Variant Mutant11.11%

Mutant Gen

Methods in Cell Biology

Volume 44, 1994, Pages 81-94

Chapter 4 From Clone to Mutant Gene

Author links open overlay panelBruce A.Hamilton*KaiZinn†

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This chapter reviews several methods for isolating mutations in cloned Drosophila genes and provide protocols for generating P-element-induced mutations at targeted loci. In these protocols, local transposition of P-elements is used to increase the probability of insertion into the site of interest. The chapter also describes flanking sequence rescue screens to identify lines bearing insertions into the gene of interest from collections of P-element transposants. Several properties of P-elements make them attractive as mutagenic agents for targeted genetic screens. Recombinant P-element derivatives whose mobility is controlled by mating to a source of transposase and whose transposition is selectable by phenotype are widely available, making the generation of single or multiple hit transposants straightforward. Because the P-element transpositions tag the inserted site with a known DNA sequence, insertions into the target region can be detected at the DNA level without a prediction of phenotype. P-element insertions isolated in or near a gene of interest can be used as substrates to generate new alleles at very high efficiencies.

Many of the basic facts about plant hormones are still obscure, including biosynthetic pathways and their regulation. Furthermore, our knowledge of the molecular steps between hormones and their action is extremely limited. The increasing collection of isogenic genotypes differing in hormone synthesis or responses offers great promise for future research.

Gene

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Y Chromosome

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International Review of Cell and Molecular Biology

Eleanor M. Maine, in International Review of Cell and Molecular Biology, 2010

3.3 Genome-wide analysis of germ line gene expression

X chromosome gene expression in the C. elegans germ line has also been explored using gene expression profiling and functional genomic analysis (Maeda et al., 2001; Piano et al., 2000, 2002; Reinke et al., 2000, 2004). Gene expression profiling has identified large sets of transcripts that are enriched in the germ line relative to the soma; these genes can be grouped into several categories based on expression pattern, including (i) germ line-intrinsic genes expressed in the XX and XO germ line, (ii) spermatogenesis-specific genes expressed in the XO and larval XX germ lines, and (iii) oogenesis-specific genes expressed in the female and adult hermaphrodite germ line (Reinke et al., 2000, 2004). These studies revealed that germ line-intrinsic and spermatogenesis-specific genes are underrepresented on the X chromosome relative to autosomes. In contrast, oogenesis-specific genes are not underrepresented on the X (Reinke et al., 2000, 2004), although essential ovary-expressed genes tend not to be X-linked (Maeda et al., 2001; Piano et al., 2000, 2002). A trend away from germ line-expressed genes on the X was borne out by subsequent genetic studies showing that, among sets of duplicated genes, those that are expressed in the germ line tend to be located on autosomes while those expressed in the soma may be located on the X chromosome (Maciejowski et al., 2005; Ohmachi et al., 2002). Taken together, these data are consistent with the pattern of H3K4me2 and other activation marks observed by Kelly et al. (2002) in the germ line: there is little X chromosome expression during mitosis, male meiosis, and spermatogenesis, but there is a burst of X-linked expression in oogenesis.

In an initial attempt to determine the functional significance of the observed patterns of dynamic chromatin modifications, Kelly et al. (2002) compared the average transcript level for all genes versus oogenesis-expressed genes on each chromosome. They found that genes whose expression remains high during meiosis tend to be located on autosomes. In contrast, the average X chromosome transcript levels were two- to threefold lower than autosomal transcript levels in the germ line. In the soma, no significant difference in autosomal versus X chromosomal transcript level was observed. These data were consistent with X-linked transcription occurring in only a small subset of germ cells. Consistent with this hypothesis, when in situ hybridization analysis was used to visualize transcript distributions, X-linked transcripts were observed during the late-pachytene/diplotene window (Kelly et al., 2002). For each gene examined, mRNA was first visible in late pachytene nuclei, consistent with the appearance of chromatin activation marks on the X chromosomes at that stage.

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Canine Genetics

E.A. Ostrander, R.K. Wayne, in Encyclopedia of Genetics, 2001

Linkage Analyses and Genetic Maps

While chromosome gene maps are necessary for determining the evolutionary relationship between genomes, and for determining the syntenic relationships between mammals, a genetic map is needed for identifying loci which contribute to traits of interest. A genetic map is one for which the distance between markers is measured as a function of genetic recombination. A marker is a short segment of DNA that varies between homologous chromosomes in the population. Because any given individual has two copies of each chromosome, each individual must have, by definition, two alleles for every marker. If identical alleles are inherited from each parent, individuals are homozygous for that marker. Markers are considered informative if there are sufficient alleles in the population that most couplings allow the inheritance of chromosomes (or regions of chromosomes) to be tracked from grandparent to parent to offspring. If the frequency of the most common allele that appears in the population is less than 95%, then the marker is referred to as polymorphic.

If a marker and a gene are physically located close together on the same chromosome, alleles on homologous chromosomes will be coinherited in a significant number of offspring and are thus linked. If two markers are located far apart on the same chromosome or on different chromosomes their alleles will be inherited independently or randomly in offspring and are unlinked. For a given region of the genome the probability of a genetic recombination event occurring between a pair of markers or a marker and a disease gene is proportional to the distance between them. This probability is expressed as a recombination fraction or, in units called centiMorgans (cM). One percent recombination is equal to 1 cM, which roughly corresponds to a million base pairs in the human genome.

To map the gene for a trait of interest, a genomic screen of DNA from families with the trait of interest is undertaken, using markers spaced about every 5–10 cM. Figure 2 shows a schematic of a two-generation pedigree and a denaturing sequencing gel resulting from analysis of a single marker. The black bars represent alleles separated on a gel, and demonstrate Mendelian inheritance of the alleles. One allele from the father, which is circled, appears in all affected individuals. In addition, no unaffected individuals inherit this allele. Thus, it can be hypothesized that the marker indicated is close to the disease gene. Additional markers and many more families would need to be analyzed to determine if the proposed linkage is true and to determine the distance between the marker and disease gene. Odds of 1000:1 that a given marker is linked to a trait of interest are indicated by a Lod score of ≥3.0 and is generally accepted as evidence of linkage. A Lod score of less than −2.0 indicates that a given marker and trait of interest are unlinked

Figure 2. (A) Segregation of alleles in a single pedigree. Females are represented as circles, males are squares. Affected individuals are colored in black, unaffected in white. (B) The marker analyzed here is hypothesized to be linked to the disease gene in question, since all affected individuals have inherited this allele from their affected parent or grandparent.

Currently most screens utilize genetic maps composed of microsatellite markers. Microsatellites are small repetitive stretches of polymorphic DNA that can be tracked using the polymerase chain reactions (PCR). They are optimal for the construction of genetic maps for several reasons. First, they are frequent and distributed randomly; there are several thousand of the common repeat arrays (e.g., (CA)n, (GATA)n, or (CAG)n) scattered throughout the canine genome. Hence collection of large numbers of markers for map building is a relatively straightforward exercise. Second, the rate at which mutations generate new variation/length alleles is nontrivial – about 10−5 for (CA) repeats and about 10−2 for microsatellites based upon tetranucleotide repeats. This means that they are highly informative in mapping studies in relatively inbred families. Nevertheless, they are sufficiently stable that the inheritance of adjacent sections of chromosomes can be tracked through several generations of a family with reliability.

