The conditional mutations in D. melanogaster are produced by gamma-irradiation, maintained in laboratory cultures, and inherited as gene mutations. However, their manifestation differs from the conventional mutations by several specific features. The most noticeable specific feature is their conditional nature, i.e., a conditional mutation manifests itself in the individuals of a certain genotype being silent in the individuals with another genotype. A particular procedure for mutation recovery determines what these genotypes will be. An overwhelming number of mutations are conditional dominant lethals. The viable mutation carriers display a drastically decreased fertility. Early zygotic lethality is inherited according to parental type (maternal or paternal). The carriers of conditional mutations give the offspring with a high rate of monstrosities. The possibility for the offspring to form monstrosities is inherited according to a parental (maternal or paternal) type. The level of fertility of conditional mutants is altered by chromosomal rearrangements. The chromosomal rearrangements themselves cause a decrease in fertility. Lethality of the progenies produced by the parents carrying rearrangements is inherited according to a parental (maternal or paternal) type. The results allow for a set of logical arguments in favor of that 1) the genome has a specialized system of genes (ontogenes) that control the course of individual development; 2) unlike a classical gene, acting according to the scheme DNA à RNA à protein, the ontogene implements the regulation according to the scheme DNA à RNA; and 3) the course of individual development is programmed by double-strand RNAs produced by ontogenes in germline cells.
A living organism appears owing to 1) the processes of protein, fat, and carbohydrate chemical syntheses; 2) the regulatory system that controls and provides these synthetic processes in due time and due place; and 3) self-organization of the synthesized chemical molecules and structures. As for the synthesis itself, it is known that this process is under the terms of reference of genetic system being controlled by genes. The way from DNA sequence to the molecule of a structural protein or an enzyme protein is known in every detail. However, genetic aspects of the regulatory processes and self-organization are less clear.
It has been stated at different times and with different degrees of determination that the genes that control the synthesis cannot, exclusively on their own, provide 1) the course of morphogenesis [
In the year of 2000, the mutations referred to as conditional mutations were obtained in drosophila [
Characteristic of the conditional mutations is a set of unusual earlier unknown properties. As a rule, these mutations (l) are lethal, (3) are dominant, (3) transfer the genome from a stable to an unstable state, (4) display a parental effect, and (5) elevate the basal metabolism [
The defects in individual development (morphoses and modifications) emerging in the offspring of mutants have suggested that these mutations denote the earlier unknown class of genes involved in the control of development. These genes were named ontogenes [
Several other important properties of conditional mutations have been noticed. These mutations interact with chromosomal rearrangements (inversions and translocations) [
The discovered properties of conditional mutations allow us to move on, from manifestation of mutations to the specificities of mutated genes and their role in ontogenesis. The goal of this work is to suggest a logically strict transition from the phenomenology of conditional mutations to the concept of ontogene, the specialized genetic unit responsible for regulation. We consider the parts of phenomenology of conditional mutations that allow for this transition, namely, 1) the effect of chromosomal rearrangements on the manifestation of conditional mutations; 2) the phenomenon of decreased fertility in carriers of conditional mutations; 3) a paternal form of the parental effect of conditional mutations; as well as 4) some properties of the chromosomal rearrangements that unite them with the conditional mutations.
The work has been performed with Drosophila melanogaster. The collection of conditional mutations was obtained earlier [
First, the mutations in the X chromosome were studied as conditional mutations. A “conditional” character of a mutation consisted in that it did not change the male phenotype and viability but acted as a dominant lethal in yellow/+ female [
The effect of a rearrangement can be regarded either as the effect of rearrangement itself as a permutation of chromosome blocks or as the effect of a set of alleles in the rearranged chromosome. In order to exclude the latter variant, the rearrangements were generated in the yellow control strain. This strain had earlier served for detection of conditional mutations in the X chromosome.
Male yellow flies were exposed to gamma-radiation and crossed with the y ec ct cv v f females carrying a set of mutations in the X chromosome. The heterozygous daughters were individually tested with yellow males to detect the cases when crossing over between the marker X chromosome and irradiated yellow chromosome was absent. The yellow sons were co-cultivated with the females carrying linked X chromosomes to determine the presence of a rearrangement in polytene chromosome preparations. The following rearrangements were obtained in the X chromosome of yellow strain: 1) inversion In(1)5 = In(1)6F-7AB; 19E-F; 2) inversion In(1)23 = In(1)4D-6AC; translocation T(1;2)12 = T(1;2)4F; 98D; and translocation T(1;2)19 = T(1;2)2B-C; 59C. For the experiment, the female heterozygotes carrying half autosomal set of yellow strain, normal X chromosome, and rearranged X chromosome from yellow strain were produced. The females of the above listed five strains (four strains with rearrangements and one control strain) were crossed with mutant (+) males of ten strains (nos. 1-36).
The mutant (+) males of strains nos. 1-34, carrying conditional mutations in the X chromosome were crossed to the yellow females of four genotypes:
1) y/y; +/+; +/+;
2) y/y; In(2LR)CyO/+; +/+;
3) y/y; In(2LR)Pm/+; +/+; and
4) y/y; +/+; In(2LR)D/+.
