial stage, but rather the mid-ontogeny stage. This moment occurs when the first scenario is completed and switches to the second scenario. The stage at which epinucleotides are compared and epinucleotides unsuitable for further development are removed occurs at this time point. This stage consists of matching the epinucleotide developmental programs. According to the proposed scheme, lethality is shifted to the early embryonic stage because the defects at levels 5-1 are summed, and each defect prevents loading of the developmental program prior to stage 0, which is the beginning of the second scenario of individual development.
The assumed existence of an epigenetically encoded developmental program and its reload along with the existence of the individual developmental program in the form of an unchanged DNA sequence are able to provide 1) Potential developmental subprograms, for example, for different sexes; 2) Asymmetry in bilateral organisms; 3) Incomplete penetrance of conditional mutations; 4) The existence of conditional mutations; 5) The phenomenon of incomplete penetrance in general. These conditions appear to occur without any changes in the DNA nucleotide sequence.
An early study by Astaurov  noted the asymmetry of bilateral organisms, which is unexplainable in terms of classical genetics. Strunnikov  introduced the concept of "implementational variation". Currently, epigeneticists assign the listed phenomena to a broad class of phenomena referred to as epigenetic variation [42-46]. Actually, this epigenetic or epinucleotide variation is formed during the function of the genetic (in every sense of this word) system of ontogenes (multiplied ontogene regulators) and only later becomes "epigenetic" with the use of the new DNA coding language that differs from the code of nucleotide sequences.
Hypothetical discrete "factors" proposed by Mendel to explain how alternative traits are inherited with time have "transformed" into fairly material entities. Initially, "genes" were represented by chromosomal regions and later as DNA sequences encoding mRNAs and proteins. Other discrete "factors" have emerged. These factors have been identified by studying traits with variable manifestations (conditional mutations) [3,4], rather than traits manifesting in a regular manner.
In this work, we have studied the properties of conditional mutations [3,4] that directly characterize the genes responsible for these mutations, in particular 1) The parental inheritance of the properties of mutations; 2) The effects of chromosomal aberrations on the manifestation of mutations; 3) A new type of interaction between mutations, the so-called long-range interactions; 4) The phenomenon of morphoses.
The conducted analysis allowed us to generate conclusions related to ontogeny and expanding the concept of developmental genetics. Ontogenes working according to a DNA-RNA (intranuclear) script are added to known Crick's genes that follow a DNA-mRNA-protein script. Ontogenes are active in germline cells. Undoubtedly, ontogeny does not commence from the moment of syngamy, but rather from the moment when the germline cells start to develop into gametes.
Ontogenes (to which we still collectively refer to as "factors") are multiplied. Unlike Mendelian genes, ontogenes are active in some cases and are inactive in others. Thus, ontogeny is variable. Incomplete penetrance of an ontogene is a rule. Another rule is the so-called "conditionality in the manifestation" of mutations in ontogenes.
Ontogenes put into genetic practice a new genetic tool, the epinucleotide composition, which is referred to as an epigenetic conformation in modern genetic terms. The obtained data unambiguously link the epigenetic conformation to ontogenes. The epinucleotide conformation is reloaded in the germline cells with the help of ontogenes and transmitted to the zygote along with the gametic chromosome set.
The epinucleotide conformation is reloaded from the last stages of individual development to the first stages. The new biological reality is the recognition and matching of the male and female chromosome sets according to the epinucleotide conformation. Zygotic selection is a rule for each fertilization event.
In general, the genetic individual developmental program is more intricate than has been believed. This program comprises 1) Two categories of genes rather than one; 2) A specific event represented by the epinucleotide conformation; 3) The matching of the conformations in the zygote; 4) Two reads of the program (one in the germline cells and the other in somatic cells). The last process occurs concomitantly with the activation of the protein-encoding genes.
The authors thank the Federal Research Center «Institution of Cytology and Genetics of Russian Academy of Sciences» for providing financial support for this work and A.A. Fedorov for his assistance with the artwork.
- NV Timofeev-Ressovsky, VI Ivanov. Some Issues of Phenogenetics. Topical Issues of the Modern Genetics 1966;114-30.
- LI Korochkin. Biology of Individual Development. Genetic Aspect 2002.
- BF Chadov, NB Fedorova, EV Chadova, EA Khotskina. Conditional mutations in Drosophila. J Life Sci 2011;3:224-40.
- BF Chadov, NB Fedorova, EV Chadova. Conditional mutations in Drosophila melanogaster: On the occasion of the 150th anniversary of G. Mendel's report in Brünn. Mutat Res Rev Mutat Res 2015;765:40-55.
- BF Chadov. Mutations capable of inducing speciation. In: VN Stegnij, Evolution Biology 2001;Tomsk State University Press:138-62.
- BF Chadov, EV Chadova, SA Kopyl, EV Artemova, EA Khotskina, NB Fedorova. From genetics of intraspeci?c differences to genetics of intraspeci?c similarity. Russian Journal of Genetics 2004;40:945-58.
- BF Chadov, EV Chadova, SA Kopyl, NB Fedorova. A new class of mutations in Drosophila melanogaster. Dokl Biol Sci 2000;373:423-6.
- BF Chadov. Mutations in the regulatory genes in Drosophila melanogaster. Proc. Intern. Conf. Biodiversity and Dynamics of Ecosystems in North Eurasia 2000;16-8.
- BF Chadov, EV Chadova, NB Fedorova. A Novel Type of Gene Interaction in D. melanogaster. Mutation Research 2017;795:27-30.
- E Hadorn. Developmental genetics and lethal factors 1961.
- Ch Auerbach. Mutation Research, Problems, Results and Perspectives 1976.
- M Ashburner. Drosophila: A Laboratory Handbook. 1989;Cold Spring Harbor Laboratory Press.