Linkage analyses of large numbers of microsatellite markers on outbred reference families, comprised of many distinct dog breeds, have led to the production of a preliminary canine genetic map. A high-density map appears well on its way to completion, with well spaced, highly informative markers spanning several chromosomes. The map likely covers greater than 85% of the canine genome, although exact estimates are difficult to determine since the precise size of the canine genome is not known. The best estimates suggest that it is about 26.5±1.1 Morgans (95% confidence interval=24.3 M to 28.7 M). As the density and coverage of the map increases, the ability to identify loci through linkage analyses of families with traits of interest will increase proportionately. Thus far, several hundred canine microsatellites have been described and placed on the canine map (Mellersh et al., 1997), with several hundred more currently in progress. While there often appears to be a unique distribution of alleles within particular breeds, it has not yet been possible to define markers which are breed specific. This is not surprising given the discussion above about the significant genetic variation that contributed to the canine gene pool.

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Toll-Like Receptors in SLE

Terry K. Means, in Systemic Lupus Erythematosus (Fifth Edition), 2011

TLR9 Polymorphisms and Copy Number in SLE

In contrast, other studies performed on American, UK, Korean, and Chinese SLE cohorts did not detect a statistically significant association of any TLR9 gene variations with SLE susceptibility.

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The ATP7B Gene

Richard Kirk, in Clinical and Translational Perspectives on WILSON DISEASE, 2019

Abstract

The ATP7B gene (chromosome 13q14.13) spans approximately 80 kb of genomic DNA, comprises 21 protein-coding exons, encodes a messenger ribonucleic acid of approximately 7.5–8.5 kb, and produces a protein containing 1465 amino acid residues. Metal-binding transcription factors may play an important role in regulation. Genetic analysis must suit the spectrum of pathogenic variants present in the population: genotyping for common mutations and/or sequencing to identify rare or novel pathogenic variants. Other rare mutational mechanisms also occur: large-scale deletions, promoter mutations, uniparental disomy, and pseudo-dominant inheritance. Diagnostic laboratories use additional quality control systems to ensure high quality of service. Approximately 800 pathogenic variants have been published. Distinguishing between pathogenic and benign variants requires the use of multiple lines of evidence. In the United Kingdom, sequencing of the protein-coding region of the ATP7B gene, with promoter sequencing and exon deletion/duplication analysis, has a 98% pickup rate for identifying significant gene alterations. The disease prevalence may be significantly higher than 1:30,000.

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Toll-Like Receptors, Systemic Lupus Erythematosus

William F. PendergraftIII, Terry K. Means, in Systemic Lupus Erythematosus, 2016

TLR9 Polymorphisms and Copy Number in SLE

The TLR9 gene (chromosome 3p21.3) is located in one of the defined susceptibility regions for SLE. Several groups have investigated whether genetic variations of TLR9, which include (−1486 T → C, –1237 C → T, +1174 A → G, and +2848 G → A), are involved in susceptibility to SLE in different populations around the world. The presence of the nucleotide G at position +1174 in intron 1 of TLR9 was demonstrated to be significantly associated with an increased risk of SLE in a Japanese cohort.50 Interestingly, the A → G +1174 SNP downregulates TLR9 expression in reporter assays. These data would fit with the current model (discussed above) that TLR9 expression is protective against SLE. A significant association was also made with −1486 T → C in an SLE cohort in China and +1174 A → G in Brazil.51,52 In contrast, other studies performed on American, British, Danish, Korean, Chinese, and Indian SLE cohorts did not detect a statistically significant association of any TLR9 gene variations with SLE susceptibility.53 In 2012, two separate meta-analyses were performed to determine the risk of SLE with three TLR9 polymorphisms (−1486 C → T, +1174 A → G, and +1635 C → T) in Asians, and no association was identified in either report.54 To date, there has yet to be a proven and replicated association of TLR9 polymorphisms with SLE risk.

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Genes, Genomes, and Genomics

Padma Nambisan, in An Introduction to Ethical, Safety and Intellectual Property Rights Issues in Biotechnology, 2017

1.7.2 Genetic Testing and Diagnostics

Genetic analysis in humans has over time revealed a number of disorders that are inherited. Documentation of this information was published between 1966 and 1998 in 12 editions of Mendelian Inheritance in Man by Victor McKusick of Johns Hopkins University School of Medicine (McKusick, 1966, 1994). Since 1987, the information became available online under the direction of the Welch Medical Library with financial support from the NCBI (McKusick, 2007). Since 2010, the website of Online Mendelian Inheritance in Man (http://www.omim.org/) is authored and edited at the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, with financial support from the National Human Genome Research Institute (NHGRI).

Changes in chromosomes, genes, or proteins correlated to disease conditions have been used to confirm or rule out a genetic condition. Since the 1950s, amniocentesis (studying free floating cells isolated from the amniotic fluid that surrounds the fetus in the womb) has been used by physicians to diagnose chromosomal aberrations, such as Down's syndrome, in the unborn fetus. More than thousand genetic tests have been developed and can be broadly classified into three categories:

•

Molecular genetic tests (or gene tests),

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Chromosomal genetic tests, and

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Biochemical genetic tests that study the amount or activity level of proteins.

In addition to diagnosis, genetic testing can be used to identify carriers [individuals who have only one copy of the mutated gene hence do not show symptoms (phenotype) of the genetic disorder], and to determine if an individual has an inherited predisposition to a disease. The advantages of genetic testing are:

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To provide an accurate diagnosis of a disease condition and to make informed decisions regarding the treatment: Genetic testing can help to identify the cause of the diseased condition and to select the appropriate treatment regimen (including whether a patient would respond to a particular treatment option). This has been found to be especially useful in the treatment of certain cancers as the therapy is expensive (thereby posing a financial burden on the patient), and could cause adverse effects (thereby further reducing the quality of life of the patient).

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To make life choices: Amniocentesis has helped to make decisions regarding the medical termination of pregnancy in cases of numerical/structural abnormalities in chromosomes known to cause disease syndromes in humans. With the development of In Vitro Fertilization techniques, in which the embryo is created in a petri dish and later implanted into the womb, genetic testing is being increasingly used to select "healthy" embryos. Preimplantation Genetic Testing is especially advocated in instances when one of the biological parents is known to be affected by (or is a carrier of) a genetic disorder.

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To make life-style choices: Several genetic disorders are known to be age-dependent and the disease is often manifested only after the reproductive age of the patient, hence is transmitted to the progeny (example, Huntington's disease). Genetic testing could help make informed decisions regarding reproductive choices. It could also help to plan for future healthcare and end-of-life decisions for debilitating diseases such as Alzheimer's, Parkinson's, and some forms of dementia.

