A evolução in vitro é um método laboratorial criado para a evolução de moléculas, principalmente de proteínas. Por meio de mutações, o método busca novas propriedades de moléculas, objetivando criar novas proteínas e, com isso, intensificar o estudo e a cura de doenças, pelo desenvolvimento de novos fármacos. O grande desafio na evolução in vitro é criar o maior número possível de moléculas de proteínas que atinjam propriedades desejadas, uma vez que apenas uma fração infinitesimal das diversidades geradas utilizando-se seqüências de DNA é aproveitada. Para se obter moléculas com funcionalidade adequada por meio dessa técnica, é requerido muito tempo e aporte financeiro. Com o objetivo de avaliar computacionalmente a funcionalidade de proteínas variantes a partir das seqüências de aminoácidos buscando reduzir o custo e o tempo desprendido em laboratório, este trabalho propõe o uso de técnicas de computação inteligentes (evolução in silicio), baseadas em aprendizado de máquina e computação evolutiva. Para o emprego de técnicas de AM, bancos de dados com elevado número de informações são fundamentais. Neste sentido, escolheu-se investigar as moléculas mutantes de hemoglobina, uma vez que a quantidade de informações disponíveis sobre a mesma é bastante extensa na literatura. Os resultados obtidos mostram que é possível desenvolver algoritmos eficientes para determinar a funcionalidade de variantes de hemoglobina. Com esses resultados, busca-se contribuir no desenvolvimento de técnicas de evolução dirigida com suporte computacional / In vitro evolution is a laboratorial method developed to molecule evolution mainly proteins. By producing mutations, this method looks for new molecule properties, aiming achieve new proteins for the development of drugs for diseases. The great challenge of in vitro evolution is the development of the highest possible number of molecules that reaches desired properties. This objective is a great challenge to be transposed, since only one infinitesimal fraction of generated proteins using DNA sequencies is usefull to obtain molecules with the desired function. Besides high financial support and time are required to apply this technique. With the objective of evaluating computacionaly and functionality of proteins mutants starting from aminoacids sequences looking for to reduce the cost and the time loosened at laboratory, this work proposes the use of intelligent computation techniques based on learning of it conspires and evolutionary computation. On the other hand, when machine learning techniques are used, it is fundamental to access data mining with high number of information. In order to reduce these difficulties, this work proposes a machine learning (ML) based on approach to evaluate computationaly hemoglobin variants. ML techniques require, in general, large data base. In order to supply this requirement, hemoglobin variants were used because there is a large number of hemoglobin variants available in the literature. The obtained results shown that is possible to develop efficient algorithms to determine hemoglobin variant function. These results can contribute for development of molecule evolution techniques

This study is an analysis of the effects of formaldehyde, a chemical which is both recombinogenic and mutagenic, on white-ivory (wi) a highly mutable allele of the white locus of Drosophila. Formaldehyde is shown to significantly increase the reversion frequency of wi. The results of this investigation differ from earlier observations in three respects: (1) there is a positive correlation between RNA (ribonucleic acid) concentration and reversion frequency in the presence of formaldehyde, but reversion does not appear to be totally RNA dependent; (2) female germ cells are more sensitive than those of the male; and ( 3) a cluster of revertants has been recovered. These indicate that formaldehyde-induced wi reversion may involve a different mechanism than that proposed for formaldehyde-induced sex-linked lethals. Different mechanisms have been proposed to account for the induction of sex-linked lethals and for the increase in recombination. Formaldehyde-induced reversion of white-ivory follows the parameters of recombination.

