Ghost Mutant

Genetic Backgrounds Used for Conditional Lethality

Special classes of conditional lethal mutants are created when the condition is not environmental, but rather is the specific genetic background of the host strain. In this situation, when a mutation is introduced into one genetic background the strain remains viable, but when introduced into another genetic background, it is not viable. Viruses that could infect one strain but not another led to the discovery of this type of conditional lethality.

Several types of genetic background mutations can allow the growth of mutant viruses that cannot grow in the wild-type host. A host carrying a nonsense suppressor mutation allows growth of a virus carrying a nonsense mutation. Other types of suppressor mutations may compensate for an otherwise lethal mutation. Generally known as extragenic or second-site suppressors, these mutations allow a strain carrying an otherwise lethal mutation to grow. Genetic crosses must demonstrate that the original lethal mutation is still present, and that the second mutational change suppresses the original lethal effect and allows growth.

Other types of genetic background mutations prevent the viability of a strain carrying a mutant protein. In this situation, mutations are found to be lethal when combined, while the individual mutations are not lethal. Such mutations are termed synthetically lethal, because the mutations are only lethal when they are put together in the same strain. Figure 1 shows the phenotypes and genotypes of these different cases of conditional lethal mutations.

Figure 1. (a) Ts-1 is a heat-sensitive conditional lethal mutant; cs-1 is a cold-sensitive conditional lethal mutant. The growth phenotypes at different temperatures are shown: (+) Growth; (–) No Growth. (b) Synthetic lethality occurs when two non-lethal mutations are combined, and found to have a lethal phenotype when in combination. (c) An extragenic suppressor is a mutation in another gene that can suppress the phenotype of the first mutation. In this case, a temperature-sensitive mutation (ts-1) was found to be suppressed by an extragenic suppressor, sup-1. In the strain with the two mutations ts-1 and sup-1, growth can occur at 42 °C, which is non-permissive for strains carrying the ts-1 mutation alone.

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Clever Mutant Isolation

J. Beckwith, in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Cell Division and Synthetic Lethal Mutations

Many of the genes encoding proteins of E. coli's cell division machinery have been defined by diverse approaches including screening for conditional lethal mutants and accidental discoveries. However, as the complexity of the cell division process and its regulation became clearer, new genetic methods for identifying the genes involved were required. Tom Bernhardt and Pete DeBoer initiated a synthetic lethal mutant hunt that succeeded in illuminating both a mechanism that ensures septum formation at mid-cell and the complexities of peptidoglycan synthesis specific to cell division.

Their starting strain contained a deletion of the min genes that encode proteins responsible for preventing septation at the poles rather than at mid-cell. Deletion of min genes does not prevent growth of the cells but causes budding off of DNA-less minicells, the result of unwanted polar divisions. The original min mutation was discovered by chance. The researchers reasoned that there were additional genes involved in the regulation of cell division which, like the min genes, were not by themselves essential for growth, but mutations of which in combination with a min mutation would be lethal to the cell – a synthetic lethal phenotype. Based on an approach for the isolation of synthetically lethal mutations developed in yeast, the researchers examined colonies from min mutant cells that had been mutagenized with a transposon and identified colonies in which the additional mutation caused by transposon insertion was synthetically lethal with the min mutant. This genetic screen was made possible by including in cells to be mutagenized a plasmid that carried the wild-type min genes and wild-type Lac genes and that tended to be lost frequently from cells. When cultures of this transposon-mutagenized strain were plated on agar media containing the XGal indicator for β-galactosidase (giving a blue color), colonies carrying a transposon insertion in a gene unrelated to cell division readily segregate the plasmid, appearing either blue-and-white-sectored or fully white (Figure 2). The researchers presumed that if the transposon inserted in some genes involved in cell division, the min deletion, transposon insertion combination would be lethal. Then, the survival of the colony would require maintaining the Min-Lac plasmid and colonies would present as fully blue.

Figure 2. A genetic screen for mutants affecting cell division. (a) A full screening plate. (b) A section of the plate amplified gives an example of one colony that maintains the plasmid because its loss would prevent growth (the blue colony with not sectors), colonies that are in the process of losing the plasmid (colonies that contain blue and white sectors) and colonies that have entirely lost the plasmid (fully white colonies). An 'all blue colony' has a mutation in a cell division gene that is synthetically lethal with the min mutation.

