Model Organisms and Mutagenesis

#### **Chapter 5**

## Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage

*Kiran Lalwani, Caroline French and Christine Richardson*

#### **Abstract**

Maintenance of genome integrity is critical to prevent cell death or disease. Illegitimate repair of chromosomal DNA breaks can lead to mutations and genome rearrangements which are a well-known hallmark of multiple cancers and disorders. Endogenous causes of DNA double-strand breaks (DSBs) include reactive oxygen species (ROS) and replication errors while exogenous causes of DNA breaks include ionizing radiation, UV radiation, alkylating agents, and inhibitors of topoisomerase II (Top2). Recent evidence suggests that a growing list of environmental agents or toxins and natural dietary compounds also cause DNA breaks. Understanding the consequences of exposure to a broad spectrum of DSB-inducing agents has significant implications for understanding mutagenicity, genome stability and human health. This chapter will review *in vivo* mouse models designed to measure DNA damage and mutagenicity, and illegitimate repair of DNA DSBs caused by exposure to environmental agents.

**Keywords:** mutagenicity, double-strand breaks, illegitimate repair, genome rearrangement, transgenic mouse model, genome instability

#### **1. Introduction**

The faithful repair of DNA lesions is central to the maintenance of genomic integrity [1]. Illegitimate repair of chromosomal DNA breaks can lead to mutations and genome rearrangements which are a well-known hallmark of multiple cancers, aging, and disease [2, 3]. DSBs can occur in a programmed manner during a metabolic process such as DNA replication, during meiosis, and the development of the immune system during V(D)J recombination and immunoglobulin class switch recombination [4] or endogenous agents such as ROS and replication errors [5]. DSBs also occur as a result of exposure to exogenous agents such as ionizing radiation, UV radiation, alkylating agents, topoisomerase inhibitors, and chemotherapeutic drugs [6–8]. Evidence shows that a growing list of natural compounds in the human diet or the environment also causes DNA breaks [9].

Mammalian cells have evolved sophisticated mechanisms to detect the damage via the DNA damage response (DDR) and signaling pathway which then activates repair pathways to maintain genome integrity [10]. Major mammalian processes to detect

and repair DNA DSBs include homologous recombination (HR) and non-homologous end joining (NHEJ) (**Figure 1**). Both of these repair pathways are cell cycle-specific and differ based on their requirement for a donor DNA template with significant DNA sequence similarity. Studies suggest NHEJ is most prevalent in non-cycling somatic cells during the G1 stage, while HR is particularly active during the S, G2, and M stages due to its requirement for a homologous sequence as a donor template [11, 12]. In NHEJ the broken ends are processed and ligated together without

#### **Figure 1.**

*The DNA double-strand breaks (DSB) are repaired by the two pathways; These are—(A) non-homologous end joining (C-NHEJ) which modifies the ends and allows ligation of the broken ends to repair the DSB; (B) homologous recombination (HR) that uses a homologous sequence from sister chromatid or homologous chromosome or a homologous sequence within the genome.*

*Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage DOI: http://dx.doi.org/10.5772/intechopen.103929*

requiring homology. By contrast, HR uses an undamaged homologous sequence from a sister chromatid, allelic locus, or an ectopically located sequence from a heterologous chromosome as a template to initiate HR or break-induced replication repair at the broken site [13].

Laboratory mice (*Mus musculus*) have been key to most *in vivo* studies on DNA damage and mutagenicity or illegitimate repair that take into account the complex environment of the mammalian system including tissue architecture, cellular differentiation programs, chromatin landscape patterns, and aging [14–18]. Multiple *in vivo* models have been developed to examine the potential for the repair of DNA DSBs [19, 20]. Furthermore, specific cell types within tissues and organs encounter a diverse set of DNA damaging insults that produce distinct types of DNA damage. Individual cells differ in their capacity for sensing, responding, and repairing specific DNA lesions [17].

#### **2. Induction and assessment of mutagenicity by endogenous sources**

#### **2.1 Programmed DNA DSBs**

Endogenous DNA DSBs can occur as deliberate, cell-required mechanisms. DNA DSBs drive the non-sister chromatid HR events responsible for genetic diversity in meiotic cells [21]. These events can lead to rearrangements including deletions, tandem duplications, inversions, and translocation of chromosomes which are not always favorable for the cell [22]. Analogous to topoisomerase II (Top2), the Spo11 enzyme initiates DSBs during prophase I of meiosis. The locations Spo11-mediated DSBs are not random and are referred to as DNA hotspots expected to occur somewhere between 10,000 and 40,000 times within the mammalian genome [23]. These DSBs initiate meiotic HR via gene conversion and crossover events. Spo11−/− knockout mice have errors in normal meiotic chromosome synapsis formation [24]. PRDM9 methyltransferase and its associated binding specificity determine the DSB hotspot locations in mice by generating nucleosome-depleted regions, allowing for the programmed DSBs to occur via Spo11 cleavage [25].

#### **2.2 Reactive oxygen species and replication stress**

DNA DSBs can occur due to the accumulation of ROS-induced oxidative stress or as the result of replication or transcription stress. ROS are often linked to neurological diseases and cancer, although they result from endogenous cellular metabolism. Some examples of endogenous ROS include the superoxide radical anion (O2˙ − ), hydroxyl radical (OH˙), peroxynitrite (ONOO<sup>−</sup> ), and hypochlorous acid (HOCl) [26]. ROS cause DNA damage through their ability to alter the overall reductionoxidation (redox) cell conditions to cause oxidative stress. By changing redox conditions of the cell, important cellular processes including signal transduction and proliferation may not be able to occur. The failure of these processes can be lethal for the cell or promote mutagenesis through GC to TA changes [27]. Guanine lesions that lead to a miscoding error change the structural integrity of the DNA by weakening hydrogen bonding between bases [26]. These guanine mutations are associated with ROS-related oxidative stress and can promote cancer development [27]. 8-Oxo-7,8-dihydroguanine (8-oxoG) is a common output of guanine oxidation. It is an

important compound because of its susceptibility to further oxidation and overall genotoxicity [28].

The base excision repair (BER) pathway is a mechanism deployed to resolve DNA lesions, as the presence of 8-oxoG, and has three major steps: (1) recognition of the lesion by DNA glycosylases, (2) base excision, (3) resynthesis and replacement of the removed base [29]. DNA glycosylases initiate BER through cleaving the *N-*glycosidic bond between the damaged base and sugar. DNA glycosylases can be either monofunctional or bi-functional whereas bi-functional DNA glycosylases include a *β*-elimination or *β, δ*-elimination step after *N-*glycosidic bond cleavage [30]. Defects in the BER pathway's mechanism can lead to the accumulation of BER intermediates, unrepaired lesions, point mutations, and DNA DSBs. DNA polymerase *β* (Pol *β*) is one of the most active DNA polymerases involved in BER. A single nucleotide polymorphism (SNP) on the gene coding for Pol *β* results in proline residue 242 becoming arginine (P242R). This mutation is suggested to cause chromosomal aberrations, and therefore, genome instability. P242R was associated with an increase in SSBs and DSBs compared to wildtype cells, and cellular transformation in mouse and human cells. An observed increase in cellular proliferation with the expression of the P242R suggested this mutation may induce a carcinogenic phenotype [31].

Replication stress is any event causing changes to the replication rate and can include halting replication. Unrepaired DNA lesions contribute to replication stress by acting as a physical block of the replication fork and its motion [32]. Single strand breaks (SSBs) generated by replication stress can further generate DSBs by nucleases, deamination, or spontaneous hydrolysis [22]. These DSBs, as well as meiotic-related DSBs, will use NHEJ or HR for repair. Errors in HR, which are less common than in NHEJ, can lead to mutagenesis and overall genome instability [33]. Phosphorylation of target proteins by ATM also triggers DDR. Chk2 has protein kinase activity allowing it to phosphorylate several effector proteins in the cell cycle checkpoint including p53 which can be modified by either ATM or Chk2 (or ATR or Chk1). ARF protein (p14) seems to stabilize TIP60 interactions with ATM for better activation and is associated with maintaining genome stability [2].

#### **2.3 Spontaneous DNA breaks**

A reporter fluorescent yellow direct repeat (FYDR) mouse model was developed to assess DSB-induced intra-chromosomal recombination events in multiple tissues including skin [34, 35]. In this model, spontaneous DSBs or DSBs induced by replication fork collapse can lead to unequal sister chromatid exchange between tandem truncated enhanced yellow fluorescent protein (EYFP) sequences resulting in gene conversion and expression of EYFP quantifiable by flow cytometry. This model showed the *in vivo* frequency of spontaneous intra-chromosomal HR in multiple tissues calculated at approximately 10−5 to 10−6 per base pair per cell division. A modification of the model using a direct repeat-GFP (RaDR-GFP) inserted in the Rosa*26* locus contains two truncated EGFP sequences in tandem [14, 34–36]. This model detected spontaneous and DNA damage agent-induced intra-chromosomal HR in most gastrointestinal organs and respiratory organs. Cell-type-specific immunohistochemistry staining of the lung [36] and pancreata [35] demonstrated cell-type and tissue-type specificity of intra-chromosomal HR recombinant populations. This model also demonstrated that older mice show an order of magnitude increase in the accumulation of recombinant cells.

#### **3. Induction and assessment of mutagenicity by exogenous agents**

Exposure of mice to nonspecific agents such as IR, Top2 inhibitors and chemotherapeutic drugs induce DSBs more broadly across the genome and in physiologically relevant contexts (**Figure 2**).

#### **3.1 Ionizing and non-ionizing radiation**

Ionizing radiations such as X-rays and gamma rays can cause direct damage by depositing energy or indirect damage by ionization of water molecules to produce free radicals that influence SSBs or DSBs [37–40]. The complexities of the damage vary according to the linear energy transfer of the radiation [37]. Alpha particles are high LET radiation and directly cause breaks [41] while non-ionizing radiations such as UVA and UVB create indirect DSBs and SSBs [7]. Several DSB repair pathway-specific proteins have been examined on bases of the IR sensitivity such as MRE1 resection protein [42], BRCA1 [43], Ku 70 [44], and Pol θ [45–47]. Exposure of mice to irradiation can cause a variety of DNA lesions including base damage, SSBs and DSBs. However, DSBs have been deduced to be amongst the toxic lesions and contribute to cell death [37]. Erroneous repair of the DSBs causes chromosomal aberrations and influences carcinogenesis [38].

The earliest methods for detection of DSBs induced by irradiation included physical separation of the broken DNA from undamaged DNA by pulse-field gel electrophoresis and comet assays [47, 48]. However, these methods were not efficient for mouse studies because of their low reproducibility and limited approximation of DSB levels [50]. More recently, micronuclei scoring is more commonly used as these cytogenetic biomarkers are easily detectable through microscopy. Micronuclei are cytoplasmic chromatin masses resulting from damaging agents such as IR [49, 50]. Another prominent and widely used method for the detection of DSBs include the identification of DSB downstream biomarkers such as γH2AX that binds to DNA at sites flanking DSBs [51, 52]. This protein is a variant of H2AX histone and forms a focus at the sites of DSBs which further signals DDR and repair response [50, 51]. The γH2AX foci can be analyzed by immunohistochemical staining and visualization under fluorescent or confocal microscopy.

To determine the repair pathway choice of DSB repair association of DSBs with proteins specific for one pathway or another is typically employed. For example, HR requires resection of the broken DNA ends from ssDNAs that are recognized and covered by replication protein A (RPA) which can be detected through immunofluorescence. To monitor the length and speed of resection per DNA molecule, BrdU antibody is used which binds to the ssDNA and forms fibers visible under a fluorescent microscope. To increase the resolution of DNA fibers, Single-Molecule Analysis of Resection Tracks (SMART) can be used [53, 54].

#### **3.2 Radiomimetic drugs**

Commonly used chemotherapeutic drugs are categorized into 5 different types based upon their chemical composition and mode of action. Widely used anti-cancer drugs for DNA damage include alkylating agents such as temozolomide (TMZ) melphalan, and cyclophosphamide [55, 56]. These agents act by attaching the alkyl groups onto the DNA and interfering with the cell cycle and transcription process.

#### **Figure 2.**

*Exogenous exposure DNA double strand break induction, damage response pathway and repair. (A) The schematic figure shows induction of DNA damage via chemotherapeutics, radiation and environmental compounds. (B) The preliminary assessment of the DSB can be done by techniques such as comet assay, pulse electrophoresis and micronuclei staining. (C) Detection DNA damage response proteins such gamma H2AX, 53BP1 and BRCA1 foci using immunofluorescence staining. (D) Hr specific techniques such as SMART assay and brdu staining. (E) To determine repair frequencies several reporters are developed. For example, GFP recombinant cells shown in bottom right.*

They can also cross-link two double-strand DNA molecules creating inter-strand cross-links (ICLs). ICLs are dangerous lesions if not repaired. Alkylating agents can also add mismatched nucleotides which can cause genome instability [56]. Studies targeting DDR and DSB repair proteins that can alter the sensitivity of chemotherapeutic drugs are used for cancer treatment modalities. Recent research proposed that deficiency of the NHEJ protein DNA ligase4 significantly enhanced the sensitivity of cells to TMZ [57]. Mouse embryonic fibroblasts (MEFs) of DNA ligase 4 knockout mice treated with a D50 dose of TMZ have higher numbers γH2AX foci

#### *Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage DOI: http://dx.doi.org/10.5772/intechopen.103929*

and significantly reduced cell survival when compared to wild-type suggesting that Ligase4 protects the cells against lesions from TMZ [57].

