A targeted screen to detect recessive mutations that have quantitative effects

A key problem in mutagenesis research is developing methods that are sufficiently sensitive to detect a wide range of abnormal phenotypes. Major variants may be easy to identify, but it can be difficult to detect mutations that have subtle effects, particularly on a complex genetic background. This paper describes a targeted mutagenesis protocol with enough sensitivity to detect recessive mutations that have modest quantitative effects. The procedure relies on consomic inbred strains of mice—strains in which one homologous pair of chromosomes of an inbred strain has been replaced with the corresponding pair from a donor strain. Mice that carry the desired donor chromosome—the target of the screen—are mutagenized and bred back to the original recipient strain. The first-generation progeny (G1) that are heterozygous only for the donor chromosome are also bred back to the recipient strain. G2 animals that inherit nonrecombinant donor chromosomes are identified by genotyping. These animals may be backcrossed repeatedly to the recipient strain to dilute off-target mutations, but ultimately, nonrecombinant G2 animals are bred to each other. Their G3 progeny are genotyped at markers spaced at 5- to 10-cM intervals to identify mating pairs that are homozygous for shared segments of the mutagenized donor chromosome. Entire litters of G4 progeny that are homozygous for defined intervals are screened. By comparing phenotypes within and among litters of nearly isogenic G4 animals, mutations can be verified and simultaneously mapped with a precision of 5–10 cM. This method has the potential to consistently detect mutations that have effects on trait means of well under one standard deviation. Received: 29 December 1998 / Accepted: 17 March 1999     

Phenotypic and Genetic Analysis of a Mutant of Heterorhabditis bacteriophora Strain HP88

Induction and characterization of a morphological mutant are described for Heterorhabditis bacteriophora strain HP88. A homozygous inbred line was used as the base population for mutagenesis and genetic analysis of mutations. Mutagenesis was induced by exposing young hermaphrodites to 0.05 M ethyl methanesulfonate. A dumpy mutant (designated Hdpy-l) was isolated from the F₂ generation of the mutagenized population. Morphological studies with light and scanning electron microscopy revealed that the head region of the adult stage was compressed. The head region of the infective juvenile was distorted and the mouth open. Backcross with the original population was successful only between mutant hermaphrodites and wild type males; 50-100 percent of the progeny of this cross maintained the dumpy phenotype, indicating that the ratio between self- and external fertilization of the eggs is > 1 and that the dumpy mutation is recessive.     

Nullisomic Tetrahymena. II. a Set of Nullisomics Define the Germinal Chromosomes

Crosses of a diploid Tetrahymena thermophila to a strain with a haploid germinal nucleus result in chromosome loss during meiosis in the haploid. The resulting monosomics can be made nullisomic by a special cross that induces homozygosis of a meiotic product of the germinal nucleus, but retention of the parental somatic nucleus. The creation and testing of single nullisomics for three of the five chromosome pairs and a triple nullisomic missing another pair is presented. Taken together, these strains make possible a series of crosses in which all but one of the chromosomes is missing in one parent. This set of nullisomics can, therefore, be used to map any mutation in Tetrahymena to a specific chromosome.     

Temperature-Sensitive Lethal Mutations on Yeast Chromosome I Appear to Define Only a Small Number of Genes

A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N -nitrosoguanidine and screened for temperature-sensitive (Ts^- ) mutants capable of growth on rich, glucose-containing medium at 25° but not at 37°. Recessive mutations induced on chromosome I are expressed, whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely. — Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss. — The available information about the size of chromosome I suggests that it should contain approximately 60–100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts^--lethal phenotype. — During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.     

Genomewide two-generation screens for recessive mutations by ES cell mutagenesis

Forward genetic mutation screens in mice are typically begun by mutagenizing the germline of male mice with N-ethyl-N-nitrosourea (ENU). Genomewide recessive mutations transmitted by these males can be rendered homozygous after three generations of breeding, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of embryonic stem (ES) cells. Here we demonstrate the feasibility of performing genomewide mutation screens with only two generations of breeding. Mice potentially homozygous for mutations were obtained by crossing chimeras derived from ethylmethane sulfonate (EMS)–mutagenized ES cells to their daughters, or by intercrossing offspring of chimeras. This strategy was possible because chimeras transmit variations of the same mutagenized diploid genome, whereas ENU-treated males transmit numerous unrelated genomes. This also results in a doubling of screenable mutations in a pedigree compared to germline ENU mutagenesis. Coupled with the flexibility to treat ES cells with a variety of potent mutagens and the ease of producing distributable, quality-controlled, long-term supplies of cells in a single experiment, this strategy offers a number of advantages for conducting forward genetic screens in mice.     

