Visible Mutations Research Articles

Post-Copulatory Sexual Selection in an Insect With High Levels of Mating Failure.

Sexual selection is not a single process. Instead, multiple processes of sexual selection can interact with respect to a given phenotype, in either reinforcing, independent, or conflicting directions. Here we consider how different processes of sexual selection interact in the seed bug Lygaeus simulans. This species is characterised by limited pre-copulatory sexual selection, but the potential for rather strong post-copulatory sexual selection. In particular, mating failure is common in this species, with around 40%-60% of copulations failing to result in the successful transfer of sperm. Mating failure is negatively correlated with female size, with smaller females being less likely to end up inseminated. We have recently argued that this pattern is best explained by cryptic male mate choice for large, more fecund females. Males therefore preferentially inseminate larger females. Here we explore how this potential cryptic male choice interacts with another component of post-copulatory sexual selection: sperm competition. We first manipulated male and female size variation, generating large and small, male and female, size classes. Using a visible mutant marker to assign paternity, we then double-mated females with males, in all combinations of male and female size. Our results showed that sperm competition outcomes were primarily driven by copulation duration, with longer copulations leading to greater paternity share for a male. We also confirmed that larger females are more likely to produce offspring than smaller females, as predicted by cryptic male choice for large females. This effect was again linked to copulation duration, with longer copulations less likely to lead to mating failure. While larger males tended to be more successful in sperm competition, especially if copulating second, female size had little effect on paternity, suggesting that cryptic male choice and sperm competition are acting relatively independently in this species.

Identification of Transposon Insertion Sites in MaizeMu-Tagged Mutants Using Mu-Seq

Mutator(Mu) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as theBonnMuresource, aMu-tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping ofMuinsertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) fromMu-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location ofMuinsertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maizeMu-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.

Muller and mutations: mouse study of George Snell (a postdoc of Muller) fails to confirm Muller's fruit fly findings, and Muller fails to cite Snell's findings.

In 1931, Hermann J. Muller's postdoctoral student, George D. Snell (Nobel Prize recipient--1980) initiated research to replicate with mice Muller's X-ray-induced mutational findings with fruit flies. Snell failed to induce the two types of mutationsof interest, based on fly data (sex-linked lethals/recessive visible mutations) even though the study was well designed, used large doses of X-rays, and was published in Genetics. These findings were never cited by Muller, and the Snell paper (Snell, Genetics 20:545-567, 1935) did not cite the 1927 Muller paper (Muller, Science 66:84, 1927). This situation raises questions concerning how Snell wrote the paper (e.g., ignoring the significance of not providing support for Muller's findings in a mammal). The question may be raised whether professional pressures were placed upon Snell to downplay the significance of his findings, which could have negatively impacted the career of Muller and the LNT theory. While Muller would receive worldwide attention, and receive the Nobel Prize in 1946 "for the discovery that mutations can be induced by X-rays," Snell's negative mutation data were almost entirely ignored by his contemporary and subsequent radiation genetics/mutation researchers. This raises questions concerning how the apparent lack of interest in Snell's negative findings helped Muller professionally, including his success in using his fruit fly data to influence hereditary and cancer risk assessment and to obtain the Nobel Prize.

Evaluation of Preclinical in vitro Cytotoxicity, Genotoxicity, and Cardiac-Toxicity Screenings of Hydrogenated Cannabidiol