Due to the rather sensitive nature of the information revealed by the tests, genetic testing is invariably coupled with genetic counseling to help patients understand how the test results would affect them and to guide them to make informed choices. The field however is mired in ethical conundrums. The ELSI initiative of the HGP is an attempt to address issues of privacy and psychological impacts of genome information revealed by genetic testing. Some of the ethical aspects associated with genetic testing are discussed in Chapter 7, Genetic Testing, Genetic Discrimination, and Human Rights of this book.

Several privately held personal genomics companies have been established, for example, the California based company 23andMe (https://www.23andme.com/en-int/) founded by Linda Avey, Paul Cusenza, and Anne Wojcicki in 2006 that has been offering direct to customer genetic testing since November 2007. (See International Society of Genetic Genealogy Wiki (2016) for a country-wise listing of personal genomics companies.)

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Chromatin Remodelling and Immunity

J.S. Rawlings, in Advances in Protein Chemistry and Structural Biology, 2017

4 Classical Functions of SMC Complexes

The first SMC gene, SMC1, was identified in Saccharomyces cerevisiae and was shown to be required for proper chromosome segregation (Strunnikov, Larionov, & Koshland, 1993). Later, it was discovered that this protein was part of the Cohesin complex (Losada, Hirano, & Hirano, 1998; Sumara, Vorlaufer, Gieffers, Peters, & Peters, 2000; Toth et al., 1999). This complex consists of a Smc1a/Smc3 heterodimer and two non-SMC components: a Kleisin family protein known as Scc1 (Rad21 in humans) and Scc3 (either STAG1 or STAG2 in humans) (Table 2). Cohesin acts as a molecular glue that holds sister chromatids together during the cell cycle beginning with DNA synthesis until anaphase of mitosis when one of the cohesion subunits, Scc1, is cleaved by Separase, a cysteine protease. This cleavage results in the release of Cohesin from sister chromatids, permitting chromosome segregation to occur (for review, see Peters, Tedeschi, & Schmitz, 2008). Because of its role in sister chromatid cohesion, it is not surprising that the Cohesin complex also has roles in DNA damage repair, specifically postreplicative double-strand break repair (Sjogren & Nasmyth, 2001).

In addition to a Smc2/Smc4 heterodimer, the Condensin I and Condensin II complexes contain two HEAT repeat containing proteins and one Kleisin subunit (Table 2). Although first discovered in 1982 as a chromosome scaffolding protein (then termed ScII), Smc2 was not characterized as a Smc protein until 1994 (Lewis & Laemmli, 1982; Saitoh, Goldberg, Wood, & Earnshaw, 1994). By 1997, the other subunits of the Condensin I complex were discovered and the complex itself was described in Xenopus egg extracts (Hirano, Kobayashi, & Hirano, 1997). The Condensin I complex functions to condense chromosomes during mitosis and is also required for proper chromosome segregation. The function of the Condensin I complex is regulated, in part, by the fact that the complex is sequestered in the cytosol during interphase. Once the nuclear envelope breaks down during prophase, the Condensin I complex can access DNA and condense it in preparation for completion of mitosis (Hirota, Gerlich, Koch, Ellenberg, & Peters, 2004; Ono, Fang, Spector, & Hirano, 2004). A second Condensin complex, found only in higher eukaryotes, was discovered more recently (Ono et al., 2003; Yeong et al., 2003). This complex, termed Condensin II, possesses the same Smc2/Smc4 heterodimer as the Condensin I complex; however, utilizes different non-SMC components (Table 2). It was immediately discovered that Condensin I and Condensin II bind different regions of chromosomes, suggesting that they contribute to chromatin architecture in distinct ways (Ono et al., 2003). Like Condensin I, Condensin II also functions in chromatin compaction and in segregation; however, unlike Condensin I, Condensin II can be found in the nucleus during interphase (Hirota et al., 2004; Ono et al., 2004). This observation signaled the possibility that higher-order chromosome condensation mediated by Condensin II is not limited to cell division and that Condensin II could play roles in interphase biology in higher eukaryotes.

The third Condensin complex, termed Condensin IDC, is a part of the dosage compensation complex (DCC) found only in Caenorhabditits elegans and unlike the other Condensins, it contains a heterodimer of DPY-27 and Smc2. As its name implies, the primary role of the DCC is to mediate dosage compensation of X chromosome genes in hermaphrodites (Chuang, Albertson, & Meyer, 1994; Csankovszki et al., 2009; Lieb, Albrecht, Chuang, & Meyer, 1998; Lieb, Capowski, Meneely, & Meyer, 1996; Tsai et al., 2008). Unlike dosage compensation in mammals which is achieved by the random silencing of a single X chromosome, the DCC interacts with both X chromosomes to downregulate gene expression by half (Ercan & Lieb, 2009; Heard & Disteche, 2006).

The Smc5/6 complex is perhaps the least understood of the SMC complexes. The first component of this complex (Smc6) was originally identified because it was able to rescue a radiation-sensitive mutant strain of Schizosaccharomyces pombe (Nasim & Smith, 1975; Phipps, Nasim, & Miller, 1985). In addition to the Smc5/6 heterodimer, this complex contains four non-SMC components. Sequence analysis of these subunits reveals that the complex may have E3 ubiquitin ligase activity as well as SUMO ligase activity (Lehmann, 2005). Other than its known roles in DNA repair, the functions of the Smc5/6 complex remain elusive (Lehmann et al., 1995; Verkade, Bugg, Lindsay, Carr, & O'Connell, 1999). However, recent work suggests that there is at least crosstalk between the Smc5/6 complex and the Cohesin complex during chromosome segregation (for review, see Tapia-Alveal, Lin, & O'Connell, 2014).

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Normal and Aberrant Growth in Children

David W. Cooke, ... Sally Radovick, in Williams Textbook of Endocrinology (Thirteenth Edition), 2016

POU1F1.