Genetic Characterization of the Region between 86f1,2 and 87b15 on Chromosome 3 of DROSOPHILA MELANOGASTER

The region between 86F1,2 and 87B15 on chromosome 3 of Drosophila melanogaster, which contains about 27 polytene chromosome bands including the 87A7 heat-shock locus, has been screened for EMS-induced visible and lethal mutations. We have recovered 268 lethal mutations that fall into 25 complementation groups. Cytogenetic localization of the complementation groups by deficiency mapping is consistent with the notion that each band encodes a single genetic function. We have also screened for mutations at the 87A7 heat shock locus, using a chromosome that has only one copy of the gene encoding the 70,000 dalton heat-shock protein (hsp70). No lethal or visible mutations at 87A7 were identified from 10,719 mutagenized chromosomes, and no female-sterile mutations at 87A7 were recovered from the 1,520 chromosomes whose progeny were tested for female fertility. We found no evidence that a functional hsp70 gene is required for development under laboratory conditions.     

Elevated 3-hydroxypropionaldehyde production from glycerol using a Citrobacter freundii mutant

A mutant strain of Citrobacter freundii capable of elevated 3-hydroxypropionaldehyde production from glycerol was isolated using chemical mutagenesis and a screening protocol. The protocol involved screening mutagenized bacterial cells on solid minimal medium containing 5 % (v/v) glycerol. Colonies were picked onto duplicate solid minimal medium plates and one plate was stained with 1 % (w/v) phloroglucinol. Those colonies staining red were further screened and a mutant, HPAO-1, was identified. The mutant strain produced a several-fold higher 3-hydroxypropionaldehyde concentration than did the parent strain when grown on 5 % (v/v) glycerol. The ratio of culture volume to flask volume influenced 3-hydroxypropionaldehyde production by the mutant cells compared to the parent cells. Aldehyde production was highest when the mutant strain was grown on 5 % (v/v) glycerol at a ratio of culture volume to flask volume of 1:3 or 1:12.5.     

Karyotypic normalcy and quasi-normalcy of developmentally totipotent mouse teratocarcinoma cells

Malignant mouse teratocarcinoma cells are, in some cases, able to undergo normal, complete differentiation after injection into blastocysts. Thus far, only three lines—of unrelated origin—have been found (all in this laboratory) to be developmentally totipotent in blastocyst tests. The karyotypes of these lines, and their somatic- and germ-cell derivatives, were investigated by G-banding methods, as a possible clue to their developmental superiority. The first, OTT 6050 (129 strain), is an embryo-derived induced tumor maintained as an ascites transplant line. Its stem cells (from embryoid body “cores”) have 40 chromosomes in the modal class, which comprises two subclasses: one all normal and one with a metacentric chromosome (isochromosome-8). However, mosaic animals from injected blastocysts have only the normal subclass in their teratocarcinomaderived cells; all are of $\text{X}\text{Y}$ male sex chromosome type. Presence of the Y chromosome was verified after transmission through the germ line of two fertile mosaic males, in their F₁ male progeny. The second teratocarcinoma line, 72484-395 (LT strain), is a spontaneous ovarian solid tumor maintained by subcutaneous transplantation. Karyotypes of cells from the tumor, and also of teratocarcinoma-derived cells in mosaic animals, were normal and of $\text{X}\text{X}$ female sex chromosome type. Karyotypes of the F₁ progeny, from tumor-strain germ cells of a fertile mosaic female, were also normal. The third line, NG 2 (129 strain), is a mutant clonal in vitro line deficient in hypoxanthine phosphoribosyltransferase. It originated from an embryo-derived experimental tumor (OTT 5568) that was established in culture (PSA1 line); the culture was then mutagenized and selected for 6-thioguanine resistance. The NG 2 line proved to be quasi-normal, with only two karyotypic anomalies: trisomy of chromosome 6 and $\text{X}\text{O}$ female sex chromosome constitution. Thus, developmental totipotency in all three lines, including one maintained in vitro, is accompanied by karyotypic normalcy or near-normalcy. Other culture lines reported to be aneuploid have not yet given evidence of totipotency. Karyotypic normalcy may therefore have predictive value useful in choosing teratocarcinoma lines with relatively high developmental prospects. This is of importance in identifying those mutant lines that would be promising candidates for introduction, via blastocyst injection, of specific mutant genes into mice.     

The characterization of color mutations in Bangiaceae (Bangiales, Rhodophyta)

The color mutations in Bangiaceae were investigated by treating the blades, conchocelis and conchospores phase of Bangia sp., Porphyra yezoensis, and P. haitanensis sampled in China with mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). A high percentage of mutation in different expression characteristics in all three phases were shown within optimum mutagen concentrations. Among mutagenized blades, mutations occurred on single cells, which is a direct outcome of mutation of haploid cells. The mutation of mutagenized conchocelis resulted in a two-step process: low-level expression in conchocelis phase, and high-level expression in progeny, explaining that mutation took place in diploid cells. The mutations of conchospores were expressed immediately at germination of spores, indicating a change in ploidy. This paper reports the process of meiosis and its effect on frond development, and the relation between color mutations and morphological characteristics expressed by mutations in Bangiaceae.     