ICL-inducing agents, such as mitomycin C (MMC), nitrogen mustards, and platinum can create cross-links that hinder DNA replication, thus preferentially targeting highly proliferative cells. Thus, these agents are widely used in the treatment of cancers and several skin conditions [55, 58]. The repair of ICLs involves both translesion break repair and HR proteins, and mutation of HR genes leads to sensitivity to ICL agents [59, 60]. Brca1 mutant mice ear fibroblasts and MEFs treated with MMC showed significantly reduced HR frequency and increased sensitivity to MMC. Interestingly, ATM mutant mice did not have a significant change in HR frequency even with higher MMC doses suggesting that ATM is dispensable for HR [59].

Molecular studies indicate the necessity of Top2 in the maintenance of genome integrity. The ability to halt Top2 function and generate enzyme-mediated DNA damage is a key reason why it is used in secondary cancer chemotherapy such as therapyrelated acute myeloid leukemia (t-AML) [61, 62]. Top2 enzyme acts by catalyzing the interconversion of topological DNA isomers through the generation of a transient DSB on one DNA helix ("gate" strand) while remaining covalently linked to the 5′ end of the DNA, followed by passage of a second DNA helix ("transfer" strand) through the DSB, and then religation of the DSB [63]. Mammals have two isoforms of Top2—α and β [61]. Chemotherapeutic drugs doxorubicin and etoposide inhibit the catalytic activity of Top2 after generating the DSB resulting in high levels of trapped Top2:DNA complexes and unrepaired DSBs [9, 61]. Such agents are also referred to as Top2 "poisons" [61]. A novel insight into secondary malignancies induced by these Top2 targeting drugs has come from studies using a transgenic mouse model with a skin-specific ablation of Top2β [63]. These skin-specific *top2*β-knockout mice were exposed to etoposide to evaluate the role of the two isozymes of DNA, Top2α and Top2β. The results demonstrated that in the absence of Top2β, there was a reduction in NHEJ induced by etoposide, suggesting a potential role of NHEJ repair in promoting the malignancies created by improper repair of these lesions [64, 65].

#### **3.3 Pollutants and environmental compounds**

Chemical compounds including air and water pollutants, pesticides and some dietary compounds are genotoxic and linked to carcinogenesis. Air pollutants such as benzene and sulfur oxide are released by the combustion of fossil fuels are often linked with leukemias [65, 66]. An *in vivo* study demonstrated how benzoquinone (BQ ) environmental agent-induced recombination in fetal hematopoietic cells in pKZ1 transgenic mice [67]. BQ potentially induced ROS measured by a significant increase in the ROS product 8-OH-2′-dG. This was followed by DSB induction that was detected by a significant increase in γH2AX foci in the BQ treated cells. The widely used pesticide endosulfan is speculated to cause chromosomal abnormalities in humans [68, 69]. Adult wild-type BALB/c mice fed endosulfan and analyzed for DSBs and ROS-mediated damage showed an increase in γH2AX foci and a significant increase in the levels of the NHEJ-associated protein 53BP1 in lungs and testes. Furthermore, elevations of several other proteins involved in the alternative end joining (Alt-EJ) pathway were evaluated by Western blot. This study provided compelling insight on the mechanism of action of endosulfan pesticide [69].

Bisphenol A (BPA) is a hormonally active environmental xenoestrogen widely found in food products. It is an epigenetic toxicant that can alter the DNA by the generation of ROS [70]. Bioflavonoids are polyphenolic compounds found in various dietary products such as soy, coffee, fruits, and vegetables [71]. These compounds have been characterized to be mechanistically and biochemically similar to the Top2 inhibitor and chemotherapeutic drug etoposide [72, 73]. In addition, bioflavonoids have been shown to cross the placental barrier and can induce *MLL* breakpoint cluster region cleavage suggesting an association with the initiation of infant leukemia [74]. A study reported prenatal exposure to flavonoids genistein or quercetin can increase the risk for leukemia onset, as assessed by the frequency of *MLL* translocations in an ATM mutant mouse model prone to develop cancer [75]. Prenatally exposed fetuses were examined at gestation day 14.5 by inverse-PCR to detect *MLL* translocations and their frequency in the fetal liver. Additionally, mice prenatally exposed to flavonoids genistein or quercetin were euthanized at 12-weeks and inverse PCR was performed to determine the presence of *MLL* translocations. These prenatally exposed mice developed leukemia albeit at later ages [75]. These results are further supported by an embryonic stem cell GFP-NHEJ model to identify chromosomal translocations between *MLL* and *AF9* breakpoint cluster regions analogous to those observed in infant leukemia [76]. Upon damage induced by etoposide or a large panel of flavonoids, DSBs in the two loci and repair by NHEJ produced a chromosomal translocation resulting in a functional full-length GFP at least partly dependent on Top2 [76, 77]. Another study examined epigenetic effects of genistein on hematopoiesis in mice; mice prenatally exposed to genistein showed the significant increase in erythropoiesis. Furthermore, transcriptional microarray analysis suggested that genistein exposure was associated with hypermethylation of certain repetitive elements which coincided with a significant down-regulation of genes involved in hematopoiesis in bone marrow cells and estrogen-responsive genes of genistein-exposed mice [78].

Another reporter system assesses mutagenic events through the *Escherichia coli*-derived LacZ gene, which codes for the production of *β*-galactosidase. *β*-Galactosidase cleaves lactose forming galactose and glucose, but is receptive to substrate 4-bromo-5-chloro-3-indolyl *β*-D-galactopyraniside (X-Gal) and produces blue precipitate when bound to *β*-galactosidase. The blue precipitate is observable through light microscopy [79]. Shuttle vectors carrying the bacterial reporter gene include micro-injection of bacteriophages and electroporation of plasmids for the development of transgenic mice for mutagenetic assay. Transgenic LacZ+ mice have been dosed with different mutagenic chemical compounds, like ethyl nitrosourea, chlorambucil, and benzo[*α*]pyrene, to observe changes in the production of X-Gal's blue precipitate as an indicator of mutagenicity [80]. The Mutamouse and Big Blue transgenic mouse models were developed via bacteriophages. Mutamouse utilizes bacteriophage *λ* DNA (*λ* gt10) as a vector for LacZ insertion at an *Eco*RI restriction site. Excision of the LacZ gene for analysis and a positive agar selection system is used with scoring of the clear plaques to identify mutants. Big Blue also has a *λ* bacteriophage shuttle vector for LacZ, but a non-selectable color screening assay to provide a ratio of blue plaques to white plaques and consequently a mutation frequency [81]. In the 35.5 transgenic mouse system, the LacZ transgene concameter is in a particularly unstable chromosomal region near the pseudo-autosomal region on the X-chromosome resulting in an increased potential for germinal and somatic mutations [80].

#### **4. Induction and assessment of mutagenicity by site-directed cleavage**

Nonspecific DNA damaging agents including chemotherapeutic drugs, environmental agents and radiation provide a global understanding of cell function during the response to DNA damage and DSBs. Molecular analysis of specific repair is

*Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage DOI: http://dx.doi.org/10.5772/intechopen.103929*

difficult as spontaneously occurring DNA breaks occur in unknown locations. Offtarget effects on the genome can be limited by using tools such as endonucleases and retroviruses. In addition to site-specific damage induced by specific endonucleases, a defective selectable marker or a defective fluorescent protein such as green fluorescent protein (GFP) can be added to develop a reporter system [82]. The endonuclease induces DSBs, and repair can result in a fluorescent or selectable active marker that was previously defective (**Figure 3**).

#### **4.1 Recombinase cleavage and repair reporters**

Development of conditional and inducible *in vivo* reporter assays allows for manipulation of gene expression, and molecular identification of deletion or addition of DNA sequence at specific loci. Generally, a DNA recombinase enzyme is involved in the development of conditional reporter systems. Recombinase enzymes such as Cre and FRT catalyze a concerted recombination reaction between two target sequences (loxP for Cre and FRT for FLP). Depending on the relative orientation of the target sites, catalysis results in the excision of the DNA gene sequences between the target sequences [83, 84]. A conditional Nbs1 null mouse MEF system developed with cre-lox recombinase provided insight regarding the role of the MRE11, RAD50 and NBS1 (MRN) complex with other repair proteins in DSB processing and HR. Nbs1 null MEFs treated with MMC or IR followed by Western blotting and immunohistochemistry of Brca1 and Rad51 indicated that loss of Nbs1 affected single-strand annealing via Rad51 suggesting its role in promoting HR. In support of this, PCR and Southern blotting suggested that loss of Nbs1 in an embryonic stem cell line promoted NHEJ repair [85].

#### **Figure 3.**

*Schematic of a GFP reporter system. This cassette consists of a ISceI-GFP is a modified GFP gene, which contains an 18 bp long ISceI recognition site and in-frame termination codons and a downstream a GFP donor fragment. Addition of an ISceI gene donor to this system induces DNA DSB at the ISceI site. Homologous recombination by gene conversion results in a functional GFP gene.*

#### **4.2 Endonuclease cleavage and repair reporters**

Restriction enzymes can induce site-specific DSBs with their sequence specificity to target DNA. Intron specific encoded endonuclease 1 (ISceI) derived from *Saccharomyces cerevisiae* is one of the first endonucleases used for the study of HR in mammalian cells and, subsequently, *in vivo* [82, 86].

The G2S mouse model was developed to determine the potential for DSB-induced inter-chromosomal HR repair *in vivo* [84]. This G2S mouse model was genetically engineered to contain three distinct transgenes—two non-functional green fluorescent protein (GFP) reporter transgenes and a bi-cistronic doxycycline (Dox) inducible ISceI transgene. Each GFP reporter construct contains an ISceI recognition site that renders it non-functional and provides for the induction of specific DSBs. Repair of the ISceI-induced DSBs by inter-chromosomal HR generates a functional GFP gene. While no GFP+ cells were detected without Dox (<1 × 10−8), following the addition of Dox to mouse chow or drinking water, fluorescent GFP+ cells were detected in a large spectrum of tissue types and hematopoietic progenitor cell populations visualized by fluorescent microscopy and quantitated by flow cytometry. Similar to results with RaDR mice and intra-chromosomal HR, aged G2S mice showed reduced numbers of inter-chromosomal HR cell populations [87].

Another study examined the genetic interactions between ATM, BRCA1, and 53BP1 in mice using a hypomorphic mutant, Brca1S1598F (Brca1SF) [88]. To study the role of these proteins in intra-chromosomal HR, primary fibroblasts from Brca1SF/SF mice and Atm−/− mice were integrated with a direct repeat GFP (DR-GFP) reporter and a Dox-inducible ISceI endonuclease. The DR-GFP contains a full-length nonfunctional GFP gene containing an ISceI endonuclease site followed by a downstream GFP homologous donor sequence; DSBs induced by ISceI cleavage can promote intrachromosomal HR repair to result in GFP+ cells. While spontaneous GFP+ cells were minimal (<0.01%), Dox addition to wild-type cells resulted in detection of GFP+ cells indicating HR repair (3–4%). Both Brca1SF and Atm−/− models showed a 3- and 2-fold reduction in GFP+ cells, respectively. ATM inhibition in wild-type cells only reduced HR by 1.6-fold, while ATM inhibitor exacerbated the generation of GFP+ cells in Brca1SF/SF fibroblasts as compared to wild-type and Atm−/− fibroblasts. PCR-based assay with the DR-GFP reporter was used to quantify the SSA pathway which suggested significant reduction. Interaction of Atm, Brca and 53 bp1 in HR, was demonstrated by the appearance of RAD51 foci from ear fibroblasts. Examination of triple mutants indicated the plausible role of ATM in generating end-resected intermediates for RAD51 filament formation in cells with compromised BRCA1 and 53BP [88].

A recent age-dependent study developed a knock-in R26BHEJ model to determine the efficiency of frequency of intrachromosomal NHEJ for repair. R26BNHEJ knock-in is a GFP-based NHEJ reporter inserted into the ROSA26A locus. The DSBs are created using ISceI and repair by NHEJ was analyzed in several tissues using flow cytometry. This model demonstrated that there was a 1.8 to 3.8-fold decline of NHEJ efficiency with increased age [89].

In the past two decades, new approaches of gene editing have enormously expanded mutagenesis studies. Use of artificial nuclease like zinc-finger (ZFN), transcription activator-like effector (TALEN) nuclease, and the latest clustered regularly interspaced short palindromic repeat (CRISPR)/associated (Cas9) system has enhanced precision of gene editing [90, 91]. ZFN and TALEN nucleases consist of sequence-specific DNA-binding domains that are fused to a nonspecific DNA cleavage module such as FokI endonuclease. These systems readily search for sequence

*Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage DOI: http://dx.doi.org/10.5772/intechopen.103929*

homology and the endonuclease cleaves at the recognition site, removing the target gene. Several development studies use ZFN and TALEN for gene editing [90]. A powerful approach for gene alteration is the CRISPR-Cas9 system. This system was initially observed in bacteria as an immune response against viruses. It consists of a single-guide RNA (sgRNA), that targets a palindromic region in the specific location of the genome, which is recognized by Cas9 nuclease generating a DNA DSB that subsequently activates the cellular DNA repair machinery. HR or NHEJ repair would result in alteration of the target gene by indel mutations [92, 93].