This approach yielded mutations in the slmA gene that was then shown to encode a DNA-binding protein that prevents septation in the region of the cell where the nucleoid is located. Although the min, slm double mutant is lethal, knocking out one or the other of these protective mechanisms is not, thus illustrating the power of the synthetic lethal approach. The researchers also identified mutations in the envC gene, which encodes a lysostaphin, an enzyme involved in peptidoglycan remodeling at the cell septum. This finding led the investigators to the discovery of a family of lysostaphins involved in cell division and initiated an understanding of how peptidoglycan synthesis is organized during the formation of the septum and separation of daughter cells. Mutations in the envC gene had been obtained previously, but the synthetic lethality with min mutations enhanced interest in this gene.

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Reproduction and Development

B. Loppin, T.L. Karr, in Comprehensive Molecular Insect Science, 2005

1.6.4.3.3 ms(3)K81

ms(3)K81 (K81) is the third mutant that produces haploid gynogenetic embryos but, in contrast to sésame and maternal haploid, K81 is a strict paternal effect embryonic lethal mutant. Homozygous K81 males do not present any apparent spermatogenesis defects: they produce motile sperm that fertilize eggs but the resulting embryos die. Similar to mh, most embryos from K81 fathers (∼90%) die after a few nuclear cycles but very few (<1 in 105) hatch as haploid gynogenetic embryos (Fuyama, 1984). The original K81 allele (K811) was collected in the field in Japan but new deletion alleles were subsequently recovered (Yasuda et al., 1995). At the cytological level, the K81 phenotype at fertilization is undistinguishable from the mh phenotype: paternal chromosomes are integrated in the gonomeric spindle but only maternal chromosomes divide correctly. It is interesting to note that the very same phenotype can be obtained from a maternal (mh) and a paternal effect (K81) mutant (Figure 6). It is reasonable to think that the K81 phenotype is most probably due to a defect in sperm chromatin organization.

Figure 6. Comparison of mitotic phenotypes observed in ms(3)K81 and sesame (ssm) mutants using anti-tubulin antibodies to label microtubules (green) and a DNA-specific flurochrome to label DNA (red). (Upper panels) Mitotic defect resulting from fertilization by ms(3)K81 sperm displaying highly condensed paternal DNA of the male pronucleus (arrow). (Lower panels) ssm eggs fertilized by wildtype sperm displaying highly condensed maternal DNA of the female pronucleus (arrow). (Adapted from Loppin, B., Docquier, M., Bonneton, F., Couble, P., 2000. The maternal effect mutation sesame affects the formation of the male pronucleus in Drosophila melanogaster. Devel. Biol. 222, 392–404.)

We have recently identified the gene responsible for the K81 phenotype (B. Loppin, P. Couble and T. Karr, unpublished data). It is a small gene that contains a single exon of 555 bp with no intron that is specifically expressed in the male germline. Surprisingly enough, we have not found any ortholog of K81 in the available database, even in the recently sequenced D. pseudoobscura and Anopheles gambiae genomes. K81 has only been found in the closest relatives of D. melanogaster and the gene appears to rapidly evolve within the melanogaster subgroup.

K81 is, thus, a gene that is absolutely required for male fertility but that is paradoxally restricted to a few species. It is now established that many male-specific genes involved in spermatogenesis are among the most rapidly evolving genes (Vacquier et al., 1997; Ting et al., 1998; Tsaur et al., 2001; Swanson and Vacquier, 2002). The K81 mutant phenotype is unique and suggests a defect in sperm chromatin organization or an aberrant epigenetic marking of the paternal genome during spermatogenesis.

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Synthetic Biology, Part A

Kimberly L. Keller, ... Swapnil Chhabra, in Methods in Enzymology, 2011

3.5 Varying the electron donor/acceptor

The most commonly used electron donor:acceptor for genetic manipulation of D. vulgaris is lactate:sulfate medium (60 mM:30 mM). Alternative electron donors or acceptors provide the opportunity of obtaining conditional lethal mutants in other pathways (Zane et al., 2010). Commonly used electron donors:acceptors for D. vulgaris and Desulfovibrio G20 are found in Table 22.2.

Table 22.2. Concentrations (mM) of different electron donors and electron acceptors currently being used for growth of Desulfovibrio strains

Electron donor:electron acceptorD. vulgaris HildenboroughDesulfovibrio G20Lactate:sulfate60:3060:30Lactate:sulfite30:2015:10Pyruvate:sulfate60:1560:15Pyruvate:sulfite30:1030:10Pyruvate6060FumarateNGa60

aNG, no growth observed.

Warning: To obtain and test mutants of genes in various metabolic pathways, it may be necessary to grow Desulfovibrio in fermenting conditions (pyruvate only) or dismutating fumarate (Desulfovibrio G20). Caution needs to be used in growing mutants in these conditions while maintaining selective pressure, because many antibiotics (including kanamycin and G418) are supplied only as sulfate salts that could supply enough sulfate to interfere with establishing growth capabilities.

