Efficient Mutation Research Articles

Effectiveness and efficiency of electron beam in comparison with gamma rays and ethyl methane sulfonate mutagens in cowpea

Cowpea is the poor man's crop that lacks variability due to its autogamous nature. Induced mutation serves as a potential source in the induction of variability in crops. On the other hand, the effectiveness and efficiency of mutagens will vary between species and even varieties. In the present study, a novel mutagen electron beam was used in cowpea for the first time along with commonly used mutagens gamma rays (physical) and ethyl methanesulfonate (chemical). The biological damages on eight quantitative characters in M1 generation and chlorophyll mutants in M2 generation were recorded. Two popular varieties viz., P 152 and VBN 1 constituted as the biological material of study. The rate of reduction in biological damage on quantitative characters was directly proportional to the dose of mutagen irrespective of the varieties and mutagens used. Physical mutagens showed the highest biological damage (EB- 37.5% and G- 37.3% overall reduction from control) than chemical mutagen (EMS- 30.4%). Comparing the physical mutagens at similar doses, 200 Gy or 300 Gy of electron beam showed more biological damage than 200 Gy or 300 Gy of gamma rays. Eleven different types of chlorophyll mutants were identified in the M2 generation. Xantha is the most occurred chlorophyll mutants (44.44%), while aurea and yellow viridis have least occurred mutants. Chemical mutagen (EMS) is considered to be the most effective (6.47%) and efficient mutagen (27.09%) based on the chlorophyll mutants and it was followed by an electron beam and gamma rays. Among the physical mutagens, electron beam showed the highest biological damage (37.50% overall reduction from control) and higher effectiveness and efficiency (3.80% and 23.38%) compared to gamma rays (1.87% and 13.38%). Hence, the electron beam can also be used as an effective mutagen in creating variation in cowpea and other crops as it is highly effective, cost less and safe mutagen.

INDUCED GENETIC VARIABILITY IN SESAME (Sesamum indicum L): A COMPARATIVE STUDY ON THE MUTAGENIC EFFECTS OF RADIATION AND EMS IN SEED GERMINATION, POLLEN VIABILITY AND CHLOROPHYLL MUTANTS

The present study envisaged the effects of two mutagens, gamma rays and EMS on the phenotypes of two sesame varieties viz., TMV7 and SVPR1. A known quantity of dry, uniform, and healthy seeds of TMV7 and SVPR 1 were irradiated using Co60 (Cobalt 60) with different doses (250, 300, 350, 400, 450 Gy) of gamma rays. For chemical mutagenesis, different concentrations of EMS @ 0.20%, 0.40% and 0.60% was used and treated for 8 h. The dose-response curve of the probit analysis showed that the optimal lethal dose for SVPR1was lower than TMV7. The expected LD50 values of gamma radiation for TMV 7 and SVPR1 were 403.91Gy and 343.84Gy, respectively. For EMS, the expected LD50 values are 0.525 % and 0.276% for TMV7 and SVPR1 respectively. Germination and pollen fertility declined linearly with an increase in dose or concentration of the mutagens. Three classes of chlorophyll mutants viz., xantha, chlorine, and viridis in M2 generation reveals a dose dependent relationship between mutagens and frequency of chlorophyll mutants. Mutagenic effectiveness was higher at lower doses whereas mutagenic efficiency was observed higher at extremity doses in both the varieties. The overall considerations on M1 generation effects showed that SVPR1was highly sensitive to gamma rays and TMV7 produced more viable mutationsthan SVPR1. The current studies suggest gamma rays as an efficient mutagen to induce essential mutations in TMV7 for the further crop improvement program.

Comparative Studies on Mutagenic Effectiveness and Efficiency in Horsegram [Macrotyloma uniflorum (Lam) Verdc.