#### **5. Induction and assessment of mutagenicity** *in utero*

*In utero* studies can provide valuable insight into the physiological processes that make mammalian models unique. Although, the single-cell *Saccharomyces cerevisiae* has a large number of genes with homologs in mammals that are involved in DNA damage, signaling and repair [94], it is important to consider the mouse model's advantage to understanding DNA damage and repair in multiple organ systems that a single-cell model cannot provide. Oogenesis, embryogenesis, and spermatogenesis are processes that give valuable insight to mutagenicity because of their roles in development and meiotic recombination and their potential to lead to trans-generational mutational consequences.

#### **5.1 Gametocyte-based assays**

Understanding the mammalian recombination pathway is useful for developing mouse models that can be used to appropriately study meiotic recombination stress and DSB repair. Because knockout of MRN complex components causes embryonic lethality, conditional disruption of NBS1 has been utilized in germ cells to assess how the MRN complex is functioning during meiotic DSB repair in mice [95]. A germ cell-specific transgenic mouse model inactivates targeted gene expression utilizing *Vasa-cre* [95, 96]. In *Nbs1*flox/−*;Vasa-Cre (Nbs1 vKO)* transgenic mice, NSB1 was conditionally knocked out preceding the time in meiotic development when Spo11-mediates DSBs. In this system, male mice were infertile. Zhang *et al.* observed improper chromosome synapsis using SYCP3 and γH2AX immunostaining of spermatocytes. Immunostaining also showed nuclear localization of MRE11 in the spermatocytes was disrupted by the depleted NBS1. Development of the Nbs1 vKO transgenic mouse model allowed for the assay of NBS1 as an indicator of MRN function, and in turn, meiotic recombination stress [95].

#### **5.2 Applications**

As modern healthcare concerns center around fetal development, mouse models can be used to understand how meiotic recombination is affected by compounds in our environment. Oogenesis is particularly important because the events of meiotic prophase I are highly influential on fetal survival. An *in utero* model has been used to assess fetal exposure to supplemented estrogen and how meiotic prophase I progression is altered in response. 17-ß-estradiol (E2) was administered to pregnant mice. The meiotic outcomes were analyzed through γH2AX staining and examination by super-resolution structured illumination microscope where γH2AX presence would signify whether meiotic recombination occurred via the initiation of a DSB.

Quantifying γH2AX *in utero* is a valuable tool for assessing meiotic mutagenicity and then later influences fetal development and success [97].

*In utero* exposure to other environmental agents that cause DNA damage can be valuable for understanding carcinogenesis. The absence of the P53 tumor-suppressor gene is linked to spontaneous tumorigenesis [27, 98]. P53 knockout mice can be used as a model for assessing tumor development when exposed to cancer-causing agents. An *in utero* study evaluated the effects of high-dose vitamin E, hypothesized to have antioxidative properties, on tumorigenesis. Pregnant P53 knockout mice were fed high-dose vitamin E until gestation day 13 or gestation day 19. The addition of vitamin E altered the redox state of the *in utero* environment. Furthermore, the oxidative stress on the ROS-dependent embryonic and fetal pathways was evaluated. DNA isolation was performed for the fetal and embryonic tissues and high-performance liquid chromatography was used to quantify the formation of 8-oxo-dG which would be used as an oxidation marker. Vitamin E dosing was associated with an increase in tumorigenesis in the p53 knockout mice; however, further studies are needed to explore the relationship between vitamin E and the tumorigenesis pathway [27]. Assessing vitamins sold commercially is valuable to improving our understanding of what supplements are safe during pregnancy and how ROS may influence *in utero* cancer development*.*

Oxidative stress has important connections to ovarian aging because these ROS lesions in ovarian follicles increase with age. Oocytes remain dormant in the diplotene stage until they are released for fertilization providing time for ROS-induced oxidative damage lesions to accumulate, and an increase of these lesions in ovarian follicles with age [99]. Pol *β* and BER, a pathway for repairing DNA lesions caused by ROS and oxidative damage, have been associated with the aging process. As rats age, Pol *β* levels decline, and BER becomes less efficient [100]. Injection of small interfering RNA (siRNA) targeting Pol *β* into young murine oocytes resulted in decreased numbers of normal oocytes, reduced oocyte survival, and an increase in detectable 8-oxoG levels, as compared to controls. In a complementary study, injection of Pol *β* complementary DNA (cDNA) into aged murine oocytes resulted in overexpression of Pol *β*, increased oocyte survival, and a decrease in detectable 8-oxoG levels, as compared to controls. These studies suggest that Pol *β* function is important for oocyte survival and aging. There is a potential to apply the overexpression of Pol *β* in clinical settings to improve oocyte survival and potentially slow the damaging effects of DNA lesions on aging oocytes. This is a potentially important finding for improving fertility and pregnancy outcomes as aging signs of progress [99].

#### **6. Conclusion**

Genomic instability plays a prominent role in the initiation of pathologies such as aging, immunodeficiencies and carcinogenesis. To combat the lethal effect of DNA damage and strand breaks, cells have evolved multiple, often overlapping DNA repair pathways efficiently and accurately repair DNA. Induction and assessment of genotoxicant DNA damage are particularly important *in vivo*. Further, these mouse models to assess DNA damage and repair can be combined with traditional mouse genetics to determine the impact of genetic modifications or polymorphisms with a focus on molecular analysis of DNA damage repair. As the number of designed and widely used synthetic environmental agents increases, understanding their impact on DNA integrity and downstream potential to promote mutagenicity is increasingly significant.

*Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage DOI: http://dx.doi.org/10.5772/intechopen.103929*

#### **Acknowledgements**

CR was funded in part by NIH/NIGMS and a Faculty Research Grant (UNC Charlotte). KL was funded in part by Proposal Development Summer Fellowship (UNC Charlotte).

#### **Conflict of interest**

The authors indicate no conflict of interest.

#### **Author details**

Kiran Lalwani, Caroline French and Christine Richardson\* University of North Carolina at Charlotte, Charlotte, NC, USA

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Lalwani K, Goodenow D, Richardson C. Eukaryotic Recombination: Initiation by Doublestrand Breaks. In: Encyclopedia of Life Sciences. Hoboken, New Jersey: John Wiley & Sons, Ltd. 2020;**1**:69-76

[2] Ciccia A, Elledge SJ. The DNA damage response: Making it safe to play with knives. Molecular Cell. 2010;**40**(2):179

[3] Hoeijmakers JHJ. Molecular origins of cancer DNA damage, aging, and cancer. The New England Journal of Medicine. 2009;**361**:1475-1485

[4] Jung D, Alt FW. Unraveling V(D) J recombination: Insights into gene regulation. Cell. 2004;**116**(2):299-311

[5] Lindahl T, Barnes DE. Repair of endogenous DNA damage. Cold Spring Harbor Symposia on Quantitative Biology. 2000;**65**:127-133

[6] Wogan GN, Hecht SS, Felton JS, Conney AH, Loeb LA. Environmental and chemical carcinogenesis. Seminars in Cancer Biology. 2004;**14**(6):473-486

[7] Limoli CL, Giedzinski E, Bonner WM, Cleaver JE. UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, -H2AX formation, and Mre11 relocalization. PNAS. 2002;**99**(1):233-238

[8] Vítor AC, Huertas P, Legube G, de Almeida SF. Studying DNA doublestrand break repair: An ever-growing toolbox. Frontiers in Molecular Biosciences. 2020;**7**:24

[9] Goodenow D, Lalwani K, Richardson C. DNA damage and repair mechanisms triggered by exposure to

bioflavonoids and natural compounds. In: DNA-Damages and Repair Mechanisms. London, UK: IntechOpen; 2021. DOI: 10.5772/intechopen.95453

[10] Harper JW, Elledge SJ. The DNA damage response: Ten years after. Molecular Cell. 2007;**28**:739-745

[11] Richardson C, Jasin M. Eukaryotic Recombination: Initiation by Doublestrand Breaks. In: Encyclopedia of Life Sciences. Hoboken, New Jersey: John Wiley & Sons, Ltd. 2001;**1**. DOI: 10.1038/ npg.els.0000577

[12] Richardson C, Moynahan ME, Jasin M. Double-strand break repair by interchromosomal recombination: Suppression of chromosomal translocations. Genes & Development. 1998;**12**(24):3831-3842. DOI: 10.1101/ gad.12.24.3831

[13] Elliott B, Jasin M. Human genome and diseases: Review double-strand breaks and translocations in cancer. Cellular and Molecular Life Sciences. 2001;**59**:373-385

[14] Wiktor-Brown DM, Hendricks CA, Olipitz W, Engelward BP. Age-dependent accumulation of recombinant cells in the mouse pancreas revealed by in situ fluorescence imaging. PNAS. 2006;**103**(32):11862-11867

[15] Tratar UL, Horvat S, Cemazar M. Transgenic mouse models in cancer research. Frontiers in Oncology. 2018;**8**:268

[16] White RR, Milholland B, de Bruin A, Curran S, Laberge RM, van Steeg H, et al. Controlled induction of DNA double-strand breaks in the mouse liver induces features of tissue ageing. Nature Communications. 2015;**6**(1):1-11

*Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage DOI: http://dx.doi.org/10.5772/intechopen.103929*

[17] Simonatto M, Latella L, Puri PL. DNA damage and cellular differentiation: More questions than responses. Journal of Cellular Physiology. 2007;**213**:642-648

[18] Pandita TK, Richardson C. Chromatin remodeling finds its place in the DNA double-strand break response. Nucleic Acids Research. 2009;**37**(5):1363-1377

[19] Specks J, Nieto-Soler M, Lopez-Contreras AJ, Fernandez-Capetillo O. Modeling the study of DNA damage responses in mice. Methods in Molecular Biology (Clifton, N.J.). 2015;**1267**:413-437

[20] Hakem R. DNA-damage repair; the good, the bad, and the ugly. EMBO Journal. 2008;**27**(4):589-605

[21] Mehta A, Beach A, Haber J. Homology requirements and competition between gene conversion and breakinduced replication during doublestrand break repair. Molecular Cell. 2017;**65**:515-526

[22] Tubbs A, Nussenzweig A. Endogenous DNA damage as a source of genomic instability in cancer. Cell. 2017;**168**(4):644-656

[23] Coopera TJ, Garcia V, Neale MJ. Meiotic DSB patterning: A multifaceted process. Cell Cycle (Georgetown, Tex). 2016;**15**(1):13-21

[24] Romanienko PJ, Daniel C-OR. The mouse Spo11 gene is required for meiotic chromosome synapsis. Molecular Cell. 2000;**6**:975987

[25] Paiano J, Wu W, Yamada S, Sciascia N, Callen E, Paola Cotrim A, et al. ATM and PRDM9 regulate SPO11 bound recombination intermediates during meiosis. Nature Communications. 2020;**11**:1-15

[26] Jena NR. DNA damage by reactive species: Mechanisms, mutation and repair. Journal of Biosciences. 2012;**37**(3):503-517

[27] Chen CS, Wells PG. Enhanced tumorigenesis in p53 knockout mice exposed in utero to high-dose vitamin E. Carcinogenesis. 2006;**27**(7):1358-1368

[28] Shafirovich V, Kropachev K, Anderson T, Liu Z, Kolbanovskiy M, Martin BD, et al. Base and nucleotide excision repair of oxidatively generated guanine lesions in DNA. Journal of Biological Chemistry. 2016;**291**(10):5309-5319

[29] Marsden CG, Dragon JA, Wallace SS, Sweasy JB. Base excision repair variants in cancer. Methods in Enzymology. 2017;**591**:119-157

[30] Talhaoui I, Matkarimov BT, Tchenio T, Zharkov DO, Saparbaev MK. Aberrant base excision repair pathway of oxidatively damaged DNA: Implications for degenerative diseases. Free Radical Biology and Medicine. 2017;**107**:266-277

[31] Yamtich J, Nemec AA, Keh A, Sweasy JB. Germline polymorphism of DNA polymerase beta induces genomic instability and cellular transformation. PLoS Genetics. 2012;**8**(11):1003052

[32] Zeman MK, Cimprich KA. Causes and consequences of replication stress. Nature Cell Biology. 2014;**16**(1):2

[33] Guirouilh-Barbat J, Lambert S, Bertrand P, Lopez BS, Porro A, Chang M, et al. Is homologous recombination really an error-free process? Frontiers in Genetics. 2014;**5**:175

[34] Wiktor-Brown DM, Olipitz W, Hendricks CA, Rugo RE, Engelward BP. Tissue-specific differences in the accumulation of sequence

rearrangements with age. DNA Repair. 2008;**7**(5):694-703

[35] Sukup-Jackson MR, Kiraly O, Kay JE, Na L, Rowland EA, Winther KE, et al. Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo. PLoS Genetics. 2014;**10**(6):1-16