Introduction Cannabidiol (CBD) is one of many naturally biosynthesized compounds produced by Cannabis sativa. There is limited information available in the literature on hydrogenated CBD (tetrahydro cannabidiol or H4CBD) ( Adams et al., 1940b ). As hydrogenated derivatives of tetrahydrocannabinol (THC) and CBD become increasingly popular in consumer markets, toxicological assessments are vital in identifying toxic characteristics, if any, caused by hydrogenated cannabinoids. Objectives Assessment of the preclinical toxicology of hydrogenated CBD is provided through the in vitro safety study of racemic H4CBD in hepatocytes, normal human lung fibroblasts (NHLF), and primary human neural progenitor (NPC) cell lines. The importance of these cell lines is related to major organs and is the primary focus in determining any major toxic characteristics when consuming products. The inclusion of the human ether-a-go-go related gene (hERG) patch clamp test, observes any inhibition of sodium and potassium ion channels related to the arrhythmia of the heart. Also, the AMES test was conducted to determine any carcinogenic characteristics that H4CBD might impose. Materials and Methods Plated NHLF, hepatocytes, and NPC were used in a preclinical 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay for cytotoxicity observations with the visible color change of cellular use of formazan, while a plated AMES test was conducted to monitor any visible mutations within Escherichia coli for carcinogenic activity. Plated cloned HEK293 cells were given set voltages to determine ion channel activity to determine if H4CBD causes inhibition within these pathways, which would mimic any arrhythmia potential in cardiomyocytes. Results Screening of the MTT assay had a median calculated 3.25 micromolar concentration where cell viability remained high in NHLF and NPC, with higher concentrations leading to decreased cell viability. A 3.25 micromolar concentration is also the median for hepatocytes, with a discrepancy in some of the data that could be accounted for by miscounting colonies. The hERG patch clamp test provided a zero net inhibition with values adding up to zero, determining that the compound did not inhibit normal processes within the ion channels of the plated HEK293 cells. The analysis of the different cell types revealed varying responses to H4CBD. NHLF exhibited a concentration-dependent reduction in cell viability, with sustained concentrations over 24 h at 6.25 µM resulting in a significant loss of viability. Conversely, hepatocytes showed a trend of decreased viability at longer exposure times and higher concentrations, but severe cytotoxicity was not observed. This suggests that hepatocytes are less susceptible to the cytotoxic effects of H4CBD compared to NHLF. In the hERG assay, H4CBD did not inhibit the action potentials within cardiomyocytes, indicating no inhibition of ion channels involved in cardiac function. This finding is important for assessing the potential cardiovascular effects of H4CBD. The AMES test results were negative, indicating that H4CBD did not demonstrate mutagenic activity in the tested strains. Conclusion This supports the conclusion that H4CBD does not possess carcinogenic potential based on this assay. Both human NPC and NHLF showed a median concentration of 3.25 µM before exhibiting a significant reduction in cell viability. This information is valuable for determining research or consumer usage limits for H4CBD. It is important to note that this study represents a preclinical assessment, and further studies are required. The experimental design followed protocols typically used for preclinical assessments of novel pharmaceuticals. These findings provide insight into the cytotoxic effects of H4CBD and contribute to establishing research and safety parameters as these compounds continue to gain attention.

Spectrum of chlorophyll mutations and morphological variations in Abelmoschus esculentus L. induced through gamma radiation

Okra [Abelmoschus esculentus (L.) Moench], is an economically exploited important traditional vegetable crop of the world. The present investigation examined the variability in induced chlorophyll mutants and othermorphological variations in okra. Seeds of two open pollinated popular varieties of okra namely Arka Anamika and Arka Abhay were irradiated with gamma doses of 30, 50 and 80 kR. The treatment 50 kR enhanced plant height, number of fruits per plant, fruit length, single fruit weight and total fruit yield per plant. Spectrum of several chlorophyll mutants were observed in the M1 generation. Other macro-mutants such as early and late flowering types, dwarf statured plants, leaf and flower mutants were also noticed at different doses of gamma radiation. The total number of visible mutation followed a trend of increasing frequency with the increase in dose of radiation.

Polar and non-polar fraction from Origanum vulgare spp. hirtum methanolic extract – differences in their bioactivity on Chlamydomonas reinhardtii test system