Background: Two horsegram varieties viz., PAIYUR 2 and CRIDA1-18R were mutated with gamma rays (G), electron beam (EB), G+EB and G+EMS (ethyl methane sulphonate) to determine the mutagenic potency in breeding programme.Methods: Uniform seeds treated with different mutagenic doses were raised in randomized block design which constituted M1 generation. Each plant was harvested individually and forwarded to M2 generation following plant to progeny row method.Result: A dose dependant decline was observed for seed germination, plant survival, root length, shoot length, plant height, pollen fertility and seed fertility in M1 population. Wide spectrum of chlorophyll mutants was induced in M2 generation with maximum frequency at EB followed by combination treatments (G+EMS and G+EB) in both varieties. The mutagenic effectiveness ranged from 0.14 per cent to 1.45 per cent in PAIYUR 2 and 0.15 per cent to 1.71 per cent in CRIDA1-18R. The high mutation rate for effectiveness was exhibited by G and G+EB. With reference to sterility, EB was found to be efficient mutagen in both varieties whereas varied efficiency was noted for lethality (EB - PAIYUR 2; G+EMS - CRIDA1-18R) and injury (G - PAIYUR 2; G+EMS - CRIDA1-18R).

Ems and gamma radiation induced mutation in grasspea (Lathyrus sativus L.)

Present investigation has been undertaken to study the induction of mutation by individual and combination treatments of EMS and gamma rays in three diverged genotypes of grasspea viz. Nirmal, Biol-212 and Berhampur Local. Combination treatments caused the more biological damage (lethality, injury and sterility) followed by gamma rays and EMS alone. The LD50 dose of gamma rays for Nirmal, Biol-212 and Berhampur Local corresponded to 590 Gy, 630 Gy and 539 Gy respectively. Above 0.5% EMS for Nirmal and Biol-212 and above 1% EMS in case of Berhampur Local was found to be lethal. Average total mutation frequency of three varieties together was highest (6.32 %) in gamma radiation followed by both EMS and combination treatment (5.8%). The most efficient dose/concentration of gamma rays and EMS were found to be 400 Gy and 0.5% EMS respectively. Mutagenic efficiency increases and effectiveness decreases with increasing concentration of EMS but the consequence was showed almost inverse relationship in case of gamma rays. Overall, irrespective of genotypes, gamma rays and EMS respectively were found to be most efficient and effective mutagen.

Isolation of desirable mutants in safflower for crop improvement

The present investigation was undertaken to isolate mutants having promising traits for safflower (Carthamus tinctorius L.) improvement. Two varieties of safflower viz., AKS-207 and Bhima were mutagenised with ethyl methanesulphonate, sodium azide and gamma rays. All the mutagens decreased pollen fertility in M1 generation with the increase in their dose. The screening of M2 population led to the isolation of several putative mutants. The frequency of mutations varied according to the mutagen, its dose and the variety concerned. The evaluation of these putative mutants in M3 generation resulted in selection of early bolting, dwarf, highly branched, large head, high test weight, thin hull, high oil content and high oleic acid mutants on the basis of their utility in breeding for safflower improvement. Amongst the three mutagens used, sodium azide proved to be most effective and efficient mutagen for induction of mutations in safflower.

Tnt1 retrotransposon as an efficient tool for development of an insertional mutant collection of Lotus japonicus

The Tnt1 retrotransposon of tobacco (Nicotiana tabacum) has proven to be a very efficient mutagen for the model legume Medicago truncatula ecotype 108 and cultivar Jemalong 2HA and for economically important plants, such as soybean and potato. In this study, the activity of Tnt1 in the model legume Lotus japonicus L. was tested. First, a new regeneration and transformation protocol was developed for L. japonicus that represents a new tool for legume mutagenesis and reverse genetics. Using this protocol, the Tnt1 retrotransposon was introduced into L. japonicus by Agrobacterium tumefaciens-mediated transformation, and primary transgenic lines, named starter lines, were constructed. In vitro regeneration via indirect somatic embryogenesis using starter lines harboring two to eight copies of the transgene resulted in new Tnt1 transposition events. The Tnt1 retrotransposon remained inactive during plant growth and in the T1 progeny, indicating that it is well suited for insertional mutagenesis in L. japonicus.