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.     

On the Genetic Control of Genes Located in the Sex-Chromosome Heterochromatin of DROSOPHILA MELANOGASTER

The genetic properties of a recessive autosomal point mutant, "daughterless" (symbol: da), are described. They are: (1) da maps in the euchromatin of 2L at about position 39 on the genetic map and between 27D and 31E on the salivary map; (2) when homozygous in females, da causes the production of unisexual male progenies owing to sex differential zygote mortality in the egg stage; (3) da has no effect on the progeny of mutant males; (4) female zygotes die, while at least some male zygotes survive, irrespective of the number of Y chromosomes or the amount of X-chromosome heterochromatin carried by either the mutant female or her progeny; (5) homozygous da progeny of heterozygous parents also show sex differential survival favoring males indicating a da effect in development as well as in oogenesis, but with the developmental effect much less pronounced; (6) extra heterochromatin from either the X or Y chromosome in either the mother or her progeny can reduce the mortality caused by da in development (and, therefore, conceivably can also reduce the abnormality in oogenesis, although this is obscured by the severity of the maternal effect.)—From these results, it is suggested that (1) da regulates either the activity of structural genes in the sex chromosome heterochromatin or the activity or stability of their products, and (2) it is a defective product of sex chromosome heterochromatic genes that causes the abnormalities resulting in the observed mortality of heterozygous progeny of homozygous mutant mothers and of homozygous mutant progeny of heterozygous mothers.—The striking parallels with the properties of the gene, "abnormal oocyte" are noted as is the close linkage between the two loci. The possibility of a special sex-chromosome-heterochromatin-regulator region on chromosome 2 is considered.     

A yeast strain for simultaneous detection of induced mitotic crossing over,mitotic gene conversion and reverse mutation

A diploid yeast strain is described which can be used to study induction of mitotic crossing over, mitotic gene conversion and reverse mutation.Mitotic crossing over can be detected visually as pink and red twin sectored colonies which are due to the formation of homozygous cells of the genotype ade240/ade240 (deep red) and ade-2-119/ade2-119 (pink) from the originally heteroallelic condition ade2-40/ade2-119 which forms white colonies.Mitotic gene conversion is monitored by the appearance of tryptophan non-requiring colonies on selective media. The alleles involved are tryp5-12 and trp5-27 derived from the widely used strain D4.Mutation induction can be followed by the appearance of isoleucine non-requiring colonies on selective media. D7 is homoallelic ilv1-92/ilv1-92. The isoleucine requirement caused by ilv1-92 can be alleviated by true reverse mutation and allele non-specific suppressor mutation.The effects of ethyl methanesulfonate (EMS), nitrous acid, ultraviolet light and hycanthone methanesulfonate were studied with D7 stationary phase cells. Mitotic crossing over as monitored by red/pink twin sectored colonies was almost equally frequent among normal and convertant cells. This showed again that mitotic recombination is not due to the presence fo a few cells committed to meiosis in an otherwise mitotic cell population.The dose-response curves for induction of mitotic gene conversion and reversion of the isoleucine requirement were exponential. In contrast to this, the dose-response curve for induction of twin sectored red and pink colonies reached a plateau at doses giving about 30% cell killing. This could partly be due to lethal segregation in the progeny of treated cells.None of the agents tested would induce only one type of mitotic recombination, gene conversion or crossing over. There was, however, some mutagen specificity in the induction of isoleucine prototrophs.     

Localization of Structural and Regulatory Genes for Phosphodiesterase in Wheat (TRITICUM AESTIVUM)

Genes (Pde-A3; Pde-B3; Pde-D3) for phosphodiesterase (PDE; E.C. 3.1.4.1.) isoenzymes in hexaploid wheat were located on the three homoeologous chromosomes of group 3 by testing the electrophoretic banding pattern of monosomic, nullisomic and nullisomic/tetrasomic compensation lines of "Chinese Spring" variety. In plants nullisomic for chromosome 5B, the 3D structural gene is not expressed and this lack of expression can be overcome by four doses of either homoeologous chromosome 5A or 5D. Our data conclusively indicate that there are genes on group 5 chromosomes which positively control the expression of the 3D structural gene. In addition, the expression of the "regulatory genes" is dosage dependent. Thus, our study reveals a complex interaction of the three genomes of wheat for regulation of PDE gene expression.     