Aim: To compare the bioactivity of both polar and non-polar fraction of Origanum vulgare spp. hirtum methanolic extract on Chlamydomonas reinhardtii. Material and methods: The polar and non-polar fractions were derived from aerial parts of Origanum vulgare ssp. hirtum, collected during the flowering stage from the ex-situ collection of IBER-BAS. GC/MS analysis of both fractions was done following the standard protocol. The measured mass spectra were deconvoluted by the Automated Mass Spectral Deconvolution and Identification System (AMDIS), before comparison with the databases. Chlamydomonas reinhardtii 137C+ (WT) was used as a test system. Spot-test, cell survival fraction (SF), test of “visible mutations” and CFGE (for measurement of induced DNA double-strand breaks (DSBs)) were applied. Results: The polar fraction did not possess genotoxic, mutagenic as well as DNA-damaging effect. The situation with the non-polar fraction was quite different. Even at the lowest concentration of 250 ppm, cell survival was decreased by 60% (SF = 0.41 ± 0.08). Treatment with concentrations equal to/or greater than 500 ppm resulted in around 100% lethality. A mild mutagenic effect was obtained for the concentration of 250 ppm non-polar fraction (IM = 4.83 ± 0.004). Well-expressed and concentration-dependent induction of DSBs for even the strong DNA fragmentation was observed after the treatment with the non-polar fraction. Conclusions: The different bioactivity of the two fractions correlated well with their different chemical composition. The polar fraction, rich in sugars, organic acids and flavonoid glycosides, did not possess genotoxic and mutagenic potential. The strong genotoxic potential of the non-polar fraction might be related to carvacrol content (37.08%), which is not present in the composition of the polar fraction. To the best of our knowledge, this study provides the first information that the carvacrol-rich non-polar fraction of Origanum vulgare spp. hirtum methanolic extract possesses genotoxic, mutagenic and DNA damaging effect on some low eukaryotes, such as C. reinhardtii. Further experiments with carvacrol should be done in order to clarify the exact mechanism of action.

On the mode of action of Origanum vulgare spp. hirtum methanolic extract and essential oil on Chlamydomonas reinhardtii

Aim: To reveal whether methanolic extract and essential oil from Origanum vulgare subsp. hirtum in doses causing even low levels of mortality in aphids, would have harmful effects on plants-genotoxic, mutagenic and/or DNA damaging. Materials and methods: Aerial parts of Origanum vulgare ssp. hirtum from the ex-situ collection of IBER, BAS during flowering were collected. Extraction and isolation procedures, as well as GC/MS analysis of essential oil and methanolic extract were performed by standard protocols. The components were identified by comparing their relative retention times to the retention times of authentic standards, and with mass spectra with the NIST. Test system: Chlamydomonas reinhardtii strain 137 C+ (WT). Endpoints: “clonal” assay, the test of “visible mutations”, constant field gel electrophoresis. Statistics: GraphPad Prism version 6.04 (San Diego, USA) and One-way Analysis of Variance ANOVA with multiple comparisons using the Tukey method. Results: A good correlation was observed between chemical composition of essential oil and methanolic extract, and their mode of action. Our genotoxic and double strand breaks results demonstrated mild genotoxic and statistically non-significant DNA damaging potential of methanolic extract and concentration-dependent well - expressed genotoxic and DNA damaging potential of essential oil. A good relationship between increased double strand breaks levels and decreased survival might be related to one of the main constituents of essential oil, suspected to be carvacrol. No mutagenic effect for ME and EO was found. Conclusion: Well-expressed toxic/genotoxic capacity of essential oil, as well as its capacity to damage DNA inducing double strand breaks, but the absence of mutagenic potential, could be considered as a good reason to recommend Origanum vulgare subsp. hirtum essential oil as a promising candidate for purposes of “green” technologies.

An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes

BackgroundGenome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies.ResultsWe sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used ‘half-seed’ explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75–100% mutation efficiencies. Indel frequencies varied widely among plants (3–100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27–100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants.ConclusionsUsing simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.

CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

BackgroundGene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in various filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™).ResultsWe present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0–40% of isolates. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum.ConclusionsUsing an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.

Induction of breeding-valuable mutations in Triticum aestivum L. under the influence of physical and chemical mutagenic factors of the environment

Aim. The use of induced mutagenesis opens greater possibilities to drastically improve cultivated plants genetically. The areas of local and mass anthropogenic pollution have unique conditions of the effect of mutagenic factors, and they require the studying of their possible use while developing parental material for winter wheat breeding. The induction frequency of breeding-valuable mutations in Triticum aestivum L., affected by the contamination with physical and chemical mutagenic factors of the environment, was studied. Methods. The choice of mutants was made in М2 and М3 generations, using record techniques and separating visible mutations, phonological observations, biometric indicator analysis. Results. The pollutants of the environment cause the frequency increase of breeding-valuable mutations in T. aestivum L. by 7.5–12.5 times under the effect of the contamination with natural radioisotopes of the area of uranium ore mining, by 2.1–19.7 times – with heavy metals of industrial enterprises, and 4.1–9.8 times – with pesticides, prohibited and not suitable for use, and toxic wastes in the places of their storage. Spectrum is predominantly represented with low-grown forms, forms with intensive growth, a long-cylindrical spike, and it depends on the nature of a polluting agent and a genotype of a plant. Conclusions. The increase of frequency and the broadening of spectrum of breeding-valuable mutations, due to the induced mutagenesis with the environmental pollution, make it possible to use it for the implementation of breeding-genetic programs aimed at improving wheat cultivars.
 Keywords: Triticum aestivum L., mutagenic factors, mutative variability, breeding-valuable mutations.