[36] Kimoto T, Kay JE, Li N, Engelward BP. Recombinant cells in the lung increase with age via de novo recombination events and clonal expansion. Environmental and Molecular Mutagenesis. 2017;**58**(3):135

[37] Li W, Li F, Huang Q, Shen J, Wolf F, He Y, et al. Quantitative, noninvasive imaging of radiation-induced DNA double-strand breaks in vivo. Cancer Research. 2011;**71**(12):4130-4137

[38] Thompson LH. Recognition, signaling, and repair of DNA doublestrand breaks produced by ionizing radiation in mammalian cells: The molecular choreography. Mutation Research—Reviews in Mutation Research. 2012;**751**(2):158-246

[39] Vignard J, Mirey G, Salles B. Ionizing-radiation induced DNA doublestrand breaks: A direct and indirect lighting up. Radiotherapy and Oncology. 2013;**108**(3):362-369

[40] Little JB. Genomic instability and bystander effects: A historical perspective. Oncogene. 2003;**22**:6978-6987

[41] Stanley FKT, Berger ND, Pearson DD, Danforth JM, Morrison H, Johnston JE, et al. A high-throughput alpha particle irradiation system for monitoring DNA damage repair, genome instability and screening in human cell and yeast

model systems. Nucleic Acids Research. 2020;**48**(19):e111. DOI: 10.1093/nar/ gkaa782

[42] Buis J, Wu Y, Deng Y, Leddon J, Westfield G, Eckersdorff M, et al. Mre11 nuclease activity has essential roles in DNA repair and genomic stability distinct from ATM activation. Cell. 2008;**135**(1):85-96

[43] Brodie SG, Xu X, Qiao W, Li W-M, Cao L, Deng C-X. Multiple genetic changes are associated with mammary tumorigenesis in Brca1 conditional knockout mice. Oncogene. 2001;**20**(51):7514-7523

[44] Koike M, Yutoku Y, Koike A. The defect of Ku70 affects sensitivity to X-ray and radiation-induced caspasedependent apoptosis in lung cells. Journal of Veterinary Medical Science. 2013;**75**(4):415-420

[45] Goff JP, Shields DS, Seki M, Choi S, Epperly MW, Dixon T, et al. Lack of DNA polymerase θ (POLQ ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation. Radiation Research. 2009;**172**(2):165-174

[46] Scully R, Panday A, Elango R, Willis NA. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nature Reviews Molecular Cell Biology. 2019;**20**:698-714

[47] Langie SA, Azqueta A, Collins AR. The comet assay: Past, present, and future. Frontiers in Genetics. 2015;**6**:266. DOI: 10.3389/fgene.2015.00266

[48] Kawashima Y, Yamaguchi N, Teshima R, Narahara H, Yamaoka Y, Anai H, et al. Detection of DNA double-strand breaks by pulsed-field gel electrophoresis. Genes to Cells. 2017;**22**(1):84-93

*Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage DOI: http://dx.doi.org/10.5772/intechopen.103929*

[49] Hayashi M. The micronucleus testmost widely used in vivo genotoxicity test. Genes and Environment. 2016;**38**:18

[50] Kinner A, Wu W, Staudt C, Iliakis G. Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Research. 2008;**36**:5678-5694

[51] Revet I, Feeney L, Bruguera S, Wilson W, Dong TK, Oh DH, et al. Functional relevance of the histone gammaH2Ax in the response to DNA damaging agents. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**(21):8663-8667. DOI: 10.1073/ pnas.1105866108

[52] Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Solier S, et al. γH2AX and cancer. Nature Reviews Cancer. 2008;**8**:957-967

[53] Cruz-García A, López-Saavedra A, Huertas P. BRCA1 accelerates CtIPediated DNA-end resection. Cell Reports. 2014;**9**(2):451-459

[54] Huertas P, Cruz-García A. Single molecule analysis of resection tracks. Methods in Molecular Biology. Clifton, New Jersey: Humana Press Inc; 2018;**1672**:147-154

[55] Wolf M, Eskerski H, Bauder-Wü St U, Haberkorn U, Eisenhut M. Alkylating benzamides with melanoma cytotoxicity: Experimental chemotherapy in a mouse melanoma model. Melanoma Research. 2006;**16**(6):487-496

[56] Weber GF. DNA damaging drugs. Molecular Therapies of Cancer. 2014;**12**:9-112

[57] Kondo N, Takahashi A, Mori E, Ohnishi K, McKinnon PJ, Sakaki T, et al. DNA ligase IV as a new molecular target for temozolomide. Biochemical and

Biophysical Research Communications. 2009;**387**(4):656-660

[58] Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, et al. Molecular mechanisms of cisplatin resistance. Oncogene. 2012;**31**:1869-1883

[59] Kass EM, Helgadottir HR, Chen CC, Barbera M, Wang R, Westermark UK, et al. Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**(14):5564-5569

[60] Hinz JM. Role of homologous recombination in DNA interstrand crosslink repair. Environmental and Molecular Mutagenesis. 2010;**51**:582-603

[61] Nitiss JL, Nitiss KC. Tdp2: A means to fixing the ends. PLoS Genetics. 2013;**9**(3)

[62] Nanya M, Sato M, Tanimoto K, Tozuka M, Mizutani S, Takagi M. Dysregulation of the DNA damage response and KMT2A rearrangement in fetal liver hematopoietic cells. PLoS One. 2015;**10**(12):1-18

[63] Attia SM, Al-Anteet AA, Al-Rasheed NM, Alhaider AA, Al-Harbi MM. Protection of mouse bone marrow from etoposide-induced genomic damage by dexrazoxane. Cancer Chemotherapy and Pharmacology. 2009;**64**(4):837-845

[64] Azarova AM, Lyu YL, Lin CP, Tsai YC, Lau JYN, Wang JC, et al. From the cover: Roles of DNA topoisomerase II isozymes in chemotherapy and secondary malignancies. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(26):11014

[65] Meng Z, Qin G, Zhang B. DNA damage in mice treated with sulfur dioxide by inhalation. Environmental and Molecular Mutagenesis. 2005;**46**(3):150-155

[66] Philbrook NA, Winn LM. Benzoquinone toxicity is not prevented by sulforaphane in CD-1 mouse fetal liver cells. Journal of Applied Toxicology. 2016;**36**(8):1015-1024

[67] Tung EWY, Philbrook NA, MacDonald KDD, Winn LM. DNA double-strand breaks and DNA recombination in benzene metaboliteinduced genotoxicity. Toxicological Sciences. 2012;**126**(2):569-577

[68] Fenner K, Canonica S, Wackett LP, Elsner M. Evaluating Pesticide Degradation in the Environment: Blind Spots and Emerging Opportunities. Science. 2013;**341**(6147):752-758

[69] Sebastian R, Raghavan SC. Induction of DNA damage and erroneous repair can explain genomic instability caused by endosulfan. Carcinogenesis. 2016;**37**(10):929-940

[70] Bariar B, Vestal CG, Richardson C. Long-term effects of chromatin remodeling and DNA damage in stem cells induced by environmental and dietary agents. Journal of Environmental Pathology, Toxicology and Oncology. 2013;**32**(4):307-327

[71] Ross JA, Kasum CM. Dietary flavonoids: Bioavailability, metabolic effects, and safety. Annual Review of Nutrition. 2002;**22**:19-34

[72] Skibola CF, Smith MT. Potential health impacts of excessive flavonoid intake. Free Radical Biology and Medicine. 2000;**29**(3-4):375-383

[73] Spector LG, Xie Y, Robison LL, Heerema NA, Hilden JM, Lange B, et al. Maternal diet and infant leukemia: The DNA topoisomerase II inhibitor hypothesis: A report from the children's oncology group. Cancer Epidemiology, Biomarkers & Prevention. 2005;**14**(3):651-655. DOI: 10.1158/1055- 9965.EPI-04-0602

[74] Schro¨der JP, Schro¨der-Van Der Elst S, van der Heide D, Rokos H, Morreale De Escobar G, Ko¨hrle AJ, et al. Synthetic flavonoids cross the placenta in the rat and are found in fetal brain. The American Journal of Physiology. 1998;**274**(2):E253-E256. DOI: 10.1152/ ajpendo.1998.274.2.E253

[75] Strick R, Strissel PL,

Borgers S, Smith SL, Rowley JD. Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia. Proceedings of the National Academy of Sciences of the United States of America. 2000;**97**(9):4790-4795. DOI: 10.1073/pnas.070061297

[76] Bariar B, Vestal CG, Deem B, Goodenow D, Ughetta M, Engledove RW, et al. Bioflavonoids promote stable translocations between MLL-AF9 breakpoint cluster regions independent of normal chromosomal context: Model system to screen environmental risks. Environmental and Molecular Mutagenesis. 2019;**60**(2):154-167

[77] Goodenow D, Emmanuel F, Berman C, Sahyouni M, Richardson C. Bioflavonoids cause DNA double-strand breaks and chromosomal translocations through topoisomerase II-dependent and independent mechanisms. Mutation Research—Genetic Toxicology and Environmental Mutagenesis. 2020;**849**:503144

[78] Vanhees K, Coort S, Ruijters EJB, Godschalk RWL, Schooten FJ, Doorn-Khosrovani SB van W. Epigenetics: Prenatal exposure to genistein

*Mouse Models to Understand Mutagenic Outcomes and Illegitimate Repair of DNA Damage DOI: http://dx.doi.org/10.5772/intechopen.103929*

leaves a permanent signature on the hematopoietic lineage. The FASEB Journal. 2011;**25**(2):797-807

[79] Levitsky KL, Toledo-Aral JJ, López-Barneo J, Villadiego J. Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections. Scientific Reports. 2013;**3**:2937

[80] Gossen J, de Leeuw W. LacZ transgenic mouse models: Their application in genetic toxicology. Mutation Research. 1994;**307**(2):451-459. DOI: 10.1016/0027-5107(94)90256-9

[81] Dean SW, Brooks TM, Burlinson B, Mirsalis J, Myhr B, Recio L, et al. Transgenic mouse mutation assay systems can play an important role in regulatory mutagenicity testing in vivo for the detection of site-of-contact mutagens. Mutagenesis. 1999;**14**(1):141- 151. DOI: 10.1093/mutage/14.1.141

[82] Richardson C, Jasin M. Frequent chromosomal translocations induced by DNA double-strand breaks. Nature. 2000;**405**:697-700

[83] Huh WJ, Mysorekar IU, Mills JC. Inducible activation of Cre recombinase in adult mice causes gastric epithelial atrophy, metaplasia, and regenerative changes in the absence of "floxed" alleles. American Journal of Physiology. Gastrointestinal and Liver Physiology. 2010;**299**(2):G368-G380. DOI: 10.1152/ ajpgi.00021.2010. Epub 2010 Apr 22

[84] Matos-Rodrigues G, Martini E, Lopez BS. Mouse models for deciphering the impact of homologous recombination on tumorigenesis. Cancers. MDPI AG. 2021;**13**(9):2083

[85] Yang YG, Saidi A, Frappart PO, Min W, Barrucand C, Dumon-Jones V, et al. Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA doublestrand breaks. EMBO Journal. 2006;**25**(23):5527-5538

[86] Jasin M. Genetic manipulation of genomes with rare-cutting endonucleases. Trends in Genetics. 1996;**12**(6):224-228

[87] White RR, Sung P, Vestal CG, Benedetto G, Cornelio N, Richardson C. Double-strand break repair by interchromosomal recombination: An in vivo repair mechanism utilized by multiple somatic tissues in mammals. PLoS One. 2013;**8**(12):1-16

[88] Chen CC, Kass EM, Yen WF, Ludwig T, Moynahan ME, Chaudhuri J, et al. ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair. Proceedings of the National Academy of Sciences of the United States of America. 2017;**114**(29):7665-7670

[89] Vaidya A, Mao Z, Tian X, Spencer B, Seluanov A, Gorbunova V. Knock-in reporter mice demonstrate that DNA repair by non-homologous end joining declines with age. PLoS Genetics. 2014;**10**(7):1-9

[90] Sommer D, Peters AE, Baumgart AK, Beyer M. TALEN-mediated genome engineering to generate targeted mice. Chromosome Research. 2015;**23**(1):43-55

[91] Meyer M, Hrabé De Angelis M, Wurst W, Kühn R. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. PNAS. 2010;**107**(34):15022- 15026. DOI: 10.1073/pnas.1009424107

[92] Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;**346**(6213):1258096. DOI: 10.1126/ science.1258096

[93] Shin JJ, Schröder MS, Caiado F, Wyman SK, Bray NL, Bordi M, et al. Controlled cycling and quiescence enables efficient HDR in engraftmentenriched adult hematopoietic stem and progenitor cells. Cell Reports. 2020;**32**(9):108093

[94] Botstein D, Chervitz SA, Cherry JM. Yeast as a model organism. Science (New York, N.Y.). 1997;**277**(5330):1259

[95] Zhang B, Tang Z, Li L, Lu LY. NBS1 is required for SPO11-linked DNA double-strand break repair in male meiosis. Cell Death and Differentiation. 2020;**27**(7):2176-2190

[96] Gallardo T, Shirley L, John GB, Castrillon DH. Generation of a germ cell-specific mouse transgenic Cre line. Vasa-Cre. Genesis. 2007;**45**(6):413-417

[97] Mu X, Tu Z, Chen X, Hong Y, Geng Y, Zhang Y, et al. In utero exposure to excessive estrogen impairs homologous recombination and oogenesis via estrogen receptor 2 in mice. Frontiers in Cell and Developmental Biology. 2021;**9**:1352

[98] Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA, Butel JS, et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 1992;**356**(6366):215-221

[99] Hua K, Wang L, Sun J, Zhou N, Zhang Y, Ji F, et al. Impairment of Pol β-related DNA base-excision repair leads to ovarian aging in mice.  Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase b and DNA-ligase. Journal of Neurochemistry. 2005;**92**:818-823

#### **Chapter 6**

## Conditional Mutations and New Genes in Drosophila

*Boris F. Chadov and Nina B. Fedorova*

#### **Abstract**

A new class of mutations of *Drosophila melanogaster* has been generated with the help of *γ*-irradiation and a new selection procedure; the mutations were named *conditional.* According to the data of genetic analysis, these mutations are discrete regions in DNA but are different from the Mendelian protein-coding genes. The genes associated with these mutations are named *ontogenes.* The general pattern of mutation manifestation matches the pattern characteristic of genetic incompatibility in distant hybridization. Development of monstrosities and the observed meiotic abnormalities suggest that ontogenes control the processes providing the proper spatial cell arrangement and switch-on of protein-coding genes. Ontogenes are active at all stages of the soma's life cycle and germinal tissue. In the character of their manifestation, the ontogenes correspond to the *long noncoding RNAs* in molecular genetics. The developed methods for generating mutant drosophila strains allow the manifestation and population dynamics of the mutants for this important group of genes to be studied.