The POU1F1 gene (chromosome 3p11, OMIM 173110) encodes PIT1, a member of a large family of transcription factors referred to as POU-domain proteins that is responsible for pituitary-specific transcription of genes for GH, PRL, TSH, and the GHRH receptor.58,454-456 PIT1, a 290–amino acid protein, contains two domains, the POU-specific and the POU-homeo domains; both are necessary for DNA binding and activation of GH and PRL genes and for regulation of the PRL, TSH-β, and POU1F1 genes.457 Its expression is restricted to the anterior pituitary to control differentiation, proliferation, and survival of somatotrophs, lactotrophs, and thyrotrophs.371,456-458 PIT1 regulates target genes by binding to response elements and recruiting coactivator proteins, such as cAMP response element–binding protein (CREB)-binding protein (CBP).459 Gene expression microarray assays combined with chromatin immunoprecipitation (CHIP) were used to detect targets of POUIFI.273

Two mouse models were first reported to have GH, PRL, and TSH deficiencies associated with mutations or rearrangements of the Pit1 gene; these were the Snell (dw/dwS) and the Jackson (dw/dwJ) dwarf mice.460,461 Many different mutations of the POU1F1 gene have been found internationally in families with GHD and PRL deficiency and variable defects in TSH expression.462-465 These mutations are transmitted as autosomal recessive or dominant traits and cause variable peptide hormone deficiencies with or without anterior pituitary hypoplasia.462-468

The most common mutation is an R271W substitution that affects the POU homeodomain, encoding a mutant protein that binds normally to DNA and acts as a dominant inhibitor of transcription.468-471 Vertical transmission of the R271W mutation was shown, emphasizing the importance of diagnostic and theraputic management during pregnancy.472 Evidence from a patient with the R271W mutation suggests that PIT1 may have a role in cell survival.471 Indeed, the mutation was used to target cell proliferation tumoral model systems. A patient diagnosed with GHD, along with dysregulation of PRL and TSH, was reported to have a lysine-to–glutamic acid mutation at codon 216 (K216E).457 This mutant PIT1 binds to DNA and appears not to inhibit basal activation of GH and PRL genes; however, the mutant is unable to support retinoic acid induction of POU1F1 gene expression. Another report suggested that CBP (p300) recruitment and PIT1 dimerization are necessary for target gene activation and that disruption of this process may account for the pathogenesis of CPHD.473 All of the reported mutations involve sites affecting POU1F1 DNA-binding, dimerization, or target gene transactivation.

Phenotypic variability occurs among patients with apparently similar genotypes. It does not appear that ACTH or gonadotropin deficiencies occur, as is frequently the case with PROP1 defects,452 but adrenarche has been reported to be absent or delayed in patients with a POU1F1 mutation.474 Circulating antibodies against Pit1 have been identified to be responsible for hypopituitarism similar to that caused by mutations.475

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Biochemistry and Molecular Biology

K.J. Kramer, S. Muthukrishnan, in Comprehensive Molecular Insect Science, 2005

4.3.3.5.5 Regulation of chitin synthase gene expression

The two insect genes encoding CHSs appear to have different patterns of expression during development. The high degree of sequence identity of the catalytic domains and the absence of antibodies capable of discriminating between the two isoforms have complicated the interpretation of experimental data to some extent. In some cases, the technical difficulties associated with isolation of specific tissues free of other contaminating tissues have precluded unambiguous assignment of tissue specificity of expression. Nonetheless, the following conclusions can be reached from the analyses of expression of CHS genes in several insect species. CHS genes are expressed at all stages of insect growth including embryonic, larval, pupal, and adult stages. CHS1 genes (coding for class A CHS proteins) are expressed over a wider range of developmental stages (Tellam et al., 2000; Zhu et al., 2002). CHS2 genes (coding for class B CHSs) are not expressed in the embryonic or pupal stages but are expressed in the larval stages, especially during feeding in the last instar and in the adults including blood-fed mosquitoes (Ibrahim et al., 2000; Zimoch and Merzendorfer, 2002; Arakane et al., 2004). The finding that both classes of CHS genes are expressed at high levels 3 h after pupariation in Drosophila suggests that both enzymes are required for postpuparial development (Gagou et al., 2002).

CHS genes also show tissue-specific expression patterns. In L. cuprina, CHS1 (coding for a class A CHS) is expressed only in the carcass (larva minus internal tissues) and trachea but not in salivary gland, crop, cardia, midgut or hindgut (Tellam et al., 2000). In blood-fed female mosquitoes, a CHS gene encoding a class B CHS is expressed in the epithelial cells of the midgut (Ibrahim et al., 2000). In M. sexta, CHS1 (coding for a class A CHS) is expressed in the epidermal cells of larvae and pupae (Zhu et al., 2002). Transcripts specific for class B CHS were detected only in the gut tissue (D. Hogenkamp et al., unpublished data). As discussed above, in Drosophila, both classes of CHS genes were shown to be upregulated after the ecdysone pulse had ceased in the last larval instar, but the tissue specificity of expression of each gene was not determined. In T. castaneum, the CHS1 gene (coding for a class A CHS) was expressed in embryos, larvae and pupae, and in young adults, but not in mature adults (Arakane et al., 2004). Even though unequivocal data are not available for each of these insect species, the following generalizations may be made. Class A CHS proteins are synthesized by epidermal cells when cuticle deposition occurs in embryos, larvae, pupae, and young adults, whereas the class B CHS proteins are expressed by the midgut columnar epithelial cells facing the gut lumen in the larval and adult stages and is probably limited to feeding stages.

Gene Mutations

Gene mutations are changes in single DNA bases, or small intragenic deletions and rearrangements. The classification of gene mutations is often used interchangeably with point mutations although, genetically, they may comprise different events. Point mutations refer to single base changes, or insertions or deletions of one or a few bases, whereas gene mutations refer to base change or intragenic additions, deletions, or rearrangements that disrupt normal gene function. These are all considered to be heritable effects because they are usually not lethal to the exposed cells and are typically measured in the posttreatment generation cells. The treated cells must undergo subsequent reproductive cycles for the mutations to be expressed and scored.

Introduction

Mutagenesis is the formation of mutations in DNA molecules. There are a variety of mutations that can occur in DNA, such as changes in the DNA sequence or rearrangement of the chromosomes. Such mutations may occur spontaneously, as a result of 'mistakes' that occur during DNA replication or mitosis. Spontaneous mutations are essential to produce genetic variation necessary for natural selection. Mutations may also occur as a result of environmental exposure to genotoxins (chemicals that alter the structure of DNA). Mutagenesis is of concern because it may lead to irreversible effects that can affect fitness of organisms, which in turn may affect population-level processes.

There are potentially thousands of mutagenic and genotoxic agents to which organisms are exposed. Examples of the classes of mutagenic compounds, the DNA damage they elicit, and their sources in the environment are listed in Table 1. Each genotoxin may elicit many different types of DNA damage.

Gain-of-Function Alleles

Systematic analysis of deletion mutant alleles, both individually and in combination, have made an enormous impact on gene function discovery. However, while loss-of-function genetic analyses identify functionally coherent gene modules and the connections between them, they are not always sufficient for elucidating pathway architecture. Historically, dominant gain-of-function (GOF) mutations have provided an incredibly powerful means for determining gene position within a regulatory cascade ([89] for example). The availability of low- and high-copy plasmid libraries in which the expression of every yeast open reading frame (ORF) is controlled by the endogenous [90–92] or an inducible promoter [93–95] has enabled systematic and genome-wide investigation of GOF and gene dosage effects in wild-type and mutant strain backgrounds to complement previous genetic interaction studies [96,97].