The mutant-prevention concentration (MPC) is a novel susceptibility measurement defined by a concentration threshold that would require cells to contain two concurrent resistance mutations for growth. Pneuococcal pneumonia, infections caused by <i> Pseudomonas aeruginosa</i>, and urinary tract infections caused by Gram-negative bacilli represent three distinct clinical situations for which fluoroquinolone-resistance occurs. MPC results were defined and measured for fluoroquinolones against clinical isolates of <i>Citrobacter freundii, Enterobacter cloacae, Escherichia. coli, Klebsiella pneumoniae, P. aeruginosa,</i> and <i> Streptococus pneumoniae</i>. Against clinical isolates of <i>S. pneumoniae</i>, MPC results for six fluoroquinolones were measured. Based on their potential for restricting the selection of resistant mutants, the six fluoroquinolones, in descending order, were found to be gemifloxacin > moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of clinical isolates that lacked a known resistance mutation had a MPC value that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. These data identify gemifloxacin, moxifloxacin and gatifloxacin as good candidates for determining whether MPC can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of fluoroquinolone ¡Vresistant <i>S. pneumoniae</i>. MPC90 results for 155 clinical isolates of <i>P. aeruginosa </i>against ciprofloxacin and levofloxacin were 4 and 16 Ýg/ml, respectively. Serum drug concentrations reported previously for standard doses were above MPC90 for 5.5 hr for ciprofloxacin and 0 hr for levofloxacin. These data suggest that superior clinical performance of ciprofloxacin correlates with activity against resistant mutant subpopulations measured in vitro. MPC results were compared with minimum inhibitory concentrations (MIC) measurements preformed by agar dilution, and microbroth dilution and minimal inhibitory concentrations (MBC) for 100 clinical isolates of <i>C. freundii </i> (n=20), <i>E. cloacae</i> (n=20), <i>E. coli</i> (n=20), <i>K. pneumoniae</i> (n=20), and <i>P. aeruginosa</i> (n=20) for ciprofloxacin, levofloxacin and garenoxacin. MPC results were 2-to-8 fold higher than MIC or MBC results. Ciprofloxacin MPC results for <i>E.coli, C. freundii, E. cloacae, K. pneumoniae</i>, and <i>P. aeruginosa</i> were 0.5, 2, 1, 1, and 4 Ýg/ml, respectively. Levofloxacin, MPC results were were 1, 2, 4, 1, and 16 Ýg/ml, respectively. Garenoxacin, MPC were 1, 8, >8, 4, and >32 Ýg/ml, respectively. Garenoxacin had the highest MIC and MPC results and was the least active compound tested against isolates of <i>C. freundii, E. cloacae</i>, and <i>P. aeruginosa</i>. These data support the rational use of quinolones in the treatments of urinary tract infections and suppression of resistance. Incorporation of the MPC measurement into dosing strategies may preserve the longevity of antimicrobial compounds for future infectious diseases.

The mutant prevention concentration (MPC) is defined as the lowest antimicrobial concentration required to inhibit the growth of the least susceptible bacterial cell based on an inoculum of ≥109 colony forming units (CFUs). The current protocol for MPC testing is technically demanding and time-consuming which limits its implementation into clinical microbiology laboratories. In an attempt to simplify the current MPC protocol we developed a modified MPC method, the microbroth dilution method, which requires two fewer days to complete than the current or traditional method. MPC values were consistent for all organisms and strains tested using both the traditional MPC method and the modified microbroth dilution MPC method.<p> Tigecycline is the first of a new class of compound glycylcyclines- with potent in vitro activity against Gram-positive organisms including penicillin-resistant and multi-drug resistant <i>Streptococcus pneumoniae</i> (SP) and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). We measured minimum inhibitory concentration (MIC) and MPC values for tigecycline against 47 clinical isolates of SP and found that the MPC90 values were >500 fold higher than the MIC90 values. To determine if MPC testing of tigecycline against SP is impacted by blood in the medium, we developed a new medium able to sustain the growth of SP without the need for blood; solidified Todd-Hewitt broth (sTHB). The MPC90 values of tigecycline against SP on sTHB were only 2 fold higher than the MIC90 values. When blood was added to the sTHB, the MPC90 values again became much greater than the MIC90 values (> 256 fold higher). MPC results for <i>Staphylococcus spp.</i> against tigecycline were not impacted by blood in the medium.