The relationship of Wolbachia infection and different phenotypes in the Drosophila melanogaster natural populations from radioactively polluted and clear areas in Ukraine

Aim. The study was performed to investigate the relationship between Wolbachia infection and phenotypes that distinct from wild-type of Drosophila melanogaster from different localities in Ukraine including those from Chornobyl Exclusion Zone during 2013–2014. Methods. We have established isofemale lines from populations: Uman’, Inkerman, Odesa, Varva, Kyiv, Drogobych, Yaniv, Poliske, Chornobyl, and Chornobyl Nuclear Power Plant (NPP). The ambient radiation (µSv/h) was measured in the sample sites. The flies were reared in the laboratory through two generations. We carried out the observation of F2 flies for visibly detectable phenotypes. According to whether the trait was inherited, observations were separated into three categories: with deviations of posterior cross-vein (C2) (incomplete penetrance), visible phenotypic changes (non-inherited) and mutations (inherited). Polymerase chain reaction (PCR) with primers specific to the 16S rRNA and Wolbachia surface protein (wsp) genes were used to determine infection presence in isofemale lines of the flies established for each population. Results. Examination of different phenotypes indicates that the highest mutation rate (but not C2 and not inherited changes) is in populations from Chornobyl Exclusion Zone and, therefore, connection with ambient radiation was detected (p = 0.0241). Generalized mixed linear regression has shown evidence that the presence of phenotypes with defects of C2 vein varies with endosymbiont infection presence (p = 0.03473) in the populations from radioactively polluted areas. Conclusion. Wolbachia is not related to occurring phenotypes neither with phenotypic changes nor with mutations, at least in surveyed populations. However, C2 defected phenotypes relates to the bacterial presence in populations from the contaminated area. Nonetheless, the origin of this relationship is unknown and the mechanisms of such a connection require further research. Keywords: Drosophila melanogaster, Wolbachia, endosymbiont, ambient radiation, mutation, phenotypic change, posterior cross-vein.

Efficiency of the use of mutations, induced on radiation-contaminated areas, when improving winter wheat cultivars

Aim. Breeding of Triticum aestivum L. cultivars requires the development of generically diversified primary material. Induced mutagenesis plays an important role in the solution of the issue. Mutagenic activity of radionuclide contaminations of the alienation zone of ChNPP and industrial facilities of uranium manufacture was studied to determine the efficiency of their use when developing the material of winter wheat which is valuable for breeding. Methods. The selection of mutants was done using the methods of recording and singling out of visible mutations, phenological observations, analysis of biometric indicators, determination of total yield capacity and its components. Results. Chronic effect of the radiation of radionuclide contaminations of the alienation zone of ChNPP and the territory of uranium ore mining results in the increase of mutation variability of winter wheat by 8–14.9 and 9.8–12.6, respectively. Mutation spectrum included 24.29–49.34 % of those valuable for breeding. Productive mutants, which exceeded primary cultivars in crop capacity by 2.7–6.9 %, were developed. Mutant samples, whose high yield capacity was supplemented with grain quality enhancement or its maintenance at the level of a primary form, were identified. Conclusions. The expansion of genetic variety of primary breeding material due to mutagenesis, induced by radionuclide contaminations, creates the opportunities for its use in hybridization aimed at the implementation of breeding-genetic programs of the improvement of wheat cultivars.
 Keywords: Triticum aestivum L., radionuclide contamination, mutation variability, valuable for breeding mutations, productive forms.