Recently, a barcoded high-copy plasmid library was developed and screened for dosage suppression. Dosage suppressors of 41 temperature-sensitive different alleles of essential genes were identified [92]. An average of ~5 different genes were found to suppress the temperature-sensitive phenotype of each essential gene mutant, suggesting that, albeit more rare than LOF interactions such as synthetic lethality, dosage suppression is also a common genetic interaction. Furthermore, while they tend to connect functionally related genes, dosage suppression interactions often overlap with negative genetic interactions and physical interactions that occur within the essential pathway of the query gene; however, most interactions are novel, suggesting that dosage suppression represents a prevalent and distinct type of interaction that is rich in novel functional information [92]. Dosage suppression can identify pathway components that act downstream of the query gene, and therefore this type of genetic interaction offers the potential to decipher gene order and the directionality of biological circuits. Ultimately, we anticipate that a global dosage suppression map will be a major contributor to the construction of a complete and high-resolution cellular landscape comprising all types of genetic and physical interaction.

Deletion Mutant

Related terms:

Amino Acids

Enzymes

Plasmids

Phenotype

C-Terminus

In Vitro

Protein

DNA

Amino Terminal Sequence

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Genetic Networks

Michael Costanzo, ... Charles Boone, in Handbook of Systems Biology, 2013

Gain-of-Function Alleles

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DNA Methylation and Viral Infections

Michel Neidhart, in DNA Methylation and Complex Human Disease, 2016

6.3.4.1 AP-1/JNK

By using different LMP1 deletion mutants, it has been determined that the C-terminal activation region 2 (CTAR2) of LMP1 is the domain required for DNMT activation. CTAR2 can activate the AP-1/JNK signaling pathway [76]. A JNK inhibitor (SP60012) has been used to show that DNMT is indeed the downstream target of JNK. In addition, mutation of the AP-1 site on DNMT1 promoter, and c-Jun dominant-negative mutant blocked the LMP1-induced DNMT activation. Chromatin immunoprecipitation (ChIP) assay further demonstrated that DNMT1, 3A, 3B, MeCP2, and HDAC1 formed protein complexes on the E-cadherin promoter in the LMP1-positive cells but not in the control cells. Taken together, a single viral oncoprotein in EBV has been identified, which is capable of activating DNMTs via AP-1/JNK signaling pathway.

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Movement of Viruses Within Plants

Roger Hull, in Plant Virology (Fifth Edition), 2014

ii Function of the MP

Analysis of C-terminal deletion mutants of CaMV MP shows that the 27 C-terminal amino acids are not required for tubule formation and the 77 C-terminal amino acids are not required for targeting to peripheral punctate spots (Huang et al., 2000). Thus the organization of the domains for tubule formation and targeting to peripheral punctate structures in the C-terminus of CaMV MP is very similar to that of CPMV MP, suggesting that CaMV MP uses the same steps in tubule formation (see below).

CaMV MP interacts with a family of host PDLPs which are suggested to be the receptors at Pds for the MP (Amari et al., 2010). The MP also interacts in yeast two-hybrid assays with some Arabidopsis proteins (MPI1, -2 and -7) (Huang et al., 2001b); MPI7 is related to a rab acceptor but the significance of this interaction is unknown. The mechanisms for removal of the desmotubule, increasing the SEL and assembly of MPs into tubules, are not yet known. As can be seen from Figure 10.20A, the tubules contain CaMV particles. Although viral CP is not an integral part of the tubule, there is a coiled-coil interaction between the MP and the virion-associated protein (VAP) (Chapter 3, Section V, B, 7) encoded by ORFIII (Stavolone et al., 2005). It is not yet known how the particles move through the tubule.

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Immunotoxin Therapy for Brain Tumors

V. Chandramohan, ... D.D. Bigner, in Translational Immunotherapy of Brain Tumors, 2017

Mutant Epidermal Growth Factor Receptor Variant III

EGFRvIII is the most common EGFR deletion mutant that is present in 20–50% of GBMs with EGFR amplification.130 The mutant EGFRvIII contains an in-frame deletion of 801 base pairs of the coding region that corresponds to exons 2–7 of the EGFR gene.131 This deletion creates a novel glycine residue at the fusion junction, generating a tumor-specific protein sequence. EGFRvIII is a constitutively active receptor tyrosine kinase,132 which is widely expressed in malignant gliomas133 and carcinomas, including head and neck134 and breast cancers.135 EGFRvIII overexpression induces GBM resistance to commonly used chemotherapeutic agents136 rendering EGFRvIII a desirable target for therapeutic intervention.

The therapeutic efficacy of EGFRvIII-specific mAbs, L8A4, Y10, and H10, chemically coupled to PE35KDEL, was evaluated against a mutant EGFRvIII-expressing cell line. All three ITs were highly efficacious in in vitro cytotoxicity assays, with IC50 in the range of 15–50 pM.137 A scFv specific for EGFRvIII, MR1 (mutant receptor), was isolated from an immunized phage library and was fused to PE38KDEL. The cytotoxic activity (IC50) of MR1-PE38KDEL against EGFRvIII-transfected malignant glioma cells was in the range of 110–160 pM.138 A dose escalation toxicity study (three doses of 1–20 μg of MR1-PE38KDEL) compared the survival of the MR1-PE38KDEL treatment group with the saline group in nontumor bearing athymic rats. On the basis of survival data, 3 μg of MR1-PE38KDEL was established to be the nontoxic dose.139 The therapeutic efficacy of three doses of MR1-PE38KDEL (1, 2, and 3 μg) was compared with saline and control IT (3 μg) in an EGFRvIII-expressing (U87MG.ΔEGFR) neoplastic meningitis model in athymic rats.139 There were 75% (1-μg group) and 57% (2 or 3 μg group) long-term survivors in the MR1-PE38KDEL treatment group. The MR1-PE38KDEL treatment group failed to reach median survival by the termination of the study at 53 days. Hence, the median survival for the MR1-PE38KDEL group was estimated to be > 53 days. All saline or control IT-treated animals died, with median survival of 7 and 10 days, respectively. Histological examination of the 3 μg MR1-PE38KDEL treatment group presented evidence of demyelination. Regional therapy with three doses of 2 μg of the MR1-PE38KDEL was concluded to be the effective dose for the treatment of EGFRvIII-expressing neoplastic meningitis tumors.139

An affinity-matured variant of MR1, termed MR1-1, with increased affinity to EGFRvIII, was generated and fused to PE38, to generate MR1-1-PE38 for targeted glioma therapy.140 MR1-1-PE38 in comparison to the parental MR1-PE38 exhibited improved cytotoxicity against the EGFRvIII-expressing NR6M cell line.140 A Phase I study to determine the MTD and DLT of MR1-1-PE38KDEL delivered intracerebrally by CED in patients with supratentorial malignant brain tumors expressing EGFRvIII was initiated.141 The study design included infusion of MR1-1-PE38KDEL by CED using two intracerebral catheters. 124I-labeled HSA was coinfused with gadolinium–diethylene triamine pentaacetic acid (Gd-DTPA) to monitor drug distribution and leakage into the CSF space after infusion. A starting total drug dose of 0.5 μg (1/20th of the MTD in rats) at a fixed flow rate of 0.5 mL/h was infused from each of the two catheters. The distribution of MR1-1-PE38KDEL by CED at a concentration of 25 ng/mL was monitored by coinfusion with the low molecular weight tracer gadolinium and 124I-labeled HSA in a malignant glioma patient.142 This study established the utility of Gd-DTPA coinfusion to precisely demonstrate MR1-1-PE38KDEL distribution at the tumor site (Fig. 10.3). Monitoring of MR1-1PE38KDEL distribution will result in improved delivery of the drug at the tumor site and enhanced therapeutic efficacy. The MR1-1-PE38KDEL Phase I study was terminated due to low patient accrual.