Plant Mutation Breeding with Heavy Ion Irradiation at IMP

<p>The Heavy Ion Research Facility in Lanzhou (HIRFL) is one of the ion-beam acceleration facilities intensively used at IMP, founded as national laboratory and opened for user in world from 1992. Since then, a lot of experiments irradiated by heavy ion beam have been carried out in the HIRFL, including plant mutation breeding. In this review, the biological effects induced by heavy ions and their corresponding mechanisms were reported from the point of view of cytological, morphological and molecular levels. To date, a large number of mutants were isolated using heavy ion irradiation IMP, such as early maturity, flower color and shape, high yield and disease resistant. In conclusion, heavy ion beam irradiation is an efficient mutagen and has significant phenotypic variations in plant. Our research will be further focused on transformation of scientific and technological achievements and mutagenic mechanism of heavy ion beam on high plant at the molecular level in the recent future.</p>

Studies on Induced Physical and Chemical Mutagenesis in Groundnut (<i>Arachis hypogia</i>)

Mutation breeding has been widely used for the improvement of plant characters in various crops. It is a powerful and effective tool in the hands of plant breeders. In any mutation breeding program, selection of an effective and efficient mutagen is very essential to produce high frequency of desirable mutation. Groundnut (Arachis hypogia) var. VRI-2. was treated with different concentration of physical and chemical mutagen namely gamma rays 10, 20, 30, 40, 50 and 60 KR and Ethyl methane sulphonate (EMS). For inducing mutation various concentration of EMS such as 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 % for six hours were applied to 200 seed sample of each concentration and one respective control. The LD50 value was observed in 50% of gamma rays and 0.5 % of EMS. The morphological and yield characters were significantly reduced seed germination, seedling survival, days to first flower, plant height, number of leaves per plant, number of grains per plant, grain length and breath, 100 grains weight, grain yield per plant, fresh and dry weight per plant. The increasing doses/concentration of gamma rays and EMS decreased in phenotypic and yield characters in M1generation. The mutagenized populations showed significantly higher variability in the M2 generation. Mutant lines showing higher yield per plant than the respective parents and checks were isolated in M2 and subsequent generation were significantly more pod yield and yield components than the untreated plants.

Studies on Induced Physical and Chemical Mutagenesis in Groundnut (<i>Arachis hypogia</i>)

Mutation breeding has been widely used for the improvement of plant characters in various crops. It is a powerful and effective tool in the hands of plant breeders. In any mutation breeding program, selection of an effective and efficient mutagen is very essential to produce high frequency of desirable mutation. Groundnut (Arachis hypogia) var. VRI-2. was treated with different concentration of physical and chemical mutagen namely gamma rays 10, 20, 30, 40, 50 and 60 KR and Ethyl methane sulphonate (EMS). For inducing mutation various concentration of EMS such as 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 % for six hours were applied to 200 seed sample of each concentration and one respective control. The LD50 value was observed in 50% of gamma rays and 0.5 % of EMS. The morphological and yield characters were significantly reduced seed germination, seedling survival, days to first flower, plant height, number of leaves per plant, number of grains per plant, grain length and breath, 100 grains weight, grain yield per plant, fresh and dry weight per plant. The increasing doses/concentration of gamma rays and EMS decreased in phenotypic and yield characters in M1generation. The mutagenized populations showed significantly higher variability in the M2 generation. Mutant lines showing higher yield per plant than the respective parents and checks were isolated in M2 and subsequent generation were significantly more pod yield and yield components than the untreated plants.