The females of these genotypes were produced in the following way. The yellow females y/y; +/+; +/+, earlier used for testing the conditional mutations in the X chromosome, were crossed with the males In(2LR)Pm/In(2LR)CyO; In(2LR)D/Sb. The males y; In(2LR)Pm/+; In(2LR)D/+ and y; In(2LR)CyO/+; In(2LR)D/+ were again crossed with the females of the initial strain y/y; +/+; +/+. The offspring of the cross contained the yellow females of all the necessary genotypes. The females differed only in the rearranged chromosomes. Females 1) were identical to those used for selection of mutants; females 2) differed from the latter by the presence of inverted chromosome CyO; females 3), by the presence of inverted chromosome Pm; and so on.
The mutation or Smba (
is dominant and lethal in homozygote. The adult males and females as well as the pupae have a shortened body. The manifestation of this mutation depends on the genotype of the strain [
The method for recovering conditional mutations in chromosome 2 is based on testing the 2/In(2LR)CyO males, carrying in the compound the irradiated X chromosome and inverted chromosome In(2LR)CyO, with the help of yellow females. The irradiated chromosome 2 with mutation (2*) survived in the 2*/In(2LR)CyO offspring and failed to survive in the 2*/2 offspring [
The signs suggesting a decrease in the male fertility in the presence of a conditional mutation became evident as early as the stage of mutant selection. The tubes with the absence of (+) daughters simultaneously contained an unexpectedly low number of yellow sons in the offspring. After the recovered mutants were brought into culture, the mutant males were specially tested for fertility.
The (+) males from each culture were individually crossed to the 3-day-old yellow females. After 3 days, the tubes bearing signs of emerged larvae were selected. The parents from these tubes were grouped to record egg laying. The egg laying continued in plug tubes with a standard medium until each tube contained 100 - 200 eggs. After at least 500 eggs per mutation were laid, the parents were removed and the tubes were placed into a thermostat at a temperature of 24˚C until emergence of imagoes. The share of the eggs that developed to the adult stage among all the laid eggs was determined.
The mortality rate of the offspring in the same cross (♀yellow × ♂+) was determined for each stage (white egg, brown egg, larva, pupa, and imago). The sample of laid ages was rather small, comprising 50 eggs.
It is not obligatory to count the laid eggs to make the inference on the fly fertility. A decrease in fertility is assessable according to the adult offspring. The absence of a certain phenotypic class or several classes in the offspring suggests a decrease in fertility as well as no offspring at all. Qualitative analysis of the offspring supplemented assessment of fertility in the carriers of conditional mutations.
1) Reciprocal Crosses with Conditional Mutations in Chromosome 3
Twelve reciprocal diallelic crosses involving four cultures of conditional mutants in chromosome 3 (in total, 24 crosses) were conducted. Each of the conditional mutations (nos. 27, 34, 46, and 55) was maintained in the heterozygote with the inversion In(3LR)Dichaete = In(3LR)71F; 85C + In(3LR)80; 84A; 93F superimposed on In(3L)69D3-E1; 70C13-D1. In the cultures of mutations, all individuals had a Dichaete phenotype. Theoretically, the offspring of two crossed cultures should comprise four phenotypic classes, namely, Dichaete males and females and Dichaete+ males and females.
2) The Response of Conditional Mutations to the Absence of the Y Chromosome in Male Genome
In the norm, a D. melanogaster male contains the Y chromosome (XY type) but also may lack it (Х0 type). The Х0 type males are sterile. The mutant sons of both types were obtained from the males carrying a conditional mutation in the X chromosome. The cross with the females C(1)DX, y w f/Y gave mutant sons XY and the cross with the females C(1)y/0, the mutant sons Х0. The shares of sons in the offspring allowed us to assess whether the presence of the Y chromosome influenced the viability of male zygote.
It is possible to obtain the mutant sons of ХY and Х0 types in another way, from the mothers carrying a conditional mutation in a heterozygous condition. Such sons were obtained by crossing the females In(1)Muller-5, wa B/+ with the y males (ХY type) and C(XY), y B/0 males (Х0 type). In this variant, the share of sons in the offspring was also determined. The data for the survival rate of the ХY and Х0 males with conditional mutations were compared to the published data on the survival of ХY and Х0 males carrying conventional mutations.
The yield of live offspring that emerged from the eggs of the pr pk cn females fertilized by the males with the flowing genotypes was estimated: 1) In(2LR)ltm3/pr pk cn; 2) In(2LR)bwv32g/pr pk cn; 3) In(2LR)B162/pr pk cn; 4) pr pk cn/F(2L); F(2R), and 5) In(2R)40/F(2L); F(2R). The males with genotypes (1)-(3) carried autosome 2 with one of the paracentric inversions In(2LR)ltm3, In(2LR)bwv32g or In(2LR)B162 and structurally normal autosome 2. The inversions have the same boundaries: 1) In(2LR)lm3 = T(2; 4)lm3 = T(2; 3)40; 60D; 102F; 2) In(2LR)bwV32g = In(2LR)40F; 59E; and 3) In(2LR)B162 = In(2LR)36E-59D. The pr pk cn females carried structurally normal autosomes 2 with visible mutations purple, prickle, and cinnabar.