Peculiarities of Mutation Process in X Chromosome of Drosophila melanogaster Z3314 Line from Zvenigorodka (Ukraine) Natural Population

The Drosophila melanogaster Z3314 line isolated from a Zvenigorodka (Ukraine) natural population is characterized by the manifestation and emergence of a wide spectrum of molecular aberrations. Among them, two types (the wing venation anomaly and violation of the leg segmentation) were the most represented. It was demonstrated that the frequency of manifestation (penetrance) and the expressiveness of these phenotypic aberrations increase with an increase in the temperature. When the Z3314 line is bred in the laboratory, autosomal visible rase (ra: 3–97.3) mutation, which leads to reduction of a part of dorso-central and scutellaria macrochaetae, was detected (isolated and identified). A number of genetic peculiarities that determined the consistency and prospects of the study were found during the mutation process study in the Z3314 line. The Z3314 line is characterized by a high frequency of the emergence of visible mutations in the X-Z3314 chromosome, which persisted for a long time of the breeding under laboratory conditions (from 2003 to 2011). Locus-specific high genetic instability in the singed locus in the X-Z3314 chromosome persisted from the moment of emergence of the first mutant alleles in 2006 until the end of the study. The emergence of mutations was observed both during the line breeding “inside” (in the case of brother–sister crossings) and after the crossings of the X-Z3314 chromosome carrier males with females of the С(1)DX,ywf/Y laboratory line with linked X chromosomes.

The analysis of mutation variability of winter wheat under soil contamination with heavy metals of industrial discharges

The aim of the research was to study genetic consequences of soil contamination with heavy metals in the zones of industrial discharges according to frequency and spectrum of visible mutations of winter wheat.Methods. The identification and record of mutation frequency and spectrum of winter wheat were carried out in generations М2 і М3. All families of the plants with changed signs were carefully examined during major stages of their growth and development. The adequacy of the difference between averages of experimental variants and the control was estimated by the standard of Student and Fisher.Result. Soil heavy metals of the industrial discharges of Burshtynska TPS, SC “Poltavchimmash”, SPC “Specialized company for thermal processing of firm household garbage” in Kharkiv city, SJC “Luhansk accumulator producing company”, UC “Lubny water supply station” and near B. Khmelnytskyi Street in Kostiantynivka caused the increase of visible mutation frequency of winter wheat varieties A'lbatros odes'kyi and Zymoiarka by 2.1-4.9 times. Mutation spectrum included several inherited changes which with high frequency were induced by soil contamination with heavy metals of the areas of all the studied objects: late ripening, high- and short-grown, long, dense, loose spike. Original and rare mutations were found among them: wide leaf, lack of wax film, yellow peak of a flag leaf, antocyanin spike husk, spike with twisted axis, light-green leaf.Conclusions. Soil contamination with heavy metals of industrial discharges causes considerable increase of mutation variability of winter wheat, which can be a genetic threat to living organisms. Mutation spectrum includes typical inherited changes which together with original and rare mutations can be used as indicators of industrial contamination of the environment with heavy metals.

A Gene-Oriented Haplotype Comparison Reveals Recently Selected Genomic Regions in Temperate and Tropical Maize Germplasm.

The extensive genetic variation present in maize (Zea mays) germplasm makes it possible to detect signatures of positive artificial selection that occurred during temperate and tropical maize improvement. Here we report an analysis of 532,815 polymorphisms from a maize association panel consisting of 368 diverse temperate and tropical inbred lines. We developed a gene-oriented approach adapting exonic polymorphisms to identify recently selected alleles by comparing haplotypes across the maize genome. This analysis revealed evidence of selection for more than 1100 genomic regions during recent improvement, and included regulatory genes and key genes with visible mutant phenotypes. We find that selected candidate target genes in temperate maize are enriched in biosynthetic processes, and further examination of these candidates highlights two cases, sucrose flux and oil storage, in which multiple genes in a common pathway can be cooperatively selected. Finally, based on available parallel gene expression data, we hypothesize that some genes were selected for regulatory variations, resulting in altered gene expression.

Evidence of hermaphroditism and sex ratio distortion in the fungal feeding nematode Bursaphelenchus okinawaensis.