**Keywords:** mutagenesis, conditional mutation, ontogene, lncRNA, drosophila

#### **1. Introduction**

A living organism is a biological system working under the control of its genetic system. This genetic system is more compact but more intricate in terms of the information content: in addition, it provides ontogenesis and phylogenesis of the organisms. Gregor Mendel founded the way of knowing for both systems: this is the way "from character to gene".

The strategy "from character to gene" has emerged to be true. The examples of the inheritance that follows the Mendelian rules are most numerous. The role of chromosomes and later, the role of DNA in heredity have become clear and the DNA code for the construction of proteins of amino acids was discovered. The Mendelian gene, coding for formation of protein, acquired the status of a universal unit of heredity and, therefore, the basic element of a living organism.

However, full-scale human genome sequencing has shown that the protein-coding genes account for only several percent of the entire genome DNA [1]. This means that genetics has so far studied in detail only a small part of the genome, whereas many fundamentally important characters were omitted. Correspondingly, the concept of the protein-coding gene as a universal basic unit of all living is not completely justified and the existence of other categories of genes cannot be excluded. It is a high time to recall the opinion of Kliment Timiryazev, a prominent biologist, on the second discovery of Mendel's rules. While praising the contribution of Mendel to the understanding of heredity, he warned that the rules for inheritance of alternative characters might appear inapplicable to the inheritance of *some other* characters of an organism [2].

The traits of intraspecific similarity, which are distinguishable in terms of taxonomy of an organism, are among these *other* characteristics. Unlike the Mendelian characters varying within a species, they display no variation. The characters of intraspecific similarity are the particular characters that come to mind when speaking about the functions and structures putatively responsible for the part of the DNA molecule that is not associated with protein-coding genes. As a matter of logic, this larger share should accommodate the genes that are responsible for the conserved characters of a living organism, i.e., the characters of species, genus, class, and so on.

This chapter describes examples of non-Mendelian genes. The classical genetic strategy (from character to gene) has been utilized by the authors in this context as well but with an eye towards the putative existence of the DNA regions with the gene properties distinct from those of Mendelian genes. Hereinafter, the Mendelian genes are regarded as the genes (1) responsible for the formation of alternative characters, (2) inherited in accordance with the Mendel's rules, and (3) coding for proteins.

The detection of a gene according to traditional hybridological procedure consists in of the detection of the variants of the corresponding trait and demonstration of a Mendelian inheritance of the variants. The primary task of the experiments on artificial mutagenesis is to find an individual with a character that distinguishes it from the norm among the offspring of the exposed organism. It is impossible to find the individual carrying a mutation in the gene responsible for a conserved trait since this mutation is a dominant lethal by definition.

As has been theoretically inferred, the lethality of conserved genes is not absolute and the genomes exist where this lethality does not manifest. The procedures searching for the mutations that are dominant lethals in one genome but not dominant lethals in another genome have been designed. The new class of mutations was named *conditional mutations* and the genes responsible for their formation and carrying them, *ontogenes* [3–5]. The name "ontogenes" results from the property of these mutations to form monstrous structures (morphoses).

#### **2. Generation of mutations in ontogenes and maintenance of mutations in culture**

#### **2.1 General scheme of approach**

By definition, the character is the property in which two objects are similar or different (the categories of similarity and distinction) [6]. The living organisms belonging to the same species carry the characters belonging to both categories. All individuals of a particular species display the characters determining the intraspecific similarity. However, some representatives of a species display the characters determining the intraspecific differences and others do not [3]. The latter category of characters is also known as the *alternative characters.* They are famous for the fact that they allowed Mendel to create his genetic theory of the living.

It is currently known that the characters of intraspecific differences at a genetic level are the variants of protein-coding genes. However, it is yet unclear how the similarity characters are organized in terms of genetics. Undoubtedly, they are also encoded in DNA and most likely represent individual DNA regions (genes); however, their arrangement and function are vague. The issues of the establishment and genetic background of the characters of intraspecific similarity are subject to the genetics of individual development and evolutionary genetics. Although a large toolkit of cytological and molecular methods is available for this these areas, the corresponding solutions are still absent.

The basic information about the characters of intraspecific differences has been obtained in the hybridization experiments currently regarded as classical. The research into the characters of intraspecific similarity could have followed the same path but it has not happened. It was believed that the invariance of the characters made it impossible to conduct genetic analysis by hybridization.

With all the uncertainty of the routes by which the similarity characters have been formed, it is doubtless that they are genetically determined. If so, the similarity in a character means that (1) the genes that determine this character are homozygous, and (2) the emerging mutant alleles are eliminated in heterozygotes. The virtual portrait of a gene responsible for a similarity character is rather specific: *the mutation in a gene is viable in a homozygote but lethal in a heterozygote.* The portrait of a Mendelian gene is opposite: the mutation in a Mendelian gene is viable in a heterozygote but may be lethal in a homozygote [7]. In order to find the genes responsible for similarity, we have searched for the unusual mutations that *would be viable in a homozygote and lethal in heterozygote.*

#### **2.2 Generating mutations in drosophila**

Drosophila is a convenient organism for the search for the above-defined mutations. The sons were obtained from the *γ*-irradiated drosophila females (**Figure 1**); part of these sons presumably carried the target mutation in the X chromosome. As was assumed, the homozygosity for the mutation in the X chromosome (males carry one X chromosome) should guarantee the viability of mutant males. All produced males were individually mated with females; the males that did not give daughters (heterozygotes for the mutation in the X chromosome) were regarded as mutant [8, 9]. The obtained mutations matched the defined requirement, namely, they were viable in males (homozygous for mutation) and lethal in females (heterozygous for mutation).

The main point in this technique is to detect the genes that are lethal in heterozygote (dominant lethals). The first batch of the mutants demonstrated that the dominant lethality of the obtained mutants was *conditional.* This lethality depends not only on the mutation itself but rather of on the genome accommodating this mutation and even on the genome of the mating partner. The mutations were named *conditional* [10] and two additional methods for their generation were proposed. In the first variant, the condition for non-manifestation of a lethal in the chromosome was an inversion in the opposite chromosome [11] and in the second, the condition for nonmanifestation of a lethal in the X chromosome was a normal genetic constitution of the mating partner [12]. Once the development of monstrosities was recorded in the mutants, we started to detect the mutants in F1 according to these monstrosities [13]. Further, having found out that the conditional mutations under permissive genetic conditions are always represented by recessive lethals;, we started to select the target

**Figure 1.**

*Detection of conditional dominant lethals in the X chromosome of* D. melanogaster*. Gamma-irradiated (30 Gy) Drosophila males were mated to females containing attached-X chromosomes. Sons of this progeny were individually crossed to* yellow *females. X-chromosome of the irradiated male is hatched. Asterisk indicates the same chromosome with mutation. In contrast to sons without lethal mutation, those that received the X with dominant lethal were daughterless.*

mutation from the our collections of recessive lethals [12]. The collection of the drosophila conditional mutations maintained in laboratory at certain times reached a hundred and more variants in the X, 2, and 3 chromosomes.

#### **2.3 Maintenance of conditional mutations in culture**

Conditional mutations were maintained in cultures, depending on specificity of each conditional mutation. Conditional dominant lethals in the X chromosome were maintained in two ways (**Figure 2**). With the first way (**Figure 2A**), the culture contained females, heterozygous for the mutation and the Muller-5 inversion. Females produced In (1)M-5 sons and "+" sons with the mutation. The latter were fertile, but no +/+ females appeared in the culture because the effect of the mutation was lethal in the homozygous females. With the second way (**Figure 2B**), the mutant X chromosome was transmitted paternally only so that females in the line contained attached-X chromosomes. Conditional recessive mutations in the X, derived from typical recessive lethals by the Muller-5 method, were maintained as typical recessive lethals in the X chromosome. Conditional dominant lethals in chromosome 2 were maintained in culture containing the In(2LR)Curly inversion. Homozygotes for every one chromosomes 2 were lethal. Conditional dominant lethals in chromosome 3 were maintained in culture containing the In(3LR)Dichaete inversion. Homozygotes for every one chromosomes 3 were lethal.

*Conditional Mutations and New Genes in Drosophila DOI: http://dx.doi.org/10.5772/intechopen.103928*

#### **Figure 2.**

*Two ways for maintenance of conditional dominant mutations in the X chromosome: (a) in heterozygous state in females containing an inverted* Muller-5 *chromosome (*In(1) Muller-5*) and the mutant X (+, solid line). Daughters* In(1) Muller-5*/+ and sons + receive the mutant X. Daughters* In(1) Muller-5/In(1) Muller-5 *and sons* In(1) Muller-5 *do not receive the mutant X. (b) in culture with attached-X chromosomes (*yvf*/*yvf*). Sons, not daughters receive the mutant X chromosome.*

#### **3. Manifestation of mutations in ontogenes**

The manifestation of mutations emerged to be numerous and diverse. Some of them are completely unexpected and fantastic, such as the development of monstrosities or changes in the basic metabolism, and others are observable although rarely in common Mendelian mutations (parental inheritance and genetic instability); however, some manifestations are well known for common mutations. The conditional mutations are described in detail in reviews [14, 15]. Here, we give only a brief description to outline these manifestations.

Most of the conditional mutations are dominant lethals. These mutations are characterized by the permissive genetic conditions (genotype) under which a dominant lethal can exist in the organism without leading to its death and the restrictive conditions (genotype) under which its manifests itself. An example is the offspring of the drosophila males carrying a dominant conditional mutation in the X chromosome (**Table 1**). The mutation has no lethal effect in the organism of males but kills the daughters that would form in the crosses of these males with *yellow* females.

In this case, the factor that saves the males from death is their gender (male). In the case of some of the generated dominant conditional mutations, the dominant lethality is eliminated by a chromosome rearrangement in the opposite homolog [11], in a nonhomologous chromosome [12], or even in the genome of the mating partner [12]. The permissive conditions remove the dominant lethality of mutation; however, *recessive lethality* remains so that the homozygotes for mutation die. Recessive lethality under permissive conditions is an obligatory attribute of conditional mutations.

The fact of a recessive lethal manifestation makes it possible to test the mutations for allelism. No alleles have been detected in the large collections of the mutations in the X chromosome (about 60 mutations) and autosome 2 (about 20). The death of mutants in a homozygous state and their survival in a heterozygote with other mutations means that conditional mutations are discrete regions of DNA molecules. Ten conditional mutations in chromosome 2 that displayed recessive lethality were

#### **Table 1.**

*Progenies and fecundity of mutant (+) males crossed to yellow females [16].*

mapped with the help of a standard set of deletions. Half mutants contained two and more lethal defects. These data suggest that the regions of multiple recessive lethality lethalities emerge in a secondary manner under the effect of the earlier formed radiation-induced mutation in ontogene [17].

The conditional mutations with a visible manifestation constitute a separate group. The *Smba* (*Small barrel*) mutation has a dominant phenotype appearing as a shortened body and short pupae. The presence of the *In(2LR)Pm* inversion in the opposite chromosome 2 removed this manifestation. The group of conditional mutations with the phenotypes *scute, radius incompletus,* and *white apricot* manifests only in females, while the corresponding males have a normal phenotype. These mutations were named dimorphic [14].