The males of the last two genotypes contained metacentric autosome 2, a normal and an inverted (In(2R)40), and two arm acrocentrics, F(2L) and F(2R), which replaced the second metacentric autosome 2. The yields of viable offspring depending on the presence of the inversion In(2R)40 = In(2R)42A-57F in male metacentric autosome 2 were compared. In order to assess the yield of imagoes from the males with acrocentrics, each male was crossed to the females of four genotypes rather than a single one. The yield of imagoes was the sum of the estimates. The procedure how the total estimate was obtained is described in detail in the paper on segregation of the D. melanogaster chromosomes carrying acrocentric autosomes [
The individuals with morphoses appear in the offspring of conditional mutants [
The mutant males of wild-type phenotype (+) from 16 cultures (nos. 1-36) in the crosses with yellow females had almost no daughters in their offspring (
Male strain | Female y/y (control) | Female In(1)5, y/y | Female In(1)23, y/y | Female T(1; 2)12, y/y | Female T(1; 2)19, y/y | |||||
---|---|---|---|---|---|---|---|---|---|---|
Total number of progenies | Rate of females | Total number of progenies | Rate of females | Total number of progenies | Rate of females | Total number of progenies | Rate of females | Total number of progenies | Rate of females | |
1 | 191 | 0.00 | 169 | 0.06 | 87 | 0.01 | 138 | 0.01 | 148 | 0.03 |
2 | 435 | 0.00 | 236 | 0.12 | 76 | 0.11 | 38 | 0.05 | 173 | 0.13 |
3 | 180 | 0.00 | 469 | 0.63 | 190 | 0.43 | 128 | 0.59 | 331 | 0.58 |
4 | 293 | 0.00 | 209 | 0.08 | 162 | 0.04 | 86 | 0.00 | 213 | 0.08 |
5 | 303 | 0.02 | 107 | 0.27 | 112 | 0.01 | 46 | 0.04 | 96 | 0.22 |
6 | 283 | 0.02 | 136 | 0.23 | 106 | 0.23 | 64 | 0.00 | 112 | 0.25 |
7 | 100 | 0.00 | 154 | 0.36 | 135 | 0.21 | 13 | 0.23 | 145 | 0.39 |
26 | 89 | 0.01 | 121 | 0.24 | 210 | 0.30 | 46 | 0.02 | 69 | 0.28 |
27 | 93 | 0.00 | 123 | 0.05 | 117 | 0.02 | 79 | 0.00 | 79 | 0.06 |
29 | 61 | 0.00 | 203 | 0.49 | 122 | 0.57 | 18 | 0.00 | 128 | 0.55 |
30 | 115 | 0.00 | 142 | 0.38 | 100 | 0.17 | 93 | 0.09 | 106 | 0.19 |
31 | 83 | 0.00 | 118 | 0.19 | 123 | 0.22 | 121 | 0.04 | 195 | 0.27 |
32 | 117 | 0.00 | 183 | 0.13 | 101 | 0.19 | 80 | 0.14 | 117 | 0.35 |
33 | 90 | 0.00 | 144 | 0.34 | 123 | 0.24 | 42 | 0.17 | 100 | 0.22 |
34 | 110 | 0.00 | 115 | 0.25 | 71 | 0.07 | 31 | 0.29 | 41 | 0.15 |
36 | 110 | 0.01 | 108 | 0.25 | 127 | 0.20 | 105 | 0.01 | 323 | 0.46 |
share of daughters in the offspring of (+) males only in rare cases remained low, approaching the expected estimate of 0.5 in the majority of cases. The fact that the rearrangements in female chromosomes were produced based on the yellow strain excludes the possibility that a change in the X chromosome gene composition was the cause of the observed effect.
The mutant male of wild-type phenotype (+) from 11 cultures (nos. 1-34) in the crosses with yellow females had almost no daughters in their offspring (
The effects of chromosomal rearrangements on manifestation of the conditional mutations in the X chromosome was for the first time observed in the crosses mutant (+) males to In(1)Muller-5/y females. The offspring of these females contained the In(1)Muller-5/+ daughters and y/+ daughters [
Male mutant strain | Female y/y; +/+ | Female y/y; +/Cy | Female y/y; + /Pm | Female y/y; +/D | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Daughter+ | Son y | Daughter + | Son y | Daughter + | Son y | Daughter + | Son y | |||||||
Cy+ | Cy | Cy+ | Cy | Pm+ | Pm | Pm+ | Pm | D+ | D | D+ | D | |||
1 | ? | 230 | ? | ? | 178 | 163 | ? | ? | 107 | 57 | - | - | 115 | 8 |
2 | ? | 230 | 14 | 13 | 127 | 134 | 4 | 3 | 70 | 72 | - | - | 42 | 7 |
4 | ? | 270 | 9 | 4 | 185 | 159 | 1 | 7 | 86 | 81 | - | - | 162 | 7 |
5 | ? | 197 | 23 | 21 | 80 | 95 | 6 | 4 | 47 | 48 | - | - | 37 | 3 |
27 | 2 | 167 | 1 | 0 | 102 | 113 | 2 | 1 | 53 | 65 | - | - | 9 | 2 |
29 | 4 | 163 | 32 | 27 | 71 | 56 | 26 | 24 | 55 | 20 | 6 | 6 | 88 | 10 |
30 | ? | 184 | 15 | 13 | 81 | 76 | 9 | 12 | 60 | 47 | - | - | 38 | 6 |
31 | ? | 242 | 32 | 20 | 127 | 102 | 5 | 4 | 28 | 29 | - | - | 70 | 6 |
32 | ? | 197 | 22 | 10 | 90 | 77 | 9 | 17 | 36 | 32 | - | - | 48 | 2 |
33 | ? | 209 | 20 | 18 | 95 | 101 | 11 | 8 | 87 | 47 | 24 | 2 | 85 | 12 |
34 | ? | 140 | 11 | 14 | 88 | 101 | 25 | 20 | 68 | 54 | - | 10 | 103 | 3 |
to design a method for recovering conditional mutations in chromosome 2. According to this method, a mutation in chromosome 2 could survive only if its opposite chromosome contained the inversion In(2LR)CyO. This method allowed for recovery of eight conditional mutations in chromosome 2.