Figure 10.3. T1-weighted signal of MR1-1-PE38KDEL (A and C), compared with measured concentration profile of gadolinium–diethylene triamine pentaacetic acid (Gd-DTPA) (B and D). (A) T1-weighted signal of MR1-1-PE38KDEL at 24 h. (B) Concentration of Gd-DTPA at 24 h. (C) T1-weighted signal of MR1-1-PE38KDEL at 72 h. (D) Concentration of Gd-DTPA at 72 h. Gd-DTPA, gadolinium-diethylene triamine pentaacetic acid.

Reprinted from Sampson JH, Brady M, Raghavan R, et al. Colocalization of gadolinium-diethylene triamine pentaacetic acid with high-molecular-weight molecules after intracerebral convection-enhanced delivery in humans. Neurosurgery. 2011;69(3):668–676, with permission from Wolters Kluwer Health.

D2C7 is a unique mAb that reacts with both EGFRwt and EGFRvIII proteins.143 A novel recombinant IT, D2C7-(scdsFv)-PE38KDEL (D2C7-IT) was constructed by fusing the disulfide stabilized D2C7 scFv (scdsFv) with PE38KDEL.144 In preclinical studies, the dual-specific IT D2C7-IT demonstrated a strong antitumor response against intracranial GBM xenografts expressing either EGFRwt or both EGFRwt and EGFRvIII.144 In a preclinical toxicity study in Sprague–Dawley rats, the MTD of D2C7-IT was determined to be between 0.10 and 0.35 μg total doses, and the no observed adverse effect level of D2C7-IT was the 0.05 μg total dose (manuscript in preparation). On the basis of the preclinical toxicity study, a Phase I study has been initiated to determine the MTD of D2C7-IT delivered intratumorally by CED in recurrent malignant glioma patients.145

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Acute Coronary Syndromes

John W. Eikelboom, Jeffrey I. Weitz, in Hematology (Seventh Edition), 2018

Reteplase

Reteplase is a second-generation nonglycosylated deletion mutant of alteplase. Reteplase is less fibrin specific than alteplase but has a longer half-life (18 minutes) that enables administration by double-bolus IV injection.

The INJECT trial (n = 6010) demonstrated that reteplase and streptokinase were associated with similar 35-day rates of mortality (9.0% vs. 9.5%), in-hospital stroke (1.2% vs. 1.0%), and major bleeding (0.7% vs. 1.0%), although reteplase was associated with a twofold higher rate of intracranial bleeding (0.8% vs. 0.4%). The GUSTO-III trial (n = 15,059) demonstrated that reteplase (two 10-mg IV bolus injections given 30 minutes apart) and front-loaded alteplase were associated with similar 30-day rates of mortality (7.5% vs. 7.2%) and stroke (1.6% vs. 1.7%).

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RNA Editing

Olena Maydanovych, ... Peter A. Beal, in Methods in Enzymology, 2007

8.1 Protocol

8.1.1 Gel mobility shift assay and data analysis

Varying concentrations of the R-D deletion mutant of ADAR2 are added to ∼10 pM (8-azaN at editing site) 5′-32P end-labeled RNA duplex in 15 mM Tris–HCl, pH 7.5, 3% glycerol, 0.5 mM DTT, 60 mM KCl, 1.5 mM EDTA, 0.003% NP-40, 160 units/ml RNasin, 0.1 mg/ml BSA, and 1.0 mg/ml yeast tRNAPhe, and the reactions are incubated at 30° for 10 min. Variation in the incubation period from 10 to 30 min does not affect the measured dissociation constant. Samples are loaded onto a running 6% nondenaturing polyacrylamide gel (79:1 acrylamide:bisacrylamide) and electrophoresed in 0.5× TBE buffer at 4° for 45 min. Storage phosphorimaging plates (Kodak) are pressed flat against the dried electrophoresis gels and exposed in the dark. The data are analyzed by performing volume integrations of the regions corresponding to free RNA, protein–RNA complex, and background sites using ImageQuant software. The data are fit to the following equation: fraction bound =A*[protein]/([protein] +Kd), where Kd is the fitted dissociation constant and A is the fitted maximum fraction RNA bound at that dissociation constant.

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Interleukin 3 (IL-3) Receptor

Padmini Rao, R.Allan Mufson, in Encyclopedia of Immunology (Second Edition), 1998

Receptor subdomains mediating IL-3-dependent proliferative responses and suppression of apoptosis

Structure–function analyses using cytoplasmic domain deletion mutants of the βc subunit have revealed two regions which appear to transduce distinct signals, both of which are required for long-term survival and proliferation (Figure 4). The membrane-proximal region of the human βc subunit (amino acids 455–562), containing a Pro-X-Pro amino acid motif and an acidic residue-rich region, is required for activation of JAK2 tyrosine kinase and PI3K induction of the proto-oncogenes pim-1, c-myc, and mitogenesis. The amino acid sequence of the membrane-proximal region is highly conserved among βc, AIC2A and AIC2B (Figure 5). The membrane-distal region (amino acids 626–763) is essential for activation of Ras, Raf-1, MAPK and p70S6K and is related to induction of the c-fos and c-jun genes. A C-terminal-deleted βc, which lacks the membrane-distal cytoplasmic sequences required for activation of the Ras-Raf-1-MAPK cascade, induces DNA synthesis, but is incapable of supporting cell survival. Thus, despite a transient mitogenic response, cells expressing this mutant die by apoptosis in the presence of growth factor. Interestingly, the expression of a constitutively activated Ras protein appears to complement defective signaling through this mutant and restores the long-term proliferation response to cytokine stimulation. These data suggest that the IL-3R prevents apoptosis by stimulating a signaling pathway distinct from the induction of DNA synthesis.

Figure 4. Structure of the human IL-3 βc subunit: EC, extracellular domain; TM, transmembrane domain; gp130 homology domain, region containing the i) Pro-Asn-Pro motif that is important for tyrosine phosphorylation and cell proliferation, and ii) acid-rich domain containing 11 acidic and 1 basic amino acids which comprise a motif shared with the p75 signaling subunit of the IL-2 receptor. The membrane-proximal domain, extending between Arg456 and Asp544 is involved in mediating the proliferative signal through the IL-3 receptor. A region encompassing amino acids Val518 to Leu626 is responsible for major tyrosine phosphorylation, and the membrane-distal region (Leu626-Ser763) is required for activation of Ras, Raf-1, MAPK, and p70 S6K, as well as induction of c-fos and c-jun.