- G Landis, D Bhole, L Lu, J Tower. High-frequency generation of conditional mutations affecting Drosophila melanogaster development and life span. Genetics 2001;158:1167-76.
- BF Chadov. Facultative dominant lethals: Genetics, ontogeny and phylogeny. In: VN Stegnij, Evolution Biology. 2002;Publishing Department Tom:2:118-42.
- BF Chadov, EV Chadova, SA Kopyl, EA Khotskina, NB Fedorova. Genes controlling development: Morphoses, phenocopies, dimorphs and other visible expressions of mutant genes. Russian Journal of Genetics 2004;40:271-81.
- BF Chadov. Ontogenes in Drosophila melanogaster: Genetic features and role in onto - and phylogenesis. Modern Problems of Genetics, Radiobiology, Radioecology and Evolution. Abstracts of the Second International Conference 2005;195.
- BF Chadov. A new stage in the development of genetics and term "epigenetics". Genetika 2006;42:1261-75.
- BF Chadov. Ontogenes in Drosophila melanogaster: Genetic features and role in onto - and phylogeny. In: VL Korogodina, A Chini, M Durante, Modern Problems of genetics, radiobiology, radioecology and evolution. 2007;80-91.
- NB Fedorova, EV Chadova, BF Chadov. Genes and Ontogenes in Drosophila: The Role of RNA Forms. Transcriptomics 2016;4:137.
- R Sager. Cytoplasmic Genes and Organelles. Foreword by BN Sidorov 1975.
- BF Chadov. Modern metaphysics and the cyclic protomodel. Eco-Potencial 2015;12:53-73.
- BF Chadov. Research Topics and List of Publications of BF Chadov 2015.
- BF Chadov, NB Fedorova, EV Chadova. Conditional Mutations in Drosophila melanogaster: Concept of Genes that Control Individual Development. Working Paper 2016.
- BF Chadov, EV Chadova, SA Kopyl, NB Fedorova. Delayed activation of the maternal genome during early development of Drosophila. 2001;78:841-5.
- NB Fedorova, EV Chadova, EA Khotskina, BF Chadov. Conditional mutations: Obtainment by the method of morphoses. In: VA Kunakh, Topics in Experimental Evolution of Organisms. 2010;78-83.
- BF Chadov. The "image" of the regulatory gene in experiments with Drosophila. Genetika 2002;38:869-80.
- BF Chadov, EV Chadova, NB Fedorova. Epigenetic phenomenology in conditional mutants of Drosophila melanogaster: morphoses and modi?cations. In: SM Zakijan, SM Vlasov, EV Dement'eva, Epigenetics. 2012;499-533.
- R Rieger, A Michaelis, MM Green. Glossary of Genetics. Classical and Molecular 1991;Springer-Verlag:553.
- BF Chadov, NB Fedorova, EV Chadova. Parental Effects of Conditional Mutations and Their Explanations. Russian Journal of Genetics 2013;49:141-50.
- M Carlson. Human embryology and developmental biology. (4th edn) 2009.
- VS Baranov, TV Kuznetsova, AA Pendina, OA Efimova, ID Fedorova, IL Trofimova. Epigenetic mechanisms of normal and pathological human development. In: SM Zakijan, SM Vlasov, EV Dement'eva, Epigenetics. 2012;225-66.
- MD Golubovsky, KA Manton. A Three-generation approach in biodemography is based on the developmental profile and the epigenetics of female gametes. Front Biosci 2005;10:187-91.
- N Ya Vaisman, MD Golubovsky. Intergeneration epigenetic effects: The effect of increased temperature on the lifespan of Drosophila melanogaster. Zh Obshch Biol 2016;77:456-63.
- BF Chadov, EV Chadova, EA Khotskina, EV Artemova, NB Fedorova. The main effect of chromosomal rearrangement is changing the action of regulatory genes. Genetika 2004;40:893-902.
- HG Callan. Lampbrush Chromosomes.1986;Springer-Verlag.
- HC Macgregor, J Varley. Working with Animal Chromosomes. (2nd edn) 1988;John Wiley & Sons.
- E Gaginskaya, T Kulikova , A Krasikova. Avian Lampbrush Chromosomes: A Powerful Tool for Exploration of Genome Expression. Cytogenet Genome Res 2009;124:251-67.
- A Fire, S Xu, MK Montgomery, SA Kostas, SE Driver, CC Mello. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-11.
- MA Matzke, JA Birchler. RNAi-mediated pathways in the nucleus. Nat Rev Genet 2005;6:24-35.
- BL Astauroff. Analyse der erblichen Stὅrungsfἅlle der bilateralen Symmmetry im Zusammenhang mit der selbstἅndigen Varialitἅtanchlicher Structuren. Ztschr f Inductive Abstammungs - und Vererbungslehre 1930;55:183-262.
- VA Strunnikov, IM Vyshinsky. Implementational variation in the silkworm. Problems in Genetics and the Theory of Evolution 1991;99-114.
- E Jablonka, MJ Lamb. Epigenetic Inheritance and Evolution: The Lamarckian Dimension. 1995;Oxford University Press.
- E Jablonka, MJ Lamb. Evolution in Four Dimensions: Genetic, Epigenetic, Behavioral, and Symbolic Variation in the History of Life. J Clin Invest 2005;115:2961
- SM Zakijan, SM Vlasov, EV Dement'eva. Epigenetics. 2012;592.
- M Meloni, G Testa. Scrutinizing the epigenetics revolution. Biosocieties 2014;9:431-56.
- SI Maletsky, VA Dragavtsev, M Meloni, G Testa, SI Maletsky. "Scrutinizing the epigenetics revolution". Polit Kontseptol 2016;1:249-54.