The chromosomal rearrangements In(2LR)CyO, In(2LR)Plum, and In(2LR)D in the genome of yellow females caused emergence of a previously absent class of daughters (
The described data do not embrace all obtained results concerning the effect of rearrangements on the manifestation of conditional mutations [
Male mutant strain | Progeny 2* (+) | Progeny CyBl L4 | Total number of progenies | Rate of progeny 2* (+) | ||
---|---|---|---|---|---|---|
♀+ | ♂ y | ♀ + | ♂ y | |||
7a | ? | ? | 116 | 110 | 226 | 0.00 |
37a | ? | 1 | 124 | 130 | 254 | 0.00 |
44a | ? | 1 | 125 | 109 | 234 | 0.00 |
53a | ? | ? | 101 | 130 | 231 | 0.00 |
5a | 11 | 6 | 146 | 140 | 303 | 0.06 |
8a | 6 | ? | 135 | 98 | 239 | 0.03 |
9a | 9 | 9 | 96 | 84 | 198 | 0.09 |
62a | 1 | 1 | 154 | 144 | 300 | 0.01 |
Control | ||||||
42a | 111 | 139 | 135 | 120 | 505 | 0.51 |
26a | 128 | 107 | 106 | 106 | 447 | 0.53 |
*Chromosome 2 carrying a conditional mutation.
but are sufficient to illustrate the important principles. First, the ability to influence the manifestation of conditional mutations is the common feature of chromosomal rearrangements, which alter the order of chromosome regions in the nuclear space. The rearrangements in the mutant genome possess this ability as well as the rearrangements in the partner genome. This ability distinguishes the genes that produce conditional mutations from the typical “Mendelian” genes. The latter as a rule do not respond to the presence of rearrangements in the genome. Second, the mechanisms underlying manifestations of a conditional mutation and a rearrangement should have a common link; otherwise, the mutations and rearrangements would not interact.
The major feature that gave the name to conditional mutations is their conditional nature, that is, the dependence of manifestation-non-manifestation on the genotype of an individual or its parents. The manifestation can be visible (altered phenotype of the individual) or lethal (death of offspring), the latter being prevalent. In the majority of cases, conditional mutations are conditional dominant lethals [
The fact that a mutation results the absence of a particular phenotypic class in the offspring gives the grounds to refer to it as “conditional” mutation. However, missing of a certain class is also associated with fertility. Leaving particular classes missing in the offspring and the genotypes associated with this absence out of consideration, we see that a conditional mutation in general demonstrates the phenomenon of a decrease in fertility. Thus, the conditional manifestation is converted from an exotic phenomenon of “a conditional pattern of manifestation” to a more known although also vague in its nature phenomenon of disturbance in fertility. This allows the research into conditional mutations of drosophila to be supplemented by a traditional estimation of fertility as the ratio of emerged imagoes to the laid eggs.
In total, 21 male (+) carrying a conditional mutation in the X chromosome were examined (
Another experiment showed a low fertility of the males with a conditional mutation in the X chromosome (
As is described above, a decreased fertility of conditional mutants is suggested by the absence of an expected class of progenies or a lower size of a certain class relative to the expected value.
The crosses ♀34 × ♂46 and ♀55 × ♂46 (
This suggests that
1) A decrease in fertility is the major property of a conditional mutation;
2) “Conditionality” is only a specific form of the decrease in fertility manifesting as the absence of a certain class of progenies (“selective lethality”); and
3) Selective lethality of the offspring depends on the genotypes of both parents; the deviations in the ratio of phenotypic classes of the progenies cannot be explained by the viability of the visible mutations marking the corresponding classes.
As is shown above, the decrease in fertility takes place in both sexes. In this section, the phenomenon of decreased fertility is considered for males. As will be
Male mutant strain | Cross 2 ♀ y × ♂+ | Cross 6 ♀ y × ♂+ | Male fertility* | ||
---|---|---|---|---|---|
Total number of progenies | Rate of daughters | Total number of progenies | Rate of daughters | ||
1 | 119 | 0.00 | 191 | 0.00 | 0.02 |
2 | 650 | 0.00 | 435 | 0.00 | 0.15 |
3 | 112 | 0.00 | 180 | 0.00 | 0.12 |
4 | 114 | 0.00 | 293 | 0.00 | 0.07 |
5 | 50 | 0.00 | 303 | 0.02 | 0.14 |
6 | 47 | 0.00 | 283 | 0.02 | 0.14 |
7 | 47 | 0.02 | 100 | 0.00 | ? |
9 | 182 | 0.07 | 529 | 0.00 | 0.40 |
10 | 162 | 0.03 | 297 | 0.04 | 0.09 |
27 | 68 | 0.00 | 93 | 0.00 | 0.18 |
29 | 15 | 0.07 | 61 | 0.00 | 0.14 |
30 | 122 | 0.00 | 115 | 0.00 | 0.19 |
31 | 106 | 0.00 | 83 | 0.00 | 0.15 |
32 | 81 | 0.00 | 117 | 0.00 | 0.13 |
33 | 144 | 0.00 | 90 | 0.00 | 0.16 |
34 | 88 | 0.00 | 110 | 0.00 | 0.12 |
26 | 92 | 0.03 | 89 | 0.01 | ? |
35 | 102 | 0.03 | 115 | 0.04 | 0.35 |
36 | 95 | 0.00 | 110 | 0.01 | 0.14 |
37 | 52 | 0.02 | 68 | 0.04 | 0.14 |
38 | 54 | 0.06 | 84 | 0.01 | 0.10 |
*The male fertility is determined as the ratio of the number of imagoes that emerged from the eggs laid by yellow females crossed with the mutant males to the number of laid eggs.