Evaluation of Anti-HIV-1 Mutagenic Nucleoside Analogues

Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of "lethal mutagenesis" that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base pairs in DNA duplexes containing or not the nucleoside analogues as follows. A promising candidate should display a small destabilization of the matched base pair compared with the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine, should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesized base pairs. Once again, only 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture because only compounds 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine were found to increase the mutation frequency by 3.1- and 3.4-fold, respectively.

Comparative mutagenic efficiency, effectiveness and induced polygenic variability in mothbean (Vigna acontifoliaL.)

Induced biological damage, polygenic variability and comparative mutagenic effectiveness and efficiency of EMS, MMS, SA and HA were studied in four varieties of moth bean, namely, RMO-40, RMO-257, Jwala and CZM-1. Gradual reduction in germination, pollen fertility and survival percentage was observed with increased doses of mutagens. The frequency of chlorophyll mutants was less and were of two types i.e., albino and xantha in which albino type was less frequent. The spectrum of morphological mutations was defined in ten different classes (revertant, bushy, bifurcated stem, bolted, sterile, miniature, branched, increased peduncle length, spreading habit, and increased primary branches). The most effective treatment appeared to be 0.05% MMS, whereas least were 50mM hydroxylamine.The effectiveness of various mutagen treatments was generally higher on RMO-40 and lower on CZM-1.The effectiveness of treatments was decreased in the order of MMS > SA> EMS > HA. The highest mutagenic efficiency was observed with 1.0 mM Sodium azide in the Mothbean variety RMO-40. On the basis of estimates of pooled over genotypes and doses with in a mutagen, SA was the most efficient mutagen followed by MMS, EMS and HA. In M3 generation, 15 progenies showed significantly superior seed yield per plant with higher magnitude of yield contributing traits then the best check, RMO-225. Branches per plant and pods per plant exhibited high genetic variability in M3 generation.

Molecular analysis of point mutations in a barley genome exposed to MNU and gamma rays

We present studies aimed at determining the types and frequencies of mutations induced in the barley genome after treatment with chemical (N-methyl-N-nitrosourea, MNU) and physical (gamma rays) mutagens. We created M2 populations of a doubled haploid line and used them for the analysis of mutations in targeted DNA sequences and over an entire barley genome using TILLING (Targeting Induced Local Lesions in Genomes) and AFLP (Amplified Fragment Length Polymorphism) technique, respectively. Based on the TILLING analysis of the total DNA sequence of 4,537,117bp in the MNU population, the average mutation density was estimated as 1/504kb. Only one nucleotide change was found after an analysis of 3,207,444bp derived from the highest dose of gamma rays applied. MNU was clearly a more efficient mutagen than gamma rays in inducing point mutations in barley. The majority (63.6%) of the MNU-induced nucleotide changes were transitions, with a similar number of G>A and C>T substitutions. The similar share of G>A and C>T transitions indicates a lack of bias in the repair of O6-methylguanine lesions between DNA strands. There was, however, a strong specificity of the nucleotide surrounding the O6-meG at the −1 position. Purines formed 81% of nucleotides observed at the −1 site. Scanning the barley genome with AFLP markers revealed ca. a three times higher level of AFLP polymorphism in MNU-treated as compared to the gamma-irradiated population. In order to check whether AFLP markers can really scan the whole barley genome for mutagen-induced polymorphism, 114 different AFLP products, were cloned and sequenced. 94% of bands were heterogenic, with some bands containing up to 8 different amplicons. The polymorphic AFLP products were characterised in terms of their similarity to the records deposited in a GenBank database. The types of sequences present in the polymorphic bands reflected the organisation of the barley genome.

Efficiency and effectiveness of physical and chemical mutagens in Trigonella foenum graecum L

Trigonella foemum graecum L was utilized to study the Chlorophyll mutations, mutagenic efficiency and effectiveness by physical mutagen gamma rays and chemical mutagen EMS. The frequency of chlorophyll mutation in M1 & M2 generation were more in gamma treated plants but the mutation spectra and mutagenic efficiency of EMS was higher than gamma. The most efficient mutagens were 0. 02% EMS, 0.06% EMS and 3KR gamma radiation to induce mutation in T. foenum graceum L.