<p> Benzalkonium chloride (BAK) is a cationic surface-acting agent that acts on bacterial cells by disrupting the intermolecular interaction of the lipid bilayer. To determine if the <i>fluoroquinolones gatifloxacin</i> (Gfx) and moxifloxacin (Mfx) are more active (lower MIC values) in the presence of BAK, we conducted MIC, MPC, and time-kill assays. MIC testing showed that in the presence of 3.125 to 50 µg/ml of BAK, the MIC of Gfx and Mfx decreased by 8- to 5000-fold against clinical isolates of methicillin-susceptible <i>Staphylococcus aureus</i> (MSSA), MRSA, Coagulase-negative <i>Staphylococci</i>(CNS), SP, <i>Escherichia coli</i> (EC), and <i>Pseudomonas aeruginosa</i> (PA). MPC testing showed that the presence of 7 to 10 µg/ml of BAK, the MPC of Gfx and Mfx decreased by 32- to 1000-fold against clinical isolates of MRSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) showed that the killing activity of Gfx against clinical MRSA isolates was enhanced in the presence of BAK with a log10-reduction (percent kill) of 1.6 (76.08%) for Gfx alone at 180 minutes compared to a log10-redecution (percent kill) of 5.4 (100%) for Gfx plus BAK at 180 minutes.<p> Alexidine (Alx) is a bisbiguanide that has been used as an effective disinfectant in the dental industry and is potentially being developed for use as an antimicrobial agent for ocular infections. We conducted susceptibility testing of Alx using MIC testing, MPC testing, and time-kill assays against Gram-positive and Gram-negative pathogens. MIC testing showed that Alx is more active against Gram-positive pathogens than Gram-negative pathogens and showed better activity than the fluoroquinolones Gfx, Mfx, and levofloxacin (Lfx) against MRSA. The MPC values measured for MRSA and MSSA against Alx were non-reproducible using the traditional MPC method. Using the microbroth dilution MPC method, MPC90 values were found to be 32 fold higher than the MIC90 values. If the experimentally determined MPC values are true MPC values, initial MPC testing indicates that Alx may have a high likelihood for selecting for resistance, however, if the MPC values are not accurate it may be necessary to modify the MPC protocol in order to complete MPC testing of Alx against MRSA and MSSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) measured bactericidal activity (> 3 log10-reduction) against MRSA, MSSA, SP, and PA.

No description available.

The mutant-prevention concentration (MPC) is a novel susceptibility measurement defined by a concentration threshold that would require cells to contain two concurrent resistance mutations for growth. Pneuococcal pneumonia, infections caused by <i> Pseudomonas aeruginosa</i>, and urinary tract infections caused by Gram-negative bacilli represent three distinct clinical situations for which fluoroquinolone-resistance occurs. MPC results were defined and measured for fluoroquinolones against clinical isolates of <i>Citrobacter freundii, Enterobacter cloacae, Escherichia. coli, Klebsiella pneumoniae, P. aeruginosa,</i> and <i> Streptococus pneumoniae</i>. Against clinical isolates of <i>S. pneumoniae</i>, MPC results for six fluoroquinolones were measured. Based on their potential for restricting the selection of resistant mutants, the six fluoroquinolones, in descending order, were found to be gemifloxacin > moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of clinical isolates that lacked a known resistance mutation had a MPC value that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. These data identify gemifloxacin, moxifloxacin and gatifloxacin as good candidates for determining whether MPC can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of fluoroquinolone ¡Vresistant <i>S. pneumoniae</i>. MPC90 results for 155 clinical isolates of <i>P. aeruginosa </i>against ciprofloxacin and levofloxacin were 4 and 16 Ýg/ml, respectively. Serum drug concentrations reported previously for standard doses were above MPC90 for 5.5 hr for ciprofloxacin and 0 hr for levofloxacin. These data suggest that superior clinical performance of ciprofloxacin correlates with activity against resistant mutant subpopulations measured in vitro. MPC results were compared with minimum inhibitory concentrations (MIC) measurements preformed by agar dilution, and microbroth dilution and minimal inhibitory concentrations (MBC) for 100 clinical isolates of <i>C. freundii </i> (n=20), <i>E. cloacae</i> (n=20), <i>E. coli</i> (n=20), <i>K. pneumoniae</i> (n=20), and <i>P. aeruginosa</i> (n=20) for ciprofloxacin, levofloxacin and garenoxacin. MPC results were 2-to-8 fold higher than MIC or MBC results. Ciprofloxacin MPC results for <i>E.coli, C. freundii, E. cloacae, K. pneumoniae</i>, and <i>P. aeruginosa</i> were 0.5, 2, 1, 1, and 4 Ýg/ml, respectively. Levofloxacin, MPC results were were 1, 2, 4, 1, and 16 Ýg/ml, respectively. Garenoxacin, MPC were 1, 8, >8, 4, and >32 Ýg/ml, respectively. Garenoxacin had the highest MIC and MPC results and was the least active compound tested against isolates of <i>C. freundii, E. cloacae</i>, and <i>P. aeruginosa</i>. These data support the rational use of quinolones in the treatments of urinary tract infections and suppression of resistance. Incorporation of the MPC measurement into dosing strategies may preserve the longevity of antimicrobial compounds for future infectious diseases.