demonstrated in Discussion, this phenomenon plays a crucial role in determining its true cause particularly in males. A decrease in fertility of mutant males followed a parental type (a paternal form of the parental effect).
The fertility of the (+) males with conditional mutations in the X chromosome is drastically decreased (
The conditional mutations in the X chromosome distinctly respond to the absence of the Y chromosome in the male genome (
Male mutant strain | Total number of laid eggs | Lethality at the stage of (%) | Live imagoues (%) | |||
---|---|---|---|---|---|---|
White egg | Brown egg | Larva | Pupa | |||
1 | 50 | 92 | 2 | ? | ? | 6 |
2 | 50 | 81 | 13 | 2 | ? | 4 |
3 | 50 | 76 | 18 | - | ? | 6 |
5 | 100 | 65 | 28 | 3 | ? | 4 |
6 | 50 | 80 | 8 | ? | ? | 12 |
7 | 50 | 52 | 32 | 6 | 2 | 8 |
8 | 50 | 90 | 6 | ? | ? | 4 |
10 | 50 | 68 | 20 | 6 | ? | 6 |
11 | 50 | 56 | 30 | 2 | - | 12 |
27 | 50 | 72 | 8 | 6 | ? | 12 |
29 | 50 | 92 | 6 | ? | ? | 2 |
30 | 50 | 96 | 2 | 1 | ? | ? |
31 | 50 | 90 | 4 | 2 | - | 4 |
32 | 50 | 46 | 32 | 8 | ? | 14 |
33 | 50 | 50 | 28 | 6 | 2 | 14 |
36 | 24 | 33 | 54 | ? | 6 | 7 |
38 | 40 | 68 | 22 | 5 | ? | 5 |
41 | 50 | 90 | 6 | ? | ? | 4 |
Average | 51 | 72 | 18 | 3 | 0.6 | 7 |
Reciprocal crosses | Progeny | Total number of progenies | Dichaete progenies (%) | |||||
---|---|---|---|---|---|---|---|---|
Dichaete+ | Dichaete | |||||||
Females | Males | Total | Females | Males | Total | |||
♀27 × ♂34 | 49 | 47 | 96 | 20 | 13 | 33 | 129 | 25.58 |
♀34 × ♂27 | 56 | 41 | 97 | 114 | 67 | 181 | 278 | 65.11 |
♀27 × ♂46 | 132 | 147 | 279 | 34 | 31 | 65 | 344 | 18.90 |
♀46 × ♂27 | 63 | 68 | 131 | 102 | 135 | 237 | 368 | 64.40 |
♀27 × ♂55 | 88 | 158 | 246 | 29 | 28 | 57 | 303 | 18.81 |
♀55 × ♂27 | 37 | 30 | 67 | 97 | 59 | 156 | 223 | 70.00 |
♀34 × ♂46 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
♀46 × ♂34 | 73 | 95 | 168 | 109 | 95 | 204 | 372 | 54.80 |
♀46 × ♂55 | 145 | 166 | 311 | 264 | 279 | 543 | 854 | 63.60 |
♀55 × ♂46 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
♀55 × ♂34 | 0 | 0 | 0 | 81 | 65 | 146 | 146 | 100 |
♀34 × ♂55 | 0 | 0 | 0 | 114 | 91 | 205 | 205 | 100 |
*In each CDL(3)/In(3LR)Dichaete strain, only the heterozygotes for CDL(3) and In(3LR)Dichaete are viable. CDL(3) is a conditional dominant lethal in chromosome 3.