Figure 5. Primary amino acid sequences of regions in the human βc, which are involved in IL-3-mediated cell proliferation. Sequences from corresponding regions of mouse AIC2A and AIC2B are also aligned to highlight the extensive homology between human βc and mouse β subunits. Region (A), between Arg456 and Phe487 appears to be essential for proliferation, while region (B) enhances the growth signal. (C) Underlined residues are acidic amino acids comprising the acid-rich region of human βc; residues in bold identify a motif homologous to that present in the IL-6 receptor gp130.

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Genomic Subtraction

Don Straus, in PCR Strategies, 1995

Overview of Genomic Subtraction

Genomic subtraction purifies DNA that is missing in a deletion mutant by removing from wild-type DNA the sequences that are present in both the wild-type and the deletion mutant genomes. The DNA that corresponds to the deleted region remains. Enrichment for the deleted sequences is achieved by allowing a mixture of denatured wild-type and biotinylated deletion mutant DNA to reassociate (Fig. 1,A). After reassociation, the biotinylated sequences are removed by binding to avidin-coated beads (Fig. 1,B). This subtraction process is then repeated several times (Fig. 1,C). In each cycle, the unbound wild-type DNA from the previous round is hybridized with fresh biotinylated deletion mutant DNA. The unbound DNA from the final cycle is ligated to adaptors (Fig. 1,D) and amplified by using one strand of the adaptor as a primer in the polymerase chain reaction (PCR; Fig. 1,E). The amplified sequences can then be used to probe a genomic library (Fig. 1,F) or they can be cloned directly (Fig. 1,G).

Figure 1. A schematic representation of genomic subtraction.

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Phosphatases and Polynucleotide Kinase

Hyone-Myong Eun, in Enzymology Primer for Recombinant DNA Technology, 1996

ii. In vivo function.

The pseT gene product is not essential because pseT deletion mutant phages grow well in most E. coli strains. Apparently a host gene product can substitute for the T4 pseT gene product in vivo. Most mutations in pse T inactivate both the 5′-kinase and 3′-phosphatase activities. However, a point mutation, pseT1, inactivates only the 3′-phosphatase activity (24, 45), whereas a mutant pseT47 lacks only the 5′-kinase activity (24). Both activities seem to be necessary for probable physiological functions such as mRNA processing and the repair of apurinic/apyrimidinic DNA (37) and of tRNA(s) cleaved by anticodon nuclease (47). Neither the pseT1 nor the pseT47 mutant is able to grow on E. coli CTr5X. Furthermore, a mixed infection of pseT1 and pseT47 mutants does not promote a phage growth in E. coli CTr5X, even though both kinase and phosphatase activities are present in the infected cells. These observations suggest that both activities must be present on the same molecule for proper biological functions.

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Vaccination Against Toxoplasmosis: Current Status and Future Prospects

D. Schaap, ... J. Alexander, in Toxoplasma Gondii, 2007

24.3.3 Vaccination using gene deletion attenuated parasites

Increased knowledge of T. gondii at the molecular level, greatly facilitated by the completion of the genome project, in combination with rapid progress in the development of genetic tools to manipulate the parasite, has generated opportunities to create new attenuated vaccines. Such targeted approaches have been used to create parasites with incomplete life-cycle stages, with reduced proliferative capacity, or with reduced virulence. Mutant parasites have been generated either with an irreversible gene deletion or, more recently, by conditionally inhibiting or activating expression of an essential gene. Genetically 'crippled' parasites have been analyzed in vitro and in vivo to characterize their mutant phenotypes and to determine whether a gene is possibly redundant. At present, the infectivity of all Toxoplasma mutants that have been generated by reverse genetics has only been analyzed in mice and not in larger animals. Whether vaccine potential or its absence demonstrated in mice can be translated into success and failure in larger animals is a matter of some conjecture. For example, while both the RH strain and the incomplete S48 strain are highly lethal to mice, RH is not persistent in pigs and induces protective immunity upon challenge with oocysts (Dubey et al., 1994). Similarly, the incomplete S48 strain does not persist in any other animal so far examined, and is commercially used as a live vaccine. Thus, the potential of a live mutant parasite cannot be determined in mice only, but will ultimately have to be established in a larger animal.

In the past 7 years, various Toxoplasma gene deletion mutants have been generated – although the objective has usually been to gain further insight into the function of a particular gene rather than to generate a vaccine. However, it is likely that any biologically important gene will contribute to the fitness and/or virulence of the parasite, and consequently the vaccine potential of these mutants has been of significant interest (see Table 24.2):

TABLE 24.2. Toxoplasma knockout strains and induced immunity in mice

ReferenceTargeted geneParental strainDosageMouse strainSurvival and/or protective effectFox and Bzik, 2002CPSIIRHUp to 107 tachyzoitesBALB/cNo proliferation, all surviveProtects against lethal challengeRachinel et al., 2004SAG1RH104 tachyzoites*C57BL/630% reduced mortalityDzierszinski et al., 2000SAG3RH20 tachyzoitesBALB/c85% reduced mortalitySoldati et al., 1995ROP1RH50 tachyzoitesSwissLethalMercier et al., 1998GRA2RH10 tachyzoitesSwiss (CD1)50% reduced mortalityBohne et al., 1998BAG1PLK**104 tachyzoitesC57BL/6No reduced mortalityZhang et al., 1999BAG1PLK**105 tachyzoitesSwiss (CD1)Five-fold reduced cyst burden

*applied via surgical injection into the intestines.**PLK is a clonal derivative of ME49.C57BL/6 is a susceptible mouse, BALB/c is a relatively resistant mouse, and CD1 is an outbred, relatively resistant line.

•

By far the most promising vaccine mutant that has been generated is RH strain with a disruption of carbamoyl phosphate synthetase II (CPSII) (Fox and Bzik, 2002). CPSII is the first enzyme in the metabolic pathway for de novo pyrimidine synthesis (generating the building blocks for RNA and DNA), and disrupting this enzyme made Toxoplasma dependent on externally supplied uracil, which it can salvage. Disruption of CPSII thus created a uracil auxotroph, which only grew in vitro when host cells were supplemented with uracil. In the absence of uracil, CPSII knockout parasites invaded host cells normally but failed to replicate. No growth was observed (without added uracil) in vitro and in vivo. Injection of mice with CPSII knockout parasites did not kill BALB/c mice, and mice previously infected with CPSII knockout parasites 40 days previously were resistant to a lethal challenge with 200 pfu of RH strain tachyzoites.