C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy

Whole-genome sequencing (WGS) is becoming a fast and cost-effective method to pinpoint molecular lesions in mutagenized genetic model systems, such as Caenorhabditis elegans. As mutagenized strains contain a significant mutational load, it is often still necessary to map mutations to a chromosomal interval to elucidate which of the WGS-identified sequence variants is the phenotype-causing one. We describe here our experience in setting up and testing a simple strategy that incorporates a rapid SNP-based mapping step into the WGS procedure. In this strategy, a mutant retrieved from a genetic screen is crossed with a polymorphic C. elegans strain, individual F2 progeny from this cross is selected for the mutant phenotype, the progeny of these F2 animals are pooled and then whole-genome-sequenced. The density of polymorphic SNP markers is decreased in the region of the phenotype-causing sequence variant and therefore enables its identification in the WGS data. As a proof of principle, we use this strategy to identify the molecular lesion in a mutant strain that produces an excess of dopaminergic neurons. We find that the molecular lesion resides in the Pax-6/Eyeless ortholog vab-3. The strategy described here will further reduce the time between mutant isolation and identification of the molecular lesion.     

Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5.

Effects of p-aminobenzoic acid (PABA) and of 4-[(2-oxo-3-bornylidene)methyl]-phenyl trimethylammonium methylsulfate (OMM), two components used in sunscreen formulations, on the mutagenicity of UVB irradiation are compared in three genetic assay systems. A haploid strain of Saccharomyces cerevisiae XV185-14C was used to measure reverse mutations at three loci. The diploid strain D5 of Saccharomyces cerevisiae was used to screen for reciprocal mitotic recombination. The induction of forward mutations was measured in Chinese hamster V79 cells. Our results indicate that UVB irradiation induced HGPRT- mutants in V79 cells, reverse mutations in Saccharomyces cerevisiae strain XV185-14C, and mitotic crossing over and other genetic alterations in Saccharomyces cerevisiae strain D5. V79 Chinese hamster lung cells were the most sensitive to UVB irradiation, followed by Saccharomyces cerevisiae haploid strain XV185-14C and the diploid strain D5. PABA and OMM were both capable of protecting all three types of cells from UVB irradiation regarding both lethality and induction of various types of genetic alterations. At higher concentrations (above 10(-5) M), OMM was more effective in its photoprotective effect toward UVB irradiation than PABA.     

Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.     

Industrial Application of Artificially Induced Diploid Strains of Torulaspora delbrueckii

Diploid strains of Torulaspora delbrueckii were tested for industrial application. Because the cell volume of the diploid strain was three times as large as that of the parental haploid strain, collection and subsequent dehydration to make compressed yeast cakes were greatly improved with the diploid YL3. The time required for dehydration of the diploid strain was shortened to 1/2.5 that of the parent strain under conventional conditions. Moreover, for the diploid cells frequent filter changes for dehydration were not required, which was the case with parental cells. Fermentation activity and tolerance to freeze-thawing in dough were succesfully inherited by the diploid strains. The diploid YL3 showed nearly the same activity as the diploid F31 in bread making. However, the endurance period of yeast cakes when stored at 30°C without softening to lead to liquefaction was much longer in YL3 (199 h) than in F31 (132 h). This superiority was ascribed to the fact that YL3 was induced through direct diploidization and had no genetic defect on chromosomes because the wild-type strain was employed as the parent, whereas F31 was obtained through protoplast fusion from two auxotrophic mutants and carried at least two mutagenized genes that were masked by heterolallelism.     

Evaluating the transferability of Hapmap SNPs to a Singapore Chinese population

Background

Sexual reproduction relies on two key events: formation of cells with a haploid genome (the gametes) and restoration of diploidy after fertilization. Therefore the underlying mechanisms must have been evolutionary linked and there is a need for evidence that could support such a model.

Results

We describe the identification and the characterization of yem ¹ , the first yem-alpha mutant allele (V478E), which to some extent affects diploidy reduction and its restoration. Yem-alpha is a member of the Ubinuclein/HPC2 family of proteins that have recently been implicated in playing roles in chromatin remodeling in concert with HIRA histone chaperone. The yem ¹ mutant females exhibited disrupted chromosome behavior in the first meiotic division and produced very low numbers of viable progeny. Unexpectedly these progeny did not display paternal chromosome markers, suggesting that they developed from diploid gametes that underwent gynogenesis, a form of parthenogenesis that requires fertilization.

Conclusions

We focus here on the analysis of the meiotic defects exhibited by yem ¹ oocytes that could account for the formation of diploid gametes. Our results suggest that yem ¹ affects chromosome segregation presumably by affecting kinetochores function in the first meiotic division. This work paves the way to further investigations on the evolution of the mechanisms that support sexual reproduction.     

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.