Male mutant strain | Cross: ♀C(1) y w f/Y × ♂+ | Cross :♀C(1) y/0 × ♂+ | ||
---|---|---|---|---|
Total number of progenies | Rate of XY males in the progeny | Total number of progenies | Rate of X0 males in the progeny | |
1 | 51 | 0.35 | 131 | 0.02 |
2 | 42 | 0.60 | 118 | 0.05 |
3 | 33 | 0.39 | 90 | 0.05 |
4 | 85 | 0.38 | 137 | 0.00 |
5 | 8 | 0.25 | 97 | 0.12 |
6 | 133 | 0.50 | 37 | 0.03 |
7 | 37 | 0.57 | 142 | 0.12 |
8 | 19 | 0.47 | 123 | 0.16 |
9 | 199 | 0.55 | 123 | 0.05 |
10 | 30 | 0.40 | 107 | 0.14 |
11 | 152 | 0.51 | 95 | 0.13 |
13 | 61 | 0.49 | 54 | 0.09 |
15 | 82 | 0.50 | 70 | 0.20 |
Total | 932 | x ¯ = 0.46 | 1324 | x ¯ = 0.09 |
likely will tend to zero ( x ¯ = 0.09). This pattern is characteristic of all 13 examined mutations in the case if mutant sons receive the mutant X chromosomes from their fathers (
In the case when sons receive the mutant X chromosomes from their mothers (
For simplicity of understanding of the result, the data on the mutant XY and X0 males in
The D. melanogaster males carrying recessive visible mutations in the X chromosome can be produced in both direct and reciprocal crosses of the corresponding cultures. The absence of the Y chromosome, as is known from the literature, influences only male fertility but not their viability [
Male mutant strain | Cross ♀In(1)Muller-5, wa B/+ × ♂y | Cross ♀In(1)Muller-5, wa B/+ × ♂ C(XY), y B/0 | |||
---|---|---|---|---|---|
Total number of progenies | Rate of XY, + males in the progeny | Total number of progenies | Rate of X0, + males in the progeny | ||
2 | 317 | 0.10 | 103 | 0.13 | |
3 | 122 | 0.19 | 15 | 0.13 | |
5 | 242 | 0.22 | 122 | 0.26 | |
6 | 501 | 0.15 | 156 | 0.19 | |
7 | 377 | 0.18 | 117 | 0.33 | |
8 | 363 | 0.25 | 192 | 0.30 | |
9 | 250 | 0.12 | 139 | 0.10 | |
10 | 194 | 0.23 | 182 | 0.25 | |
11 | 291 | 0.22 | 170 | 0.29 | |
29 | 285 | 0.21 | 307 | 0.11 | |
30 | 378 | 0.15 | 165 | 0.19 | |
31 | 460 | 0.17 | 121 | 0.21 | |
32 | 226 | 0.19 | 89 | 0.21 | |
33 | 162 | 0.14 | 205 | 0.22 | |
34 | 264 | 0.10 | 193 | 0.01 | |
35 | 444 | 0.08 | 184 | 0.10 | |
36 | 481 | 0.21 | 221 | 0.28 | |
38 | 504 | 0.24 | 138 | 0.24 | |
41 | 359 | 0.23 | 227 | 0.39 | |
Total | 6220 | x ¯ = 0.18 | 3046 | x ¯ = 0.21 | |
* Mutation in the X chromosome (+).
Type of mutation in X chromosome | Chromosomal type of son | Donor of X mutation | |
---|---|---|---|
Mother | Father | ||
Mendelian visible | XY | + | + |
X0 | + | + | |
Mendelian lethal | XY | ? | ? |
X0 | ? | ? | |
Conditional lethal | XY | + | + |
X0 | + | ? |
*(+) denotes the presence of sons and (?), the absence оf sons or their extremely low number.
cells. Unlike the D. melanogaster males carrying visible mutations, the males carrying recessive lethal mutations in their X chromosome do not emerge in both the direct and recessive crosses [
The conditional mutations in the laboratory collection were maintained as heterozygotes. Half offspring of a heterozygote receives the mutation and the other half does not. Nonetheless, morphoses emerged not only in the progenies that got the mutation, but also in the progenies lacking it. In the cross of a yellow female with the mutant (+) male carrying a conditional mutation in the X chromosome, the yellow sons did not get the mutant X chromosome but still part of them formed morphoses (
One of the ways to maintain conditional mutations in the X chromosome was to keep them in the females heterozygous for the inverted chromosome In(1)Muller-5, B wa. In the culture Muller-5/mutation, the females In(1)Muller-5, B wa/In(1)Muller-5, B wa (B wa phenotype) and the males In(1)Muller-5, B wa (B wa phenotype) did not carry conditional mutation; however, some of them had morphoses (
As to the chromosomal rearrangements, it has appeared that they not only alter the manifestation of conditional mutations, but also themselves act as like conditional mutations. The chromosomal rearrangements decrease fertility of the carriers, and this decrease follows a parental type. Four inversions in chromosome
2-In(2LR)ltm3, In(2LR)bwv32g, In(2LR)B162, and In(2R)40-decrease the male fertility to 81-40% of the norm (
decrease the fertility of their carriers, and this decrease takes place in the form of a parental (paternal) effect.
The results suggest several inferences on the genetic elements giving rise to conditional mutations.
Male genotype | Female genotype | Number of laid eggs | Number of emerged imagoes | Rate of imagoes | Rate of the progenies with rearrangements relative to their total number |
---|---|---|---|---|---|
In(2LR)ltm3 pr pk cn | pr pk cn | 1987 | 1066 | 48.5 ± 5.4 | 0.45 |
In(2LR)bwv32g/ pr pk cn | pr pk cn | 1990 | 1599 | 80.7 ± 2.0 | 0.50 |
In(2LR)B162/ pr pk cn | pr pk cn | 1405 | 538 | 40.5 ± 5.9 | 0.50 |
pr pk cn/F(2L); F(2R)* | 1) pr pk cn | 2195 | 1631 | 73.7 ± 3.0 | 0.50 |
2) F(2L), pr C(2R), cn | 2286 | 145 | 12.6 ± 2.2 | 0.42 | |
3) C(2L), b; F(2R), + | 2719 | 23 | 1.4 ± 1.0 | 0.52 | |
4) C(2LR)EN, c bw | 2178 | 0 | 0.0 | ? | |
Total | 87.7 | x = 0.48 | |||
In(2R)40/F(2L); F(2R)* | 1) pr pk cn | 2193 | 867 | 42.3 ± 10.0 | 0.51 |
2) F(2L), pr; C(2R), cn | 2325 | 22 | 2.0 ± 0.3 | 0.68 | |
3) C(2L), b; F(2R), + | 2078 | 56 | 5.2 ± 0.6 | 0.52 | |
4) C(2LR)EN, c bw | 2492 | 0 | 0 | 0 | |
Total | 49.5 | x = 0.51 |
*Yield of viable progeny was determined as the sum of imagoes that emerged in four crosses with various tester females.