Induced chlorophyll mutations. I. Mutagenic effectiveness and efficiency of EMS, HZ and SA in mungbean

A systematic and comparative study on the frequency and spectrum of chlorophyll mutations induced by ethylmethane sulphonate (EMS) — an alkylating agent, hydrazine hydrate (HZ) — a base analogue and sodium azide (SA) — a respiratory inhibitor, was carried out in two mungbean varieties, namely, PDM-11 and NM-1. Awide spectrum of chlorophyll mutants was obtained in the M2 generation. All these chlorophyll-deficient mutants were lethal except maculata, viridis and virescent. Chlorina followed by xantha types were predominant in both the varieties. EMS treatments induced the highest frequency of chlorophyll mutations followed by HZ and SA. The frequency of chlorophyll mutations was dose-dependent and increased with the mutagen concentration. Based on effectiveness in both varieties, the order of mutagens was HZ > SA > EMS. Two criteria viz., pollen sterility (Mp/S) and seedling injury (Mp/I) were taken into consideration to determine the efficiency of the mutagens. EMS was found to be the most efficient mutagen followed by HZ and SA. Moderate concentrations of the mutagens were the most effective and efficient in inducing mutations.

Gene expression changes in human prostate carcinoma cells exposed to genotoxic and nongenotoxic aryl hydrocarbon receptor ligands

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) are known as efficient mutagens and ligands of the aryl hydrocarbon receptor (AhR), which has been suggested to play an important role in prostate carcinogenesis. In order to evaluate the complex relationship between the genotoxicity and the AhR-mediated activity of PAHs in prostate cells, we selected benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as model genotoxic and nongenotoxic AhR ligands, respectively, to explore global changes in gene expression in LNCaP cells by microarray analysis. We identified 112 genes that were differentially expressed in cells treated for 24h with BaP, TCDD or both compounds. Our data indicated that the impacts of BaP and TCDD on transcriptome of LNCaP cells significantly overlap, since over 64% of significantly up-regulated genes and 47% of down-regulated genes were similarly affected by both AhR ligands. This suggested that the activation of AhR played a prominent role in the nongenotoxic effects of BaP in the prostate carcinoma cell model LNCaP. Both AhR ligands suppressed expression of genes associated with cell cycle progression, DNA replication, spindle assembly checkpoint or DNA repair, which probably occurred secondary to inhibition of cell cycle progression. In contrast, we identified Wnt5a, an important regulator of prostate cancer progression, to be induced as early as 6h after exposure to both AhR ligands. The AhR ligand-induced Wnt5a upregulation, together with other observed alterations of gene expression, may further contribute to enhanced cell plasticity of prostate carcinoma cells.

Variable Leaf Epidermal Morphology in TNT1 Insertional Mutants of the Model Legume Medicago Troncatola

ABSTRACTIn this report some typical leaf morphological characteristics of M. truncatula mutants generated by a Tnt1 retrotransposon insertion mutagenesis were evaluated and summarized. It was found that all the examined leaf epidermal parameters were strongly influenced in the Tnt1 mutant lines. Epidermal cells varied in shape and size, and diversified in the patterns of cell walls. Although the leaves of all mutant plants were amphistomatic, stomata were more abundant at the lower (abaxial) leaf surfaces than the upper (adaxial) leaf surfaces. On the other hand, the number of stomata on both leaf surfaces varied widely among different Tnt1 lines. Based on these observations, we conclude that most of the observed mutant phenotypes were caused by the Tnt1 insertions. In addition, the evaluated leaf epidermal features can be reliably applied for phenotypic profiling of M. truncatula mutant lines. Morphological variables in leaf epidermis in all the screened mutants demonstrated that Tnt1 is a very efficient mutagen, confirming that Tnt1 gene tagging strategy is one of the most valuable systems for legume functional genomics.