The permissive genetic conditions allow the dominant lethal mutations in heterozygote to avoid lethality. However, this does not mean that the heterozygotes become completely normal. They have an abnormally high level of locomotor activity and

#### *Conditional Mutations and New Genes in Drosophila DOI: http://dx.doi.org/10.5772/intechopen.103928*

basic metabolism. In addition, they display genetic the instability appearing as (1) activation of the mobile element Dm 412; (2) formation of visible secondary mutations; (3) development of modifications and monstrosities (morphoses); (4) loss of dominant lethal manifestation of mutation with preservation of recessive lethality; and (5) loss of the manifestation of the visible dominant mutations in the chromosome opposite to the mutant homolog [14, 15, 18, 19].

**Figures 3** and **4** show examples of modifications and morphoses in the offsprings of mutants. The share of the individuals with morphoses in the offspring of a mutant fly can reach several tens of percent [20, 21]. Because of a strong effect on ontogenesis, the genes responsible for generation of conditional mutations were named *ontogenes* [3–5]. For the sake of brevity, the mutations in ontogenes are hereinafter referred to as *ontomutations.*

A specific feature of ontomutations is that their manifestations are inherited in a parental manner. Thus, the morphoses in a heterozygote for an ontomutation emerge not only in the offsprings that received the ontomutation but also in the offsprings that have not received it [22, 23]. An example of the parental effect is evident for the ontomutations that cause the death of daughters in the crosses with *yellow* females (**Table 1**). The share of dying eggs in the cross is very high (over 50%); this suggests the death not only of the daughters that received the ontomutation, but also of part of the sons that have not received the ontomutation. Meiotic abnormalities hold a special place among all manifestations of ontomutations. This consists in of a high level of chromosome nondisjunction and loss [24] and will be separately considered below.

#### **Figure 3.**

*Modifications in the offspring of conditional mutants: (a) inserted head capsule regions in the eye); (b) a "triangle" eye; (c) defects of the eye shape; (d) narrow wings; (e) pulled apart wings; (f) reduced unspread wings; (g) altered shape of the wings; (h) altered shape of the wings with bubbles and abnormal venation; and (i) interruptions of win veins L4 and L5.*

#### **Figure 4.**

*Morphoses in the offspring of conditional mutants: (a) two heads on one neck; (b) additional head with two eyes instead of the left eye; (c) left eye of two separate fragments; (d) bifurcated tarsus of the right front leg; (e) right wing is widened and contains a bubble; (f) small process instead of the right wing; (g) two processes instead of the right wing; (h) abdomen is turned by 180°; and (i) the upper fly lacks tergites on the abdomen and the right wing is round-shaped.*

The pattern of ontomutations manifestations suggests that ontomutations are formed in the genes dissimilar to Mendelian ones. The absence of the own morphological "face" of the majority of ontomutations, the dependence of manifestation on different genetic factors, and the development of morphoses demonstrate that the main function of ontogenes is the regulatory function. However, it has emerged rather difficult in the characterization of ontogenes to advance further than the mere statement of "dissimilarity" and "regulatory character". The range of biological phenomena to be considered and understood appeared to be considerably wider as compared with the Mendelian mutations.

Eventually, it appeared possible to approach the resolution of the question on *the nature of ontogenes,* namely, on what is their biological mission, in which tissues and at which time moments they are active, and what are the forms of this activity. Find below the step-by-step theoretical analysis of the phenomenology of conditional mutations.

#### **4. Manifestation of ontogenes and distant hybridization**

Some signs resembling the abnormalities characteristic of distant hybridization were evident in the manifestation of ontomutations. It was reasonable to perform a detailed comparison since a similarity would allow ontogenes to be regarded as the genes responsible for species specificity (membership).

*Conditional Mutations and New Genes in Drosophila DOI: http://dx.doi.org/10.5772/intechopen.103928*

Distant hybridization is the cross of the individual belonging to different taxa (species, genera, families, etc.) [25]. This hybridization is accompanied by the pattern of abnormalities that is independent of a particular cross and of the kingdom to which the parents belong (animals or plants).

The pattern of abnormalities (that is, the pattern of interspecific incompatibility) comprises (1) a high sterility of the cross; (2) parental effect when producing the hybrid; (3) phenotypic mosaicism of the hybrid; and (4) meiotic abnormalities of the hybrid leading to sterility [25, 26]. Characteristic of the ontomutations that we have generated are

1.*Sterility of the cross.* Ontomutations are conditional dominant lethals. The offspring in the crosses of ontomutants can be absent in part or at all. As an example, **Table 2** shows the results of crosses between the strains carrying ontomutations in chromosome 3 [14].

The males of strain 46 in the crosses with females 34 or 55 give no offspring at all but give offspring with the females of strain 27. The cross of strains 55 and 34 gives no normal offspring but the crosses of mutants of strains 55 and 34 with other strains give normal offspring.

2.*Parental type of inheritance:.* This type of inheritance is a character of the manifestation of ontomutations. **Table 2** clearly demonstrates this effect: two pairs of ontomutations (34 and 46) and (46 and 55) give offspring in one cross direction but do not give it in the opposite direction. Ontomutations display most different forms of the parental effect, both rare in Mendelian mutations or absent at all. This comprises paternal inheritance and paternal–maternal variant

#### **Table 2.**

*Proportion of* Dichaete *progeny in reciprocal crosses of four lines* Dichaete*/mutation [27, 34, 46, 55], containing conditional mutations in chromosome 3.*

[22, 23]. All forms of paternal effects in ontogenes have been described in detail [22, 23, 27].

As is evident, the pattern of aberrations in the ontomutants is similar to that of the interspecific incompatibility. The question is what the cause of incompatibility is. The heterozygosity in Mendelian genes cannot be the cause of incompatibility because heterozygosity does not lead to lethality in an intraspecific hybridization; moreover, it frequently leads to heterosis. In addition, the mutations in Mendelian genes do not

#### **Figure 5.**

*Mosaics in strains with conditional mutations: (a) the left half of the abdomen is gray the right half, yellow; (b) sex comb is present only on the right front leg; (c) eyes of different colors in the offspring of a* w<sup>a</sup> /+ *female; (d) colorless left half of last tergites; (e) left half of the abdomen of a female type color and, right, of a male type; (f) different shapes of eyes in the offspring of a B/+ female; (g) as spot of red ommatidia on the background of white ommatidia; (h) yellow left wing and part of the thorax of a gray fly; and (i) right half of the thorax and scutellum are hairless and have no bristles.*

#### **Table 3.**

*The effect of mutation in ontogene on the X chromosome nondisjunction in drosophila female meiosis [24].*

interfere with meiosis and the corresponding mutants are viable even in compounds with deletion. It is clear that *the heterozygosity in Mendelian genes cannot be responsible for interspecific incompatibility. Correspondingly, the cause underlying the incompatibility is the heterozygosity in the genes that determine the species membership.* In their native genome, these genes are in a homozygous state and thus properly fulfill their role.

The similarity between the manifestations of ontomutations and the pattern of interspecific incompatibility in distant hybridization suggests that (1) the ontogenes belong to the group of the genes responsible for intraspecific similarity and (2) an unusual phenomenology of ontomutations results from their heterozygosity for ontogenes. The latter is similar to the heterozygosity in distant hybridization but is reached in another way. We generate ontomutations with the help of mutagenesis and get heterozygotes for an ontogene by combining them with an initially normal ontogene. Indeed, it is necessary to take into account that all genes responsible for the species membership in an interspecific hybrid are in a heterozygous state versus only one gene (ontogene) in the experiments with ontomutations.

The observed similarity to the pattern of interspecific incompatibility considerably simplifies the understanding of the role of ontogenes in the organism. Any "incompatibility" does not exist for the Mendelian genes and the phenomenon of interspecific incompatibility is determined by the conflict of the genes that form the species specificity of organisms rather than the Mendelian genes. The ontogenes belong to the former group of genes. It is useful to recreate in mind the pattern of incompatibility in distant hybridization to enhance the understanding of the role of ontogenes. Incompatibility is the result of heterozygosity for ontogenes.

#### **5. Ontogenes and construction of cell ensembles**

The biological mission of ontogenes was clarified when studying the phenomenon of development of monstrosities (morphoses) in the offspring of an ontomutant (**Figures 6** and **7**). In genetic literature, morphosis is defined as a nonadaptive and typically unstable variation of individual morphogenesis associated with a change in the external environment [28–31]. Here, the term morphosis is used to designate the nonheritable morphological abnormalities caused by specific genetic features of the

#### **Figure 6.**

*The morphoses of the "plus tissue" type (surplus 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) and 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.*

parent itself rather than by the external conditions; correspondingly, they may be referred to as "*endomorphoses*" unlike the earlier known "*exomorphoses*" [21].

The morphoses emerging in conditional mutants are the abnormalities of different degrees of manifestation. Most of them do not prevent flies to hatch from pupae, live, mate, and even give giving offspring. An experimenter working with drosophila for a sufficiently long time has undoubtedly encountered the cases of morphosis development but such cases are very rare. However, morphoses frequently emerge in the offspring of the generated conditional mutants [20, 21]. Soon after commencement of the work with conditional mutations, the collection of colored images of morphoses became very large (about 1000). The diversity and morphological complexity of morphoses are great [32]. The morphological defects are also characteristic of Mendelian mutations but the latter are is incomparably simpler.

The asymmetry of morphoses is the decisive phenomenon in the understanding of the role of ontogenes. *Unlike a bilaterally symmetric morphological defects caused by Mendelian mutations, morphoses are asymmetric:* as a rule, they are present on one side of the body (left or right) [33]. The bilateral asymmetry can be certainly regarded as a cell-level phenomenon. The asymmetry results from an incorrect spatial arrangement of the cells formed by division. Thus, it turns out that ontogenes do control the growth of embryo, its size, and spatial symmetry; moreover, the defects in ontogenes (ontomutations) make asymmetric the normally symmetric structures. The Mendelian genes control production of proteins in cells but do not control the arrangement of

#### **Figure 7.**

*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 cap-sule and 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 lift wing and cone-like stretched left thorax.*

cells. That is the reason why Mendelian genes do not interfere with a bilateral symmetry [33].

The involvement of ontogenes in cell spatial arrangement is confirmed by the meiotic abnormalities in ontomutants. As is shown in Section 3, the ontomutations in a heterozygote significantly interfere with the normal meiosis. As is known, the heterozygotes for Mendelian mutations have normal meiosis [34]. Correspondingly, it is reasonable to assert that ontogenes control cell division (in this case, meiotic division) and Mendelian genes do not. Summing up, *the phenomenon of asymmetry of morphoses together with the phenomenon of disturbed meiosis in ontomutants suggests that ontogenes are actually responsible for the construction of cell ensembles.*

It is valid to regard that the "key players" in ontogenesis are now found: they are the ontogenes and Mendelian genes. The former (ontogenes) control the construction of cell ensembles, while the latter controls the production of protein sets in the cells forming the ensembles. To make the picture complete, it is logical to assume that ontogenes also switch on the Mendelian protein-coding genes. The patterns of morphoses in the individuals carrying ontomutations together with mutations in Mendelian genes confirm this assumption.

Consider an example when an additional small head has developed in a fly at the place of the right eye because of a mutation in ontogene (**Figure 8**). Since ontogenes switch on Mendelian genes, the mutant for the Mendelian mutation *Bar* displays the *Bar* phenotype not only for the normal left eye, but also for the aberrant right eye on the newly formed additional small head. It is evident from the available large

#### **Figure 8.**

*Morphoses and Mendelian mutation* Bar*. 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.*

collection of morphosis images that although the monstrosities are manifold and unusually located, the traits in morphoses that are definitely controlled by Mendelian genes (color of cuticle, eye color, and bristle color) are "switched on" correctly and perfectly fit the fly's genotype. This "adjustment" of the Mendelian genes to the morphological structures despite their pathologies suggests that the event of the switch-on of the structures is automatically the event of the switch-on of a certain set of Mendelian genes.

The discussion of the mechanisms underlying ontogenesis after the works by Jacob and Monod [35] necessarily includes the idea of the regulator genes that trigger the structural genes. It is believed that the regulator genes belong to the category of protein-coding genes. Our data do not contradict the existence of protein regulators but suggest ontogenes as the key players in the organization of ontogenesis. Ontogenesis is not only the production of proteins, but also the *production of the array of cells* housing the production of proteins and ontogenes there are involved in the production of the cell array.

#### **6. Activity of ontogenes in different tissues and at different developmental stages**

The Mendelian genes are active in the soma from the very beginning of somatogenesis and to the end of life. According to the experiment, ontogenes are also active in: (1) the germline before meiosis (in premeiosis), (2) during meiotic divisions, and (3) in the zygote at the stage of synkaryon formation.

*Premeiosis in germinal tissue:.* A half of the offspring of a parent heterozygous for an ontomutation receives the mutation and the other half does not. However, the overwhelming majority of manifestations of ontomutations are observed in the entire offspring. This is true for the emergence of morphoses [22, 23], lethal effect of ontomutations [27], the effect of a chromosome rearrangement on the lethal effect of ontomutation [12, 36], the effect of the Y chromosome on the lethal effect of

ontomutation [16], the effect of ontomutation on nondisjunction [24], and so on. All these cases of parental (maternal or paternal) inheritance mean that the mutant ontogenes are active in germline cells. The activity consists in the formation of the "factors" (it is not important which particular factors) that lose a physical link with the ontomutation (DNA region) whereby they originated. As a consequence, these factors after the reduction division equiprobably find themselves in both the gametes carrying ontomutation and the gamete lacking it.