The mutant prevention concentration (MPC) is defined as the lowest antimicrobial concentration required to inhibit the growth of the least susceptible bacterial cell based on an inoculum of ≥109 colony forming units (CFUs). The current protocol for MPC testing is technically demanding and time-consuming which limits its implementation into clinical microbiology laboratories. In an attempt to simplify the current MPC protocol we developed a modified MPC method, the microbroth dilution method, which requires two fewer days to complete than the current or traditional method. MPC values were consistent for all organisms and strains tested using both the traditional MPC method and the modified microbroth dilution MPC method.<p> Tigecycline is the first of a new class of compound glycylcyclines- with potent in vitro activity against Gram-positive organisms including penicillin-resistant and multi-drug resistant <i>Streptococcus pneumoniae</i> (SP) and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). We measured minimum inhibitory concentration (MIC) and MPC values for tigecycline against 47 clinical isolates of SP and found that the MPC90 values were >500 fold higher than the MIC90 values. To determine if MPC testing of tigecycline against SP is impacted by blood in the medium, we developed a new medium able to sustain the growth of SP without the need for blood; solidified Todd-Hewitt broth (sTHB). The MPC90 values of tigecycline against SP on sTHB were only 2 fold higher than the MIC90 values. When blood was added to the sTHB, the MPC90 values again became much greater than the MIC90 values (> 256 fold higher). MPC results for <i>Staphylococcus spp.</i> against tigecycline were not impacted by blood in the medium.<p> Benzalkonium chloride (BAK) is a cationic surface-acting agent that acts on bacterial cells by disrupting the intermolecular interaction of the lipid bilayer. To determine if the <i>fluoroquinolones gatifloxacin</i> (Gfx) and moxifloxacin (Mfx) are more active (lower MIC values) in the presence of BAK, we conducted MIC, MPC, and time-kill assays. MIC testing showed that in the presence of 3.125 to 50 µg/ml of BAK, the MIC of Gfx and Mfx decreased by 8- to 5000-fold against clinical isolates of methicillin-susceptible <i>Staphylococcus aureus</i> (MSSA), MRSA, Coagulase-negative <i>Staphylococci</i>(CNS), SP, <i>Escherichia coli</i> (EC), and <i>Pseudomonas aeruginosa</i> (PA). MPC testing showed that the presence of 7 to 10 µg/ml of BAK, the MPC of Gfx and Mfx decreased by 32- to 1000-fold against clinical isolates of MRSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) showed that the killing activity of Gfx against clinical MRSA isolates was enhanced in the presence of BAK with a log10-reduction (percent kill) of 1.6 (76.08%) for Gfx alone at 180 minutes compared to a log10-redecution (percent kill) of 5.4 (100%) for Gfx plus BAK at 180 minutes.<p> Alexidine (Alx) is a bisbiguanide that has been used as an effective disinfectant in the dental industry and is potentially being developed for use as an antimicrobial agent for ocular infections. We conducted susceptibility testing of Alx using MIC testing, MPC testing, and time-kill assays against Gram-positive and Gram-negative pathogens. MIC testing showed that Alx is more active against Gram-positive pathogens than Gram-negative pathogens and showed better activity than the fluoroquinolones Gfx, Mfx, and levofloxacin (Lfx) against MRSA. The MPC values measured for MRSA and MSSA against Alx were non-reproducible using the traditional MPC method. Using the microbroth dilution MPC method, MPC90 values were found to be 32 fold higher than the MIC90 values. If the experimentally determined MPC values are true MPC values, initial MPC testing indicates that Alx may have a high likelihood for selecting for resistance, however, if the MPC values are not accurate it may be necessary to modify the MPC protocol in order to complete MPC testing of Alx against MRSA and MSSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) measured bactericidal activity (> 3 log10-reduction) against MRSA, MSSA, SP, and PA.