Conditional mutations have been generated by exposure to gamma-radiation, a widely used inducer of DNA damage; they have been recovered and maintained in culture as defects in certain chromosomes. The conditional mutations display lower stability in their manifestation as compared with the conventional drosophila mutations; however, this does not interfere with their handling as mutations. The frequency of conditional mutations in the X chromosome at a radiation dose of 30 Gy taking into account a decreased penetrance amounts to 4.95% [
Six of the 80 recovered conditional mutations additionally had a visible manifestation. In laboratory cultures, the conditional mutations were maintained as heterozygotes and acted as recessive lethals [
A distinct association of the conditional mutations with DNA sequence makes them akin to classical mutations. The more drastic is their difference from the classical mutations in their manifestation. “Conditionality” is the dependence of mutation manifestation on the genetic specificity of the genome. Conditional pattern of their manifestation is not characteristic of the genes in the classical genetics. They are regarded as independent hereditary units.
Spatial factor is one of the “conditions” influencing the manifestation of conditional mutations. It has been discovered just by accident. The mutant males with a conditional mutation in the X chromosome gave no daughters in the cross with yellow females but gave daughters in the cross with the In(1)Muller-5, y wa females, also yellow in their phenotype but carrying the inversion in their X chromosome [
A decrease in the fertility of conditional mutants was noticed immediately on recovery of the first batch of such mutations [
A decrease in fertility should be recognized as the major and leading property of conditional mutations. The chromosome rearrangement, it appears, also possesses this property (
The level of fertility of the conditional mutants changes owing to additional genetic changes within a mutant genome as well as the genome changes in its partner in cross. In particular, the fertility of a male carrying a conditional lethal depends on the genotype of the female to which it is crossed (
In the classical genetics, a phene is non-alternatively “coupled” with the gene. A mutant phenotype is manifested in a cell or an individual only in the presence of the corresponding mutant gene. The inseparability of gene and phene is the basis of the classical genetics. The paternal effects of conditional mutations and chromosomal rearrangements cancel this classical statement [
The natural question arises on what is the particular factor transferred to the progenies that did not received the mutation but nonetheless simulate the presence of mutation by either a lethal effect or morphoses. The only possible answer is that 1) the genes forming the basis of conditional mutations are active in the diploid germline cells; 2) they produce regulatory products that act on the overall diploid genome; and 3) this effect is transmitted to the progeny independently of whether it got the mutation itself with the gamete or not. The germline cells house a specialized group of regulatory genes the activity of which is similar to the gene activity in the soma during organogenesis.
The genome activity in germline cells may concern only the regulatory activity. As is known, morphogenesis there is absent. The facts of formed morphoses and modifications following a parental pattern suggest the presence of a regulatory activity in germline cells. The parental effect (especially, paternal effect) irrefutable suggests that the cause is an event that takes place before the meiotic reduction division. Correspondingly, we assume that a program for successive genome activity intended for implementation in the future offspring is formed in the mitotically dividing gonial cells. Because of uniqueness of the function and action site (germline cells), the genes responsible for development of this program got a special name, ontogenes, meaning the genes that control the course of ontogenesis. Still before uniting into a zygote, the gametes of different sexes already possess the main sections constituting the program for development of the zygote. The general course of development is already determined. When the programs of the future development in gametes forming a zygote misfit, the zygotes die at the very first steps of their development.
Two chemical compounds are formed on the basis of DNA template, RNA and protein. If a protein were the regulatory product formed on the ontogene, the ontogene in its properties would not differ from a conventional gene. However, this is not the case, as is evident from the specific features of conditional mutations mentioned in Introduction and Results. Consequently, only RNA remains to be the regulatory product in question. Indeed, this does not exclude that the genome contains the regulators of a protein nature.
The inference on the role of RNA as a regulator independently follows from the data on chromosomal rearrangements. Chromosomal rearrangements influence the manifestation of conditional mutations (Results, Section 1). This effect may be implemented by RNA, for example, short RNAs, but by no means a protein. For the rearrangements to influence the regulation, the regulator should not leave the nuclei. This condition can be met if short RNAs act as such a regulator. It is reasonable to consider that the chromosomal rearrangement changes the distance between the site where RNA is produced and the site where it binds the target. The distance between the opposite alleles of the ontogene also changes. In this case, the changes in spatial arrangement of chromosome regions should influence the regulation efficiency within the nucleus.
If a protein is the regulatory product in question, the chromosomal rearrangement cannot influence the regulation. For the molecule of a regulatory protein to be formed, the corresponding mRNA has to leave the nucleus for the cytoplasm to be translated on the ribosome and then return to the nucleus to bind to the target. The procedure of exiting the nucleus and coming back to it will annul the effect of intranuclear distances. It is known that the conventional genes, with proteins as their final product, do not depend in their manifestation on the presence of rearrangements in the genome. As is evident here, three conclusions made in this work favor RNA as the regulator in question, namely, 1) the regulator should be the product of a DNA region; 2) it is not a protein; and 3) its function is to be implemented within the nucleus.