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Interpreting the Stress Response of Early Mammalian Embryos and Their Stem Cells

Y. Xie, ... D.A. Rappolee, in International Review of Cell and Molecular Biology, 2011

2.2 Using null mutants to define essential developmental events

There are three key deadlines for prenatal developmental events, one near implantation. In the rodent model, null mutants that produce lethal phenotypes elucidate essential roles for protein kinases in vivo, and analyses of many null mutant lethals established essential molecular, cellular, and organ function required at specific deadlines for embryonic, fetal, and placental/yolk sac (Copp, 1995; Rappolee, 1999; Stanton et al., 2003). Essential developmental deadlines occur at E5.5 (5.5 days after fertilization), E8.5, and E11. At E5.5, basic cellular processes must be under zygotic control after loss of maternal gene products. Also, the endoderm must acquire nutrients for the embryo. At E8.5, limited diffusion of oxygen requires gene expression that mediates production of a beating heart, closed vascular system, and red blood cells. At E11, a working placenta is required to mediate nutrient and blood-gas transport to the fetus. Thus, the early implanting embryo and its stem cells must expand cell numbers and then differentiate to mediate essential function, as previously reviewed (Rappolee, 2007; Rappolee et al., 2010).

Phenotypes of null lethals are determined in gestational females under unstressed conditions, and more studies are needed for null mutants that may generate lethality only during gestational stress. But, stress kinases may not appear as essential in mouse null mutants where females undergo gestation under a normal, low-stress environment. Under these conditions, there may be no reproductive lethality, or effects of diminished fertility may be subtle. We anticipate, however, that stressed pregnancies would reveal the essential functions of stress enzymes during reproduction. Stress enzymes have not been tested in vivo during stressed gestations, but placental hormones have. For example, null mutants of decidual prolactin-related protein (DPRP) and the placental hormone prolactin-like peptide-A (PLPA) have only small fertility problems during normal gestation. But when the female is exposed to hypobaric caging creating gestational hypoxia, these hormonal nulls become lethal (Ain et al., 2004; Alam et al., 2007). This sort of testing is needed to sort out the mechanisms by which stress enzymes mediate gestational stresses in the conceptus in vivo. Thus, gestational stresses such as low oxygen, toxic environmental compounds, or malnutrition may reveal the essential adaptive functions of stress enzymes.

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Organizational Cell Biology

F. Martin-Belmonte, ... M. Galvez-Santisteban, in Encyclopedia of Cell Biology, 2016

Polarity complexes

Two main apical and one basolateral groups of proteins have been found to be involved in the establishment of cell polarity (Figure 1). The first major apical polarity complex is the Par complex, identified in C. elegans embryonic lethal mutants and conserved in Drosophila oocytes and neuroblasts and mammalian epithelial cells. The Par complex, which establishes the apical-basal membrane border, consists of Par3 (Bazooka, in Drosophila) and Par6, which are the scaffolding proteins, and the kinase aPKC, core effector of the complex. Other proteins, including Cdc42 GTPase and Rac1, are considered part of the same group (Lin et al., 2000). The second apical polarity complex is the Crb complex, which controls the extension of the apical surface (Roh et al., 2003). Crb (Crumbs 1–3 in vertebrates) forms a complex with protein associated with Lin Seven 1 (PALS1; Stardust, in Drosophila) and PALS1-associated tight junction protein (PATJ). In both Drosophila and vertebrate epithelial cells, the Crb complex is enriched at the apical margin of the lateral domain. The Crb complex interacts with the Par complex through the interaction of PALS1 to Par6. In Drosophila and mammalian epithelia, Par6/aPKC colocalize with the Crb complex, while most Par3 localizes at TJs, slightly basally to the Crumbs complex. One exception is the primary epithelium of Drosophila (see below), where Bazooka is enriched at the level of the AJs.

The basolateral complex is the Scrib complex, composed of Scribble (Scrib), lethal giant larvae homolog (Lgl), and discs-large homolog (Dlg). The Scrib complex localizes to AJs in mammals (with SJs in Drosophila) and defines the basolateral membrane domain (Laprise et al., 2004). At least in Drosophila, another group of proteins, the Yurt/Coracle (Yrt/Cora) group, has been identified as a novel additional player in polarity establishment. The Yrt/Cora group promotes basolateral membrane stability by negatively interacting with the apical determinant Crumbs (Laprise et al., 2009). It is important to note that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not in late embryogenesis, when apical and basolateral markers can be normally segregated even in the absence of Yrt/Cora. This implies the existence of yet unknown polarity proteins that act to define lateral membrane stability in late-stage embryos. Notably, the mammalian Yrt ortholog binds to Crb and contributes to epithelial organization (Laprise et al., 2006).