The desert ant Cataglyphis fortis is one of the hyperthermophilic species of Cataglyphis. It lives in the Sahara desert and forages during the hottest hours of the day when it can get up to 70˚C in the sand. The body temperature of the ant during the foraging runs can reach a maximum of 55˚C. Since C.fortis is one of few eukaryotic hyperthermophilic species, its proteins probably have a high thermostability. Investigating the thermostability can give valuable information about the principles of protein folding and stability in hyperthermophiles.Fatty acid binding proteins (FABPs) have an important role in the cell taking up and transporting fatty acids and regulating metabolic and inflammatory pathways. FABPs have been extensively studied and structures from several species have been determined. The determined structures of all FABPs are very similar why thermostability studies of FABP from C.fortis are highly relevant.Fluorescence spectroscopy is an easy and fast method to measure intrinsic protein fluorescence. Tryptophans were genetically introduced into three different positions in FABP to be used as environmental sensitive probes. Complementing the measurement results with a model of the 3D structure of FABP from C.fortis gave additional information about the ligand binding.The (local) thermostability of the mutants can be detected by shift in wavelength maximum during temperature ramping experiments. All mutants are stabilised in the presence of fatty acids. The mutant with tryptophan positioned closest to the supposed ligand binding residues (Y11W) is most affected. The mutant with tryptophan situated farthest from the supposed binding residues (Y52W) shows a stabilisation of Tm less evident than for Y11W. Thus, the structural changes following fatty acid binding are more obvious in the environment close to the binding site.However, the third mutant C87W shows no significant stabilisation although positioned closer to the fatty acid binding site than Y52. This is probably due to the size difference between the original and introduced amino acid in the mutation. Since the high value of the starting λmax for C87W implies that C87W is quite exposed to the aqueous solvent, the residue is likely to not have subsumed in the protein tertiary structure.Further, the myristic acid stabilise the melting temperature of all the mutants while octanoic acid only has a local effect of Y11W increasing the cooperativity. This implies different binding properties and that myristic acid stabilise the entire protein while octanoic acid only has a local stabilisation effect around the ligand binding site.

Early flowering is important for maize adaptation to short-season growing environments. Dwarfism, by preventing lodging, has the potential to increase grain yield. This thesis investigates three novel mutants of maize. The early flowering mutant (EarlyF) sheds pollen 1 to 5 days earlier than wild type plants. EarlyF, was shorter and developed fewer leaves than wild type plants, suggesting an earlier transition from vegetative to reproductive development. A candidate QTL for EarlyF maps to bin 7.03. The two allelic dwarf mutants, brachytic3-1 and brachytic3-2, have short internodes at maturity, resulting in severely reduced plant height. Despite being short, days to pollen shed and number of leaves were unchanged for both brachytic3-1 and brachytic3-2. brachytic3 maps to a ~ 7 Megabase region of bin 5.04. This thesis characterizes EarlyF, br3-1 and br3-2 and sets the stage for positionally cloning the mutations causing these mutants and has potential to contribute to maize improvement.