Three large data sets favor the hypothesis on existence of the ontogenes that are active in germline cells. The first set of data, cytological, is related to the lampbrush chromosomes [
The conditional mutations manifest in a variety of ways [
The existence of a dominant lethal mutation as a real laboratory culture is possible only if the mutant gene in the genome is at least repeated. Let us illustrate this as a figure (
Activation of one or another gene from the cluster depends on genotypic conditions, such as chromosomal rearrangement in the genome, sex of an individual, and specific features of the partner genome in cross [
It is also evident from
The fact that there exist the DNA regions (genes) determining the characters of the living organism forms the basis of the modern genetics. If a progeny inherits a particular DNA region from its parent, the corresponding character appears in this individual; if not, the character does not form. The procedure typical of recovery of the conventional mutations was used when dealing with conditional mutations: the conditional mutations were put in culture using a standard method; however, the corresponding DNA damages fundamentally differ from the classical genes in their manifestation.
The first distinction consists in the mode of manifestation of the mutant character. The presence of mutation in a parent is sufficient for mutation manifestation in the offspring (parental effect). A basic principle of the classical genetics―“where there is the gene, there is the phene”―is not met. The classical genetics also describes the inheritance that follows a parental pattern [
The second distinction refers to the form of parental effect. As a rule, the parental effect appears as a maternal effect [
The third distinction is again related to the form in manifestation of conditional mutations. The mutations are referred to as “conditional” according to the method used for their detection. The mutation reveals itself under conditions of a certain genotype, being concealed under conditions of another genotype. However, this is true only for the property of dominant lethality. The fact that a mutant individual has survived by no means suggests that this individual is normal. The survived mutation carriers display a set of deviations from the norm. First and foremost, this concerns the fertility of mutants. The fertility is drastically decreased and most sensitive to the changes in the genome. Conditional mutations are entitled to be termed “the mutations with variable fertility”. The conditionality of mutations in the majority of cases is just a qualitative test for a drastic decrease in fertility. This test detects a complete absence of a certain class in the offspring in a certain type of cross.
The death of zygotes in a cross, be it solitary or massive, accompanies any crosses in living nature. A genetic understanding of this phenomenon was absent for a long time despite its biological significance. However, conditional mutations allow for explanation of this phenomenon with the specific features in the function of regulatory genes (ontogenes).
The fourth distinction consists in a clear dependence in the manifestation of an ontogene on the spatial factor. As is known, a classical gene can be transferred to any distance from its original site without any noticeable loss in its function (manifestation). As it has emerged, the chromosomal rearrangements alone also decrease the fertility according to a parental type. The spatial factor, which is so necessary for the function of a living system from theoretical considerations, asserts itself in full force by the example of ontogenes.
Our results and their consideration suggest two important conclusions. First, along with the known genetic system providing the syntheses of proteins, fats, and carbohydrates intended for construction of a living organism and its function, there is the system providing genetic regulation of these syntheses. The latter system utilizes the same genetic template, DNA, and also works according to the principle of discreteness (ontogenes) but its final product is short RNAs rather than polypeptides. It is likely that this part of genome is larger than the former one and is able to solve the basic problems of designing the ontogenesis, providing for genetic similarity, and systems-based nature of all living organisms, etc., as was stated in the Introduction. This particular system with its specific features forms the background for the phenomenology of epigenetics. It is most likely that short RNAs are involved in the regulation; although the events that take place after formation of the RNA duplex are yet to be studied.
Second, the specific features in manifestation of the elements constituting this system (ontogenes) that have been discovered so far allow for understanding why this system for a long time escaped the focus of genetic research. Genetic mutations, the major tool and for a long time span the only tool of experimental genetics, are poorly suitable for the research into the regulatory part of the genome because of lethality of the mutations in regulatory genes. Genetics had to come a long way until circumventing this difficulty. A “present” of nature itself has emerged to be fundamentally important. It appeared that the regulatory system works in the way that “lethal mutations are not always lethal”.
The known mutation Curly allows for an illustration of the ontogene and its differences from a classical gene. This mutation is linked with the inversion In(2LR)Cy. As is evident from the obtained data, Curly combines the features of a classical gene mutation and an ontogene mutation. Its visible manifestation (curled wings) is the manifestation of a classical gene mutation. Every individual that receives In(2LR)Cy has the curly wings phenotype, independently of having received it from a father or a mother. The individuals that have not received In(2LR)Cy display no curly wings. However, Curly as an ontogene mutation changes fertility of its carrier, decreasing or increasing it (
The detection of ontogenes makes it possible to monitor the work of the regulatory system of the genome at the level of a whole organism, paying attention to fertility, development of morphoses and modifications, overall genome instability, and energy metabolism. The research into conditional mutations as a tool for studying ontogenes will give the insight into the pattern of damages in the main part of genomic DNA and expand our knowledge about the mutational variation of the organism as suggested by the classical genetics. The role of genetic processes in germline cells drastically increases. It cannot now be reduced to the meiotic reduction in ploidy and recombination. In each organism, the genetic program of individual development in part of its regulatory elements is checked and adjusted during the maturation of gametes.
The authors thank the Federal Research Center “Institution of Cytology and Genetics of Russian Academy of Sciences” for providing financial support for this work (budget project no. 0324-2018-0019) and A.A. Fedorov for his assistance with the artwork.