Nematodes have many different reproductive strategies along with their divergent life histories; the ability of hermaphrodite to self- and cross-fertilize is useful for genetic manipulation. Here, we demonstrate the hermaphroditism of the fungal feeding nematode Bursaphelenchus okinawaensis, which was formerly described as a parthenogenetic nematode, and we show its other unique sexual characteristics. To determine that it is hermaphroditic, we performed the following experiments: observation of the pronuclear and chromosome behavior during oogenesis and early embryogenesis; observation of spermatogenesis during the fourth larval stage; investigation of sperm utilization; and investigation of phenotypic segregation after cross-mating using a chemically induced visible mutant. We then investigated the mating preferences and spermatid size difference between males and hermaphrodites. B. okinawaensis males successfully mated only with sperm-depleted old hermaphrodites, and the spermatid sizes of males were almost the same as those of hermaphrodites. Moreover, the sex ratio of cross-fertilized progeny was highly skewed toward hermaphrodites. B. okinawaensis is phylogenetically distant from established model nematodes such as C. elegans and is more closely related to some economically relevant parasitic nematodes. This newly discovered hermaphroditic nematode has great potential for evolutionary and parasitological research.

Chromosomal localization of microsatellite loci in Drosophila mediopunctata

Drosophila mediopunctata has been used as a model organism for genetics and evolutionary studies in the last three decades. A linkage map with 48 microsatellite loci recently published for this species showed five syntenic groups, which had their homology determined to Drosophila melanogaster chromosomes. Then, by inference, each of the groups was associated with one of the five major chromosomes of D. mediopunctata. Our objective was to carry out a genetic (chromosomal) analysis to increase the number of available loci with known chromosomal location. We made a simultaneous analysis of visible mutant phenotypes and microsatellite genotypes in a backcross of a standard strain and a mutant strain, which had each major autosome marked. Hence, we could establish the chromosomal location of seventeen loci; including one from each of the five major linkage groups previously published, and twelve new loci. Our results were congruent with the previous location and they open new possibilities to future work integrating microsatellites, chromosomal inversions, and genetic determinants of physiological and morphological variation.

Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster

The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in 1910, when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters.

Photogenic Flies—A review of Chyb and Gompel's Atlas of Drosophila Morphology: Wild-type and Classical Mutants

Photogenic Flies—A review of Chyb and Gompel's Atlas of Drosophila Morphology: Wild-type and Classical Mutants

Metastatic NSCLC: Can molecular biology guide surgical decision making?

e18045 Background: Guidelines do not recommend surgical resection of metastatic non-small cell lung cancer (NSCLC), with the exception of single brain or adrenal gland lesions, and further resection in the event of a new lung metastasis is generally not indicated. A different therapeutic approach is used in patients with multiple primary lesions. Can molecular alterations help to distinguish between independent primary tumors and metastatic lesions? Methods: A 74-year old woman underwent bilateral inferior lobectomy in May 2008 for two different synchronous NSCLC (adenocarcinoma and adenocarcinoma in situ, formally known as “bronchioloalveolar carcinoma” – J Thorac Oncol 2011;6:244–285). Three months after surgery the patient developed a single symptomatic brain metastasis. A CT scan also highlighted a suspect lingular lesion of the lung. Stereotaxic brain radiotherapy was carried out followed by systemic platinum-based chemotherapy. The brain metastasis showed very good radiological partial remission (PR) while the lung lesion remained stable after 4 cycles of chemotherapy. After two years’ follow-up the brain metastasis remained in PR whereas the lung lesion had progressed, and fine needle aspirate revealed adenocarcinoma. Results: Molecular analysis showed that EGFR gene status was wild type in all three tumors, whereas K-ras gene status differed. With regard to the two synchronous tumors, a barely visible G12C K-ras gene mutation was found in the bronchioloalveolar carcinoma, while a G12D K-ras mutation was detected in the adenocarcinoma. Moreover, K-ras gene status in the lingular node revealed a G12A mutation. On the basis of these results we hypothesized the presence of a third lung cancer and it was decided to proceed with surgery. The patient underwent laparoscopic lingulectomy which confirmed the diagnosis of adenocarcinoma with the same G12A K-ras mutation. Conclusions: This case highlights the importance of the molecular characterization of tumors in patients with multiple lesions. Different alterations within a single gene in different lesions are indicative of an independent clonal origin and suggestive of separate primary tumors rather than secondary lesions, thus facilitating treatment decision making.