*Meiotic division:.* Various meiotic abnormalities caused by ontomutations suggest that ontogenes are active in meiosis (see Section 3 and [24] for comprehensive description).

*Synkaryon formation.* The activity of ontogenes at this stage can be referred to as "the recognition of mating partner" [36]. The *yellow* females do not give daughters in the crosses with the males carrying an ontomutation in the X chromosome (**Table 4**). The prohibition for the presence of daughters in the offspring is removed if the females carry the Cy, Pm, or D inversion in autosomes 2 and 3. It is important that not only the daughters carrying the Cy, Pm, or D autosomes start appearing in the offspring but also the daughters without them. We have assumed that some *tags* appear on the chromosomes of female and male sets during the development of both the female and male gametes of ontomutants as early as the premeiosis. When the chromosome sets enter the zygote, the tags are compared and ontogenesis is triggered in the case the sets display similarity and does not in the absence of similarity [22, 23]. The zygote of drosophila dies at the stage of egg [22, 23]. Formally, this pattern is similar to that when the meeting of pronuclei is prevented, which is observed in genetic incompatibility in plants and protozoans [37, 38].

**Table 4.**

*Effect of rearranged chromosome 2 and 3 on dominant lethality of conditional mutations in the X chromosome delivered to the zygote together with sperm {cross of mutant males to females: 1)* y/y*; +/+; 2)* y/y*;+/* In(2LR)Cy*; 3)* y/y*;+/* In(2LR)Pm *and 4)* y/y*; +/* In(3LR) D*} [14].*

*Ontogenesis of the soma.* The development of morphoses suggests that ontogenes are active at this stage of individual development (see Section 5). A parental type of inheritance of these aberrations [22, 23] indicates that the genetic events in gonial cells are involved in their induction.

As is evident from the list of activities, ontogenes outdo the Mendelian genes in temporal and spatial parameters of their activity. The activity of ontogenes in germinal tissue, where Mendelian genes are inactive, is quite a surprise. The activity of ontogenes at different stages allows for the explanation of an intricate pattern of the ontomutation manifestations. For example, the combination of conditional dominant lethality with definite recessive lethality, illogical at a first glance, is explainable with that the former manifests itself during synkaryon formation and the latter, in the premeiosis of germinal tissue. The activity of ontogenes in the germline for the first time explains the radiation effects appearing as sterility and emergence of mutations in F1 [39]. The observed activity of ontogenes in the germinal tissue puts the question on the forms of activity of ontogene DNA: a typical form of gene activity is coding for protein synthesis; however, no protein synthesis has been recorded in the germinal tissue.

#### **7. Forms of activity of ontogenes**

*Activity as nRNA formation.:* The chromosome rearrangements of inversion and translocation types interact with ontomutations [12]. The rearrangements themselves act as ontomutations decreasing fertility according to the parental effect [12]. The parental effect suggests the gene activity in the premeiotic cells of the germline. Thus, we may state that a certain chromosome rearrangement in these cells is active and the change in the activity of ontogenes is the result of its presence. Any rearrangement changes the distance between individual ontogenes. If the ontogenes in these cells "communicate" via nRNA, the change in the distances between ontogenes will quite expectedly lead to a change in the function. The lengths of the ways an nRNA have has to cover from an ontogene to another ontogene in a normal genome and in the genome carrying a rearrangement are different. Thus, nRNA can be a regulator of ontogene activity in the premeiotic germline cells.

Usually, proteins act as regulators of gene activity; however, a protein cannot act as a regulator of ontogene activity in germline cells in the case of a rearrangement. The schemes of regulation with the help of a protein and an nRNA are considered in a separate paper [5]. The way of a protein regulator have has to cover in this case (DNA–mRNA–ribosome–protein–DNA) is too long and passes through the cytoplasm. Such regulator will be unable to respond to the minor changes in the distances between ontogenes in the nucleus caused by a rearrangement. On the contrary, an immediate regulation of an ontogene by another ontogene with the help of an nRNA is feasible. All events (synthesis of nRNA and migration of nRNA) and all players (inducer ontogene and receptor ontogene) in this case reside within the nucleus ([5], **Fig. 6**). Thus, the most likely regulators of ontogenes are nuclear noncoding RNAs (ncRNAs). In this case, ontogenes act as both ncRNA inducers and ncRNA recipients.

The recent studies on genome-wide annotation utilizing high-throughput transcriptomics from a single- cell embryo to differentiated tissue cell types demonstrate that over two-thirds of the transcribed mammalian genome codes for tens of thousands of different classes of small and long noncoding RNAs (lncRNAs). The lncRNAs form the largest class of ncRNA subtypes. According to some recent

estimates, there exist over 58,084 transcripts in the mammalian genome, which is larger than the number of protein-coding RNAs. In addition, lncRNAs appear to be key regulators in a wide range of biological processes, including cell proliferation, cell cycle, metabolism, apoptosis, differentiation, and pluripotency [40, 41].

It has become clear over the period from generation of the first batch of conditional mutations in *Drosophila melanogaster* in 2000 [8, 9] and a shorter time interval when lncRNA genes were studied [42, 43] that their biological functions are analogous. Both (1) are not protein-coding genes but control the operation of the latter; (2) are elements of the conserved part of the genome; (3) control the progression of ontogenesis and (4) phylogenesis; (5) are responsible for energy exchange in the organism; (6) control cell division; and (7) are inherited according to a parental type. Thus, these two groups most likely represent the same category of genes.

*Conformation (coiling and remodeling) of DNA of ontogenes.* The fact of a drastic disturbance of cell meiotic division in the presence of an ontomutation has been demonstrated (Section 3). If so, the ontogenes in meiosis are active even taking into account that the chromosomes in a meiotic cell are highly compacted. Thus, the activity is guided by highly compacted DNA of an ontogene and the parental effect on nondisjunction [24] suggests that this coiling "originated" from the premeiotic germline cells.

The previous section discusses the interaction between ontogenes in the zygote, when the parental chromosome sets meet after fertilization [36]. The parental chromosome sets are also highly compacted. The situation there is the same: the ontogenes are active although they are highly compacted. These two facts suggest that *ontogene is a DNA sequence in a state of regulated coiling.* A valid argument favoring this assumption has been earlier obtained by theoretical analysis of the pairing in a heterozygote for inversion [44].

The resulting conclusion focuses the attention on the studies that demonstrate the activity of heterochromatin blocks. Keeping in mind to do this large work in the future, see some studies indicating an important role of heterochromatin in the chromosome behavior in meiosis [45–49]. It cannot be excluded that the multilocality of some ontogenes that we have discovered by deletion mapping of ontomutations in chromosome 2 [17] is explainable with that the ontogenes are represented by coiled repeats. The pattern of somatic pairing in the regions of lncRNAs Firre in different chromosomes suggests the same inference [50].

*Biophysical aspect of ontogene activity.* The activity of ontogenes coming from compacted chromosome regions suggests that the mutual spatial arrangement of the DNA regions belonging to ontogenes is functionally significant. The studies into the effect of lncRNAs on DNA remodeling [51–53] confirms this. Having commenced the work with mutations in ontogenes, we encountered the cases of interaction of the ontogenes separated by considerable distances [44, 54, 55]. The simplest case is the interaction of the ontogenes that leads to the pairing of homologs in meiosis [44]. Note that the DNA of ontogenes in this process is in a coiled state. It is logical to assume that the forces emerging as a result of coiling of lncRNA regions are the factor that brings the homologs together, however, the mechanism of action of this factor is not clear. The new genes, which undoubtedly exist, fulfill the functions that cannot be implemented by Mendelian genes. Unlike the Mendelian genes, responsible for de novo protein synthesis, ontogenes control the template-based reproduction of DNA molecules as well as the reproduction of the cell itself via its division. In that case, the new genes must possess the capabilities that the Mendelian genes lack. Otherwise, the Mendelian genes themselves could cope with this task.

#### **8. Ontogenes and problems in genetics**

Currently, the ontogene, similar to the gene in the early days of genetics, is still hypothetical. The particular solutions will appear in the experiments; however, theoretical studies are also necessary. The specific feature of the moment is in that the concept of ontogene is introduced after a long period when the concept of gene represented by a protein-coding gene variant is a sole (universal) hereditary unit. The possible existence of other kinds of genes besides the Mendelian genes have been asserted by de Vries [56], Filipchenko [57], Timiryazev [2], Timofeev-Ressovsky in his first interpretation of the mutations with a varying manifestation [58], and in the hypothesis by Altukhov and Rychkov on the role of special (unchangeable) genes in speciation [59]. These hypotheses have not been further developed because of "objectlessness": the experimental genetics of that time did not know any other genes except for the Mendelian genes. The discovery of mutations in ontogenes, no matter how "strange" they may be, changes the situation. Theoretical discussion of the genetic problems where the concept of ontogene (or its molecular analog, lncRNA gene) can be utilized seems most important

If we admit the existence of ontogenes, the structure of biological characters becomes universal and simple. Each character comprises (1) the cellular basis and (2) the proteins filling the cells. A Mendelian (simple or monogenic) character is regarded as a virtual structure in which its cellular basis is meant to exist but does not considered, while the protein contained in it is considered. On the contrary, cellular basis of the species-, genus-, family-level, etc. characters is considered but their protein content is omitted.

#### **9. Conclusions**

Mutagenesis acts as an architect of the living. Theoretically, only mutations give the possibility to (1) expand the potential of an existing biological species and (2) create new species. Mutations in Mendelian genes actually manage to fulfill the first task but fail in the latter [10, 60]. As has emerged, the problem has a simple solution: in addition to Mendelian genes, the genome contains the genes belonging to another category. Earlier, the mutations putatively belonging to this new category have been generated for drosophila. The new mutations were named conditional and the new genes, ontogenes. Currently, it is most possible that lncRNA genes are the molecular analogs of ontogenes. Here, we attempt to construct the phenomenology of conditional mutations, described earlier, into a logically arranged pattern representing a special part of the genome composed of ontogenes. The work of Mendelian genes on the production of proteins is unfeasible without the ontogenes. The arguments favoring a common nature of ontogenes and lncRNAs are considered in the paper.

The category of genes responsible for the specific outlook of a species is not visible in the case of an intraspecific hybridization but becomes evident in distant hybridization as the syndrome of interspecific incompatibility. The pattern of ontogene manifestation repeats the pattern of interspecific incompatibility. This means that the ontogenes belong to the category of genes that determine the species' specificity. The patterns of monstrosities and meiotic abnormalities reveal the main mission of the ontogenes, namely, the control over construction of cell ensembles in ontogenesis. Concurrently, they also include the Mendelian genes that control protein synthesis.

*Conditional Mutations and New Genes in Drosophila DOI: http://dx.doi.org/10.5772/intechopen.103928*

The ontogenes are active in every living cell in a spatial aspect in the germline and soma and in a temporal aspect, starting from the gonial divisions to the renewal of differentiated somatic cells. Our data suggest us that an event of genome editing, taking place in the premeiosis and involving ontogenes, precedes the formation of every gamete. The specific features in the function of ontogenes underlie the following characteristics untypical of the Mendelian genes: (1) dominant lethal effect; (2) conditional effect; (3) parental inheritance; (4) decrease in fertility; and (5) integral forms of variation referred to as individual and epigenetic variations.

#### **Acknowledgements**

The authors thank the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, for financial support of this work (budget project no. 0259- 2021-0011).