Osmotic shock improves Tnt1 transposition frequency in Medicago truncatula cv Jemalong during in vitro regeneration

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.

Mutagen specificity for 71 lines resistant to barley powdery mildew race D1 and isolated in four highbred barley varieties

A search for practically valuable mutant genes conferring resistance to barley powdery mildew applied a screening system where four commercial varieties of barley without known genes for resistance were tested to the successful mildew race D1. Ionizing radiations and chemicals were used as mutagens. Among the 71 resistant lines isolated, 28 were recessive and belonging to locus ml-o, and 43 were dominant. In tests to a panel of 30 different races of barley powdery mildew, 27 among the dominant lines showed resistance spectra similar or identical to those characterizing 7 previously known genes. The remaining 16 lines formed 12 groups displaying new spectra of resistance to the panel of mildew races. Taking the frequency of recessive mutations in locus ml-o as a standard, ionizing radiations were significantly more efficient mutagens for producing dominant resistance to mildew. In spite of some possible sources of contaminant dominant resistance genes, the evidence points at a vast majority of the isolated lines representing genuine dominant mutations. Being produced in a single mutational step, such dominant genes for resistance offer evidence of having a regulatory function, regulating aspects of the host metabolism of importance for or interfering with the pathogen development.

Lethal mutagenesis of poliovirus mediated by a mutagenic pyrimidine analogue.

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.

Induction of Transient Cytopenia to Detect Hematopoietic Defects in the Mouse.

In an attempt to generate new mouse models of human hematopoietic diseases and increase our knowledge of the genetic networks that orchestrate hematopoiesis, we are developing a sensitized screen that will more effectively identify hematopoietic in heterozygous mutants. The chemical mutagen ethylnitrososurea (ENU) is a highly efficient mutagen in the mouse and other model organisms, generating primarily point mutations. As a part of the Centre for Modeling Human Disease (CMHD), we have used simple clinical assays such as blood morphology and automated blood cell counts to test saphenous vein collected peripheral blood from approximately 6500 offspring of ENU mutagenized mice to identify clinically relevant dominantly inherited hematopoietic disease models. Phenodeviant G1 animals are bred to wild type mice to generate G2 mice, and G2 strains demonstrating high penetrance undergo a genome scan. This strategy has yielded 14 heritable dominant mutants to date. However, many genes and pathways will not express phenotypes in a dominant manner due to the homeostatic balance maintained by complex genetic networks, possessing excess capacity or redundancy, a problem exacerbated by using a viable peripheral blood assay. For example, only one of the 14 heritable hematopoietic mutants demonstrates anemia. Because environmental sensitization can exacerbate latent phenotypes, such as the effect of a high salt diet in an individual with prehypertension, sensitized screens should identify subtle and additional phenotypes of dominant mutations. Thus, we are developing a sensitized screen to uncover subtle dominant phenotypes by inducing transient cytopenia and following the kinetics and magnitude of the recovery. We are using a variety of drugs (5-fluorouracil, phenylhydrazine, and hydroxyurea) to induce cytopenia and recruit hematopoietic stem cells and progenitors to repopulate the blood with differentiated cells. We hypothesize that animals with defects in blood development will demonstrate altered recovery kinetics. This strategy will allow us to determine if there is a defect in blood cell progenitors, while keeping the animals viable for further screening and breeding. We are testing these drugs on known dominant and recessive mutants to validate the assay, determine the best dosages, and compare the effects of each drug. However, we have already begun to use 5-fluoruracil on a cohort of G1 animals and have identified two G1s that display latent recovery in at least 1 blood lineage. The two G1s are confirmed heritable and are currently undergoing a genome scan to map position the mutation. We believe this strategy will further our understanding of the molecular mechanisms that regulate hematopoietic development, as well as generate new mouse models of human hematopoietic diseases and identify the underlying molecular defects of diseases whose etiological origins are currently unknown.