Cocoa (Theobroma cacao L.) is an important cash crop in the coastal areas of Papua New Guinea (PNG). In 2000, a mutant cocoa genotype MJ 12-226, with abnormal growth characteristics was identified at the Cocoa and Coconut Institute of PNG. The mutant, found among progenies of the cross Scavina 12 x Nanay 149, was characterized by dwarfing, small and narrow leaves, a small root system and strong branching habit. This study was designed to improve the understanding of the dwarf cocoa mutant and identify its relevance to future cocoa farming. The specific objectives are to investigate the feasibility of using a dwarf mutant of cocoa as a commercial rootstock for various hybrid cocoa clones, to study the genetics of this dwarf mutant and the role of plant hormones in differentiating this mutant from the normal phenotype. This study was carried out at the Cocoa and Coconut Institute of PNG, Rabaul, Papua New Guinea, and at the University of Queensland, Gatton Campus, Australia. The comparative study of mutant and normal cocoa seedlings revealed distinct vegetative differences between the two genotypes at the nursery stage. The largest effects of the mutant genotype are on stem elongation, short internodes, multiple stems, root growth and leaf growth. The agronomic results indicated that the mutant rootstock significantly reduced tree vigour at least in the early years. When used as a rootstock, the mutant had a significant dwarfing effect on scion growth of cocoa clones in the nursery and persisted in the field 12 months after planting. After that, the cocoa clones grafted on mutant rootstock reverted to normal growth. It was postulated that the accumulation of endogenous growth substances and cambial activity between the scion (normal) and the rootstock overcame the dwarfing effect of the mutant genotype. Yield and vegetative data collection from this trial ceased on May 2006 and recommenced in May 2007 to allow trees to recover from heavy canopy pruning due to cocoa pod borer attack. This resulted in one year loss of yield and vegetative data from this experiment. The first two years’ yield data indicated no significant differences between mutant, normal segregants and commercial rootstocks. However, cocoa clones bud grafted on these mutant rootstocks produced significantly greater yield at higher planting density compared with lower densities and this was sustained for two years. However, it might be expected that competition among cocoa trees would increase with increased size of trees, favouring low density planting and reducing the differential performance of the trees under high density planting with time. The major advantages of reducing tree vigour are to improve the harvest index, ease of harvesting, pruning and overall management of the trees. The continuation of this study to collect mature tree yield data (from fifth to eighth years) would confirm these results. This work has also demonstrated for the first time that cocoa clones grafted on mutant rootstock can produce similar yields to clones grafted on normal or commercial rootstocks. Moreover, this study has also established that cocoa clones derived from orthotropic scions can yield the same as plagiotropic scions when grafted on to either mutant or normal rootstocks. Therefore, it is proposed that mutant rootstocks and orthtropic scions should also be considered for future use in cocoa farming. The strong branching habit and dwarfing stature of the mutant suggested that the mutation affects the quantity or the balance of plant growth hormones, or both. The effect of exogenously applied gibberellic acid (GA3) on developmental processes in dwarf mutant and normal cocoa seedlings was studied. Both dwarf mutant and normal one month old cocoa seedlings responded to gibberellic acid (GA3) by accelerated longitudinal growth, especially seedling height. Paclobutrazol (PBZ) applied to normal cocoa seedlings altered the growth so that they almost resembled the mutant phenotype. It was postulated that PBZ prevented GA biosynthesis in the normal cocoa seedlings and therefore decreased bioactive auxin (IAA) which, in turn, then promoted lateral shoot growth; PBZ also slightly retarded plant growth. Synthetic auxin (IBA/NAA) significantly inhibited lateral bud sprouting and growth of intact, decapitated and nodal segments of mutant and normal cocoa seedlings compared with control and those treated with IAA. It appears that the mutant and normal cocoa genotypes have a similar capacity to metabolize IAA and this capacity was much greater than for synthetic auxins. The results suggested that auxin levels in the mutant seedlings were probably limiting, and therefore affected biosynthesis of gibberellic acid thus resulting in the dwarfing characteristics of the mutant cocoa genotype. However, analysis of endogenous IAA and GA1 levels at four months old did not reveal differences between the mutant and normal seedlings. The present genetic studies which included backcrossing, test crossing and selfing of F1 progenies confirmed earlier work on the mutant and showed that the mutant was probably heterozygous (Dd). The allele (DD) in mutant cocoa is probably lethal in nature because the phenotype of the homozygote was not observed amongst the mutant progenies. Mutant segregants were obtained in a 1:1 ratio only when the mutant clone was used as the female; however, when the mutant was used as male, very few mutant segregants were obtained. This suggested selective pollen viability (pollen grains carrying the D allele are nonviable) or an incompatible reaction between pollen tubes (D carrying pollen) and style or ovule. The selfing and test-crosses of F1 progenies resulted in deviation from the expected 3:1 segregation ratio to a 1:1 ratio.