### **Author details**

Boris F. Chadov\* and Nina B. Fedorova Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, Russian Federation

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Venter J, Adams MD, Myers EW, et al. The sequence of the human genome. Science. 2001;**291**:1304-1351

[2] Timiryazev KA. Collected Works. Vol. 2. Moscow: Sel`khozgiz; 1937-1939. pp. 5-7 [in Russian]

[3] Chadov BF, Chadova EV, Kopyl SA, Artemova EV, Khotskina EA, Fedorova NB. From genetics of intraspecific differences to genetics of intraspecific similarity. Russian Journal of Genetics. 2004;**40**(9):945-958

[4] Chadov BF. Ontogenes in *Drosophila melanogaster*: Genetic features and role in onto – and phylogeny. In: Korogodina VL, Chini A, Durante M, editors. Modern Problems of Genetics, Radiobiology, Radioecology and Evolution. Moscow, Russia: JINR; 2007. pp. 80-91

[5] Fedorova NB, Chadova EV, Chadov BF. Genes and Ontogenes in Drosophila: The Role of RNA Forms. Transcriptomics. 2016;**4**:137. DOI: 10.4172/2329- 8936.1000137

[6] Kondakov NI. Logicheskii slovar– spravochnik (Logical Dictionary– Handbook). Moscow: Nauka; 1975. p. 477

[7] Chadov BF. Mutations capable of inducing speciation. In: Stegnij VN, editor. Evolution Biology. Tomsk: Tomsk State University Press; 2001. pp. 138-162 (in Russian). Available from: http:// www.evolbiol.ru

[8] Chadov BF, Chadova EV, Kopyl SA, Fedorova NB. A new class of mutations in Drosophila melanogaster. Doklady Biological Sciences. 2000;**373**:423-426

[9] Chadov BF. Mutations in the regulatory genes in Drosophila melanogaster. In: Proceedings of International Conference on Biodiversity and Dynamics of Ecosystems in North Eurasia; 21–26 August 2000. Novosibirsk: IC@G; 2000. pp. 16-18

[10] Chadov BF. Facultative dominant lethals: Genetics, ontogenesis, and phylogenesis. In: Stegniy VN, editor. Evolutionary Biology. Vol. 2. Tomsk: Tomskogo gosudarstvennogo universiteta; 2002. pp. 118-142 [in Russian]

[11] Chadov BF, Chadova EV, Kopyl SA, Fedorova NB. Delayed activation of the maternal genome during early development of Drosophila. Doklady Biological Sciences. 2001;**378**(1-6): 294-298. DOI: 10.1023/A:1019247615297

[12] Chadov BF, Chadova EV, Khotskina EA, Artemova EV, Fedorova NB. The main effect of chromosomal rearrangement is changing the action of regulatory genes. Russian Journal of Genetics. 2004;**40**(7):723-731. DOI: 10.1023/B:RUGE.0000036520. 93159.a5

[13] Fedorova NB, Chadova EV, Khotskina EA, Chadov BF. Conditional mutations: Generation using the technique of morphoses. In: Factors of Experimental Evolution. Collection of Papers. Vol. 8. Kiev: Logos; 2010. pp. 78-83 [in Russian]

[14] Chadov BF, Fedorova NB, Chadova EV, Khotskina EA. Conditional mutations in Drosophila. Journal of Life Sciences. 2011;**5**(3):224-240

[15] Chadov BF, Fedorova NB, Chadova EV. Conditional mutations in Drosophila melanogaster: On the Occasion of the 150th Anniversary of G. Mendel's Report in Brünn. Mutation Research - Reviews in Mutation Research. 2015;**765**:40-55. DOI: 10.1016/ j.mrrev.2015.06

[16] Chadov BF. "The image" of the regulatory gene in experiments with Drosophila. Russian Journal of Genetics. 2002;**38**(7):725-734

[17] Chadov BF, Chadova EV, Fedorova NB. Conditional mutations in D. melanogaster: Specific features in localization and secondary mutation. In: Proceedings of the International Conference Chromosoma 2012; 2–7 September 2012. Russia: Novosibirsk; [in Russian]

[18] Chadov BF, Chadova EV, Khotskina EA, Fedorova NB. Conditional lethal mutations shift the genome from stability to instability. Russian Journal of Genetics. 2009;**45**(3):276-286. DOI: 10.1134/S102279540903003X

[19] Fedorova NB, Vasilyeva LA. New evidence of mass induction of transpositions of MGE 412 due to the isogeniszation of forked line of Drosophila. In: Proceedings of the III Congress of the Vavilov Society of Geneticists and Breeders. Genetics in XXI Century: Current State and Prospects of Development; 6-12 June 2004. Vol. 2. Moscow: Genetics Society; 2004. p. 61

[20] Chadov BF, Chadova EV, Kopyl SA, Khotskina EA, Fedorova NB. Genes controlling development: Morphoses, phenocopies, dimorphs and other visible expressions of mutant genes. Russian Journal of Genetics. 2004;**40**(3):271-281. DOI: 10.1023/B%3ARUGE.0000021627. 82588.41

[21] Chadov BF, Chadova EV, Fedorova NB. Epigenetic phenomenology in conditional mutants of Drosophila melanogaster: Morphoses and modifications. In: Zakijan SM, Vlasov SM, Dement'eva EV, editors. Epigenetics. Novosibirsk: SD RAN; 2012. pp. 499-533 [in Russian]

[22] Chadov BF, Chadova EV, Fedorova NB. The Genetics of Conditional Mutations and Individual developmental Programs in D. SCIOL Genetics Science. 2017;**1**:3-21. Available from: https://sciol.org/articles/ genetic-science/fulltext.php?aid=sgs-1-002

[23] Chadov BF, Chadova EV, Fedorova NB. Conditional mutations in Drosophila: Concept of genes that control individual development. Advances in Bioscience and Biotechnology. 2018;**9**:243-272. DOI: 10.4236/abb.2018.96017

[24] Chadov BF, Fedorova NB. Ontogenes and chromosome nondisjunction in the D. melanogaster meiosis. Advances in Bioscience and Biotechnology. 2021;**12**(1):31-59. DOI: 10.18103/mra.v9i4.2366. Available from: http://www.researchgate

[25] Serebrovsky AS. Animal Hybridization. Moscow–Leningrad: Biomedgiz; 1935. p. 290

[26] Lobashev ME. Distant hybridization. In: Genetics. Leningrad: Leningrad Gosudarstvennogo University; 1967. pp. 491-518 [in Russian]

[27] Chadov BF, Fedorova NB, Chadova EV. Parental effects of conditional mutations and their explanations. Russian Journal of Genetics. 2013;**49**(2):141-150. DOI: 10.1134/S1022795413020038

[28] Rieger R, Michaelis A, Green MM. Glossary of Genetics. Classical and Molecular. Berlin: Springer-Verlag; 1991. p. 553

[29] Frizen G. X-ray morphoses in Drosophila. Biology Zhurnal. 1935;**4**: 687-704 [in Russian]

[30] Rapoport IA. Specific morphoses in Drosophila melanogaster caused by chemical compounds. Bulletin of Experimental Biology and Medicine. 1939;**7**:415-417 [in Russian]

[31] Goldschmidt RB, Piternick LK. The genetic background of chemically induced phenocopies in Drosophila. Journal of Experimental Zoology. 1957; **135-136**:127-202-1201-28

[32] Chadov BF, Chadova EV, Fedorova NB. Images of morphoses and modifications in Drosophila melanogaster conditional mutants. Mutation Research/Reviews in Mutation Research. 2015;**765**:40-55. DOI: 10.13140/RG.2.1.2721.9042. Available from: http://www.researchgate

[33] Chadov BF, Fedorova NB. The Mutations Disturbing the Bilateral Symmetry in Drosophila. SCIOL Genetic Science. 2019;**2**:139-152. Available from: https://sciol.org/articles/genetic-science/ fulltext.php?aid=sgs-2-015

[34] Chadov BF. Nongomologous chromosome pairing in oogenesis of Drosophila melanogaster. Abstract of dissertation for the degree of Ph.D. Institute of Developmental Biology: Moscow; 1971 [in Russian]

[35] Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of proteins. Journal of Molecular Biology. 1961;**3**: 318-356

[36] Chadov BF, Chadova EV, Fedorova NB. A Novel Type of Gene Interaction in D. melanogaster. Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis. 2017;**795**: 27-30. DOI: 10.1016/j. mrfmmm.2017.01.002

[37] Lewis D. Gene-environment interaction: A relationship between dominance, heterosis, phenotypic stability and variability. Heredity. 1954; **8**:333-356

[38] Linskens HF. Physiology der Fortpflanzung and Sexualitat. Fort-schr Botany. 1955;**17**:791-818

[39] Fedorova NB. A mutation in ontogene and radiation-induced instability in Drosophila melanogaster. In: Proceedings of The 5th Congress on Radiation Studies (Radiobiology, Radioecology, and radiation Safety); 10–14 April 2006. Moscow: ICRP. p. 75

[40] Geisler S, Coller J. RNA in unexpected places: Long non-coding RNA functions in diverse cellular contexts. Nature Reviews Molecular Cell Biology. 2013;**14**:699-712

[41] Wang X, Song X, Glass C, Rosenfeld M. The long arm of long noncoding RNAs: Roles as sensors regulating gene transcriptional programs. Cold Spring Harbor Perspectives in Biology. 2010;**3**:a003756 a003756

[42] Akhade VS, Pal D, Kanduri C. Long noncoding RNA: Genome organization and mechanism of action. In: Rao M, editor. Long NonCoding RNA Biology. Advances in Experimental Medicine and Biology. Vol. 1008. Singapore: Springer; 2017. DOI: 10.1007/978-981-10-5203-3\_2

[43] Lakhotia SC. From heterochromatin to long noncoding RNAs in Drosophila: Expanding the arena of gene function and regulation. In: Rao M, editor. Long Non Coding RNA Biology. Advances in Experimental Medicine and Biology. Vol. 1008. Singapore: Springer; 2017. DOI: 10.1007/978-981-10-5203-3\_3

[44] Chadov BF, Fedorova NB. Ontogenes and the paradox of homologous pairing. Advances in *Conditional Mutations and New Genes in Drosophila DOI: http://dx.doi.org/10.5772/intechopen.103928*

Bioscience and Biotechnology. 2021;**12**: 1-9. DOI: 10.4236/abb.2021.121001

[45] Chadov BF. Near-centromere heterochromatin and co-orientation of chromosomes in dividing cells of Drosophila. Genetica (Russia). 1987; **23**(6):1082-1087

[46] Chadov BF. Mechanism of chromosome nondisjunction in Drosophila meiosis. In: Abbondandolo A, Vig BK, Roi R, editors. Chromosome Segregation and Aneuploidy-III. Italy. Ispra: E.C. Joint Research Center; 1996. pp. 328-343

[47] Hawley RS et al. There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology. Developmental Genetics. 1993;**13**(6): 440-467

[48] Karpen GH, Le MH, Le H. Centric heterochromatin and the efficiency of achiasmate disjunction in Drosophila female meiosis. Science. 1996;**273**(5271): 118-122

[49] Hughes SE et al. Heterochromatic threads connect oscillating chromosomes during prometaphase I in Drosophila oocytes. PLoS Genetics. 2009;**5**(1): e10000348

[50] Hacisuleyman E, Goff L, Trapnell C, et al. Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nature Structural & Molecular Biology. 2014;**21**: 198-206. DOI: 10.1038/nsmb.2764

[51] Prensner J, Iyer M, Sahu A, Asangani I, Cao Q, Patel L, et al. The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex. Nature Genetics. 2013;**45**:1392-1398

[52] Han P, Li W, Lin C, Yang J, Shang C, Nurnberg S, et al. A long noncoding RNA protects the heart from pathological hypertrophy. Nature. 2014;**514**:102-106

[53] Minajigi A, Froberg J, Wei C, Sunwoo H, Kesner B, Colognori D, et al. A comprehensive Xist interactome reveals cohesin repulsion and an RNAdirected chromosome conformation. Science. 2015;**349**:aab2276

[54] Fedorova NB, Chadov BF. Gene interactions in Drosophila without contacts and chemical intermediaries. In: Abstracts Book of: International Conference on Cell and Experimental Biology (Virtual Conference). 9-11 December 2020. Boston: CEB; 2020. p. 6. Available from: https://cellexpbiol. unitedscientificgroup.org/proceedings/ CEB-2020\_Abstracts.pdf

[55] Chadov BF, Fedorova NB. Ontogenes and the genetics of intraspecific Similarity. Medical Research Archives. 2021;**9**(4):2-10

[56] Gershenzon CN. Foundations of the modern genetics. Naukova dumka: Kiev; 1983. 220 p [in Russian]

[57] Filipchenko Yu A. The evolutionary idea in biology. Izd. Sobashnikovykh: Moscow; 1923. 287 p [in Russian]

[58] Timofeev-Ressovsky NV. The connection between the gene and external character. In: Gazenko OG, Ivanov VI, editors. Selected Works. Moscow, Meditsina; 1996. pp. 59-84 [in Russian]

[59] Altukhov YP, Rychkov YG. Genetic monomorphism of species and its potential biological significance. Zhurnal Obshchei Biologii. 1972;**33**:281-300 [in Russian]

[60] Chadov BF, Chadova EV, Fedorova NB. Ontogenes and the Problem of Speciation. Journal of Evolutionary Science. 2019;**1**(1):33-47. Available from: https://openaccesspub. org/jes/article/1013

### *Edited by Michael Fasullo and Angel Catala*

Reactive oxygen species (ROS) and DNA double-strand breaks can result from mitochondrial defects and external sources, such as ionizing radiation. If not repaired properly, pathogenic mutations are generated. Human diseases resulting from inherited mitochondrial defects manifest in organs that physiologically require a high level of ATP synthesis. These diseases are clinically challenging, but new experimental clinical therapies include gene editing and mitochondrial transplants. Pathogenic ROSassociated cellular damage includes DNA double-strand breaks, and mouse models are now available to study multiple repair pathways. This book discusses the clinical manifestations of mitochondrial diseases in both the eye and the kidney, and presents new insights into double-strand break repair pathways and developmental phenotypes of g-ray-associated ontogenic mutations of Drosophila melanogaste.

Published in London, UK © 2022 IntechOpen © SvitDen / iStock

Mutagenesis and Mitochondrial-Associated Pathologies

Mutagenesis and

Mitochondrial-Associated

Pathologies

*Edited by Michael Fasullo and Angel Catala*