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Mumps Virus☆

B.K. Rima, W.P. Duprex, in Reference Module in Biomedical Sciences, 2014

Evolution and Genetics

Human populations only became dense enough to sustain MuV from about 4000 years ago and, therefore, it has been suggested that the virus must have evolved from an animal pathogen. Recently a closely related virus has been identified in an epauletted fruit bat. However, no closely related primate or other animal pathogen has been identified. Neither temperature-sensitive nor any other conditional lethal mutants of MuV have been reported nor has recombination been described in any nonsegmented negative-strand RNA virus. Although host range mutants have not been isolated, adaptation of the virus to growth in embryonated eggs or in chicken embryo fibroblasts requires a number of blind passages. Strains adapted in this way do not readily grow and fail to generate syncytia in mammalian cells in culture. As MuV is a neuropathogenic virus some clinical isolates have been adapted to grow in the CNS of experimental animals to study this biological property. These viruses have been used in reverse genetics approaches to identify molecular determinants of neuropathogenesis. These main determinants appear to reside in the viral envelope proteins. At present neutralizing monoclonal antibody escape mutants of the HN glycoprotein are the only type of MuV mutants which have been described.

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Viral Genetics and Evolution

FRANK FENNER, ... DAVID O. WHITE, in Veterinary Virology, 1987

ABORTIVE INFECTIONS AND DEFECTIVE VIRUSES

Viral infection is not always productive, i.e., does not always lead to the synthesis of infectious progeny; some or even all viral components may be synthesized but not assembled properly. This is called abortive infection. It may be due to the fact that certain types of cells are nonpermissive, i.e., lack some enzyme or other requirement essential for the replication of a particular virus, whether it be the wild type or a host-dependent conditional lethal mutant.

Abortive infections due to genetically defective viruses are of considerable theoretical interest. For example, temperature-sensitive mutants are defective at high temperatures, but can be rescued (i.e., helped to yield infectious progeny) by coinfection of the cell with a helper virus, which is usually but not necessarily a related virus. Defective interfering particles have already been described.

Adeno-associated viruses (family Parvoviridae), which can be recovered from the throats of various animals, appear to be absolutely defective, in that they fail to replicate unless an adenovirus (or experimentally a herpesvirus) is also replicating in the same cells. In nature, they are invariably recovered with an adenovirus, from animals concurrently infected with both viruses.

The extreme example of defectiveness is seen in cells transformed by certain papillomaviruses (family Papovaviridae, see Chapter 12). Here part or all of the viral genome is integrated into the cellular genome and replicates with it, or persists in the cell as an episome. The genetic information in part of the viral genome may be expressed, for virus-specified proteins are often synthesized, but ordinarily no infectious virus is produced.

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Viral Hepatitis

M. Estée Török, in Manson's Tropical Infectious Diseases (Twenty-third Edition), 2014

HCV Structure and Genome

HCV is an RNA virus that is classified in the Flaviviridae family. The HCV genome is a positive-sense single-strand RNA molecule of approximately 9500 nucleotides. There are highly conserved 5′ and 3′ untranslated regions flanking a 9000 nucleotide single ORF, which encodes a polyprotein of approximately 3000 amino acids. The polymerase enzyme of HCV lacks proofreading ability resulting in errors in replication. Many of these nucleotide changes result in a non-functional genome or a lethal mutant. However, others persist and account for the tremendous genetic diversity that is characteristic of HCV.36 This heterogeneity influences the pathogenesis of infection, response to antiviral therapies and prevents the development of conventional vaccines.

Six major genotypes and more than 50 subtypes have been described. Sequence homology between different genotypes is less than 80%. Genotype 1 is most common in Europe and the USA (60–70% of isolates); genotypes 2 and 3 are less common in these areas and genotypes 4, 5 and 6 are rare. Genotype 3 is most common in India, the Far East and Australia. Genotype 4 is most common in Africa and the Middle East but appears to be emerging in Europe among injection drug users and MSMs. Genotype 5 is most common in South Africa and genotype 6 is most frequent in Hong Kong, Vietnam and Australia.

Quasispecies are families of highly similar strains that develop within an infected host over time; sequence homology is greater than 95%. Differences between quasispecies are usually only apparent in the most rapidly changing parts of the genome (hypervariable regions). The clinical implications of quasispecies are not fully understood although they may be important in the natural history, persistence and response to treatment of the virus.37 In one study of 59 patients with chronic HCV infection increased quasispecies heterogeneity was associated with longer estimated duration of infection, transmission by transfusion, higher HCV viral load and genotype 1. In another histological study, different quasispecies were compartmentalized in specific regions of the liver and the degree of compartmentalization was greater in histologically advanced disease.

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