•

Surface antigens (SAGs) of Toxoplasma have also been targeted for deletion. SAGs are thought to be involved in host-cell attachment and the activation of a host immune response. The major tachyzoite surface antigen is SAG1, and two types of SAG1 mutants have been generated. One was made by chemical mutagenesis and the other was recently genetically engineered (ΔSAG1). Both attach to, enter, and proliferate at approximately normal rates within host cells (Kasper and Khan, 1993; Mineo and Kasper, 1994; Rachinel et al., 2004). ΔSAG1 tachyzoites were lethal in susceptible C57BL/6 mice (although survival was slightly prolonged compared with wild-type infected mice), but deletion of SAG1 prevented an acute ileitis when tachyzoites were directly injected into the intestine.

•

SAG3 deletion mutants showed more pronounced effects than ΔSAG1 tachyzoites; these had significantly reduced adherence to host cells in vitro, and mortality was reduced 80 percent upon infection in BALB/c mice compared with wild-type organisms (Dzierszinski et al., 2000).

•

Recently, a genomic cluster containing four bradyzoite-specific SAGs (SAG2c, SAG2d, SAG2x, and SAG2y) was deleted in one knockout. Deleting these four SAGs (ΔSAG2cdxy) yielded viable tachyzoites that could still differentiate into bradyzoites in vitro. In contrast, preliminary studies in vivo showed that 9 out of 10 ΔSAG2cdxy-infected mice were negative for brain cysts when assayed 3 months after infection (J. Saeij and J. Boothroyd, personal communication). Consequently, of the SAG knockouts ΔSAG2cdxy appears the most promising as a vaccine candidate, because although it is still infective and can transform into bradyzoites it persists poorly if at all.

•

Two secretory vesicle protein (ROP1 and GRA2) gene-deletion mutants have also been generated. Disruption of either ROP1 or GRA2 resulted in no difference in growth rates or host-cell invasiveness in vitro, although ΔGRA2 was less virulent in mice (Soldati et al., 1995; Mercier et al., 1998). • Disruption of BAG1, a bradyzoite-specific heat-shock protein, was thought to interfere with the formation or viability of tissue cysts. However, in one study (Bohne et al., 1998) disruption of BAG1 had no effect on tissue-cyst formation, while in a second study (Zhang et al., 1999) disruption of BAG1 could only reduce the number of tissue cysts in mouse brains by roughly five-fold. In the latter study, the lethal dose with ΔBAG1 did increase from 2 × 106 to 5 × 107, compared with the parental PLK strain (a clonal line derived from ME49). Nevertheless, tissue cysts were still being formed and were completely normal, proving that BAG1 is not essential for bradyzoites. The authors suggested that BAG1 homologous genes may exist in Toxoplasma, generating some redundancy.

Attenuated parasites can also be generated by targeting expression of essential genes. Since deletion of such genes will immediately result in non-viable parasites, targeting the expression of essential genes should occur in a conditional way. For the generation of such conditional knockout parasites, an inducible expression system that provides stringent regulation of gene expression would be optimal. Ideally, switching on the expression of the targeted gene will result in a normal phenotype of the parasite, whereas reducing the expression of the targeted gene will result in an attenuated (lethal) phenotype. We and others developed a tetracycline-inducible expression system to allow conditional expression of essential genes. At Intervet Parasitology, Nicole van Poppel showed that efficient transcriptional control of T. gondii by tetracycline-inducible expression system could be obtained using YFPtetR, a repressor consisting of yellow fluorescent protein (YFP) fused to the N-terminus of wild-type tet repressor (tetR). YFPtetR was shown to be a functional repressor with the capacity of 88-fold repression of transcription when expressed in T. gondii (van Poppel et al., 2006). As shown in Figure 24.1, in the absence of tetracycline YFPtetR will block the expression of the reporter beta-galactosidase, whereas when tetracycline is present YFPtetR will be released from its operator elements, relieving the block on transcription and thus yielding normal expression of beta-galactosidase.

FIGURE 24.1. Tetracycline-inducible expression with YFPtetR in T. gondii. A schematic figure demonstrates how genomically integrated YFPtetR regulates tetO-containing promoters in a tetracycline-dependent manner (see text for further details).

YFPtetR was 10 times more sensitive to tetracycline compared with wild-type tetR, and as a result YFPtetR could be regulated with low non-toxic concentrations of tetracycline. During these studies we noted that T. gondii promoters often have multiple transcriptional start sites, which will reduce the level of regulation with tet-repressors. Therefore, the promoter from TgRPS13 gene (encoding small subunit ribosomal protein 13) was selected in our laboratory as a strong promoter containing only one major transcriptional start site, which upon integration of 4 repeated tet-operator elements showed a 100-fold regulation with YFPtetR. Today, these are the highest inducible levels of expression achieved in T. gondii. Currently, we are studying to what extent this inducible system can regulate the expression of an endogenous and essential ribosomal protein, TgRPS13, by integration of multiple tetO sites in its gene promoter region. Such conditional lethal parasites require that they are propagated in the presence of tetracycline. In the absence of tetracycline, such parasites are depleted of TgRPS13 and are expected to die if the concentration of TgRPS13 is too low. Such parasites are useful as invasive but non-persistent vaccine strains in animals or humans. Using conditional mutants as live vaccines requires that these parasites are safe – i.e. incapable of reversion to a virulent phenotype due to mutations. At the moment it is too early to determine the safety of such parasites, but conditional-live HIV strains containing tetracycline-regulated transcription are being considered as human vaccines, illustrating that this may have clinical potential (Verhoef et al., 2001).

Soldati and co-workers generated a similar regulation system, but they used a codon-adapted tetR named tetRS to obtain expression in T. gondii, and used the anhydrotetracyline derivative to overcome the toxic effects of tetracycline (Meissner et al., 2001). In their system, TetRS exerted a 15-fold regulation. During these studies they also showed that a fusion of tetRS with the transactivator VP16 was inactive, probably because VP16 does not connect with the Toxoplasma transcriptional machinery. In a subsequent study (Meissner et al., 2002a) an artificial transactivator was generated, where tetRS was fused to a 26 amino-acid long hydrophobic sequence, creating an anhydrotetracycline-dependent transactivator (named TATi-1) with a 15–20-fold level of regulation. TATi-1 was used to regulate expression of myosin A, when placed under control of a promoter containing 7 repeated tet-operator elements. Myosin A is thought to power the gliding motility of Toxoplasma, thereby being crucial for parasite dispersion and/or invasion. Thus parasites were generated with myosin A expression being dependent on TATi-1. In the presence of anhydrotetracycline, TATi-1 no longer transactivates and myosin A expression was stopped, leading to parasites that were unable to glide and were only 20 percent invasive compared to wild-type parasites, but that could grow at a normal rate once inside the host. These parasites were injected into BALB/c mice and were shown to be non-lethal when maintained in the presence of anhydrotetracycline (supplied via drinking water). Interestingly, after 11 days the anhydrotetracycline supplement was stopped, and the mice remained viable and were protected from a normally lethal challenge with 150 RH-strain tachyzoites. The same TATi-1 system was recently used to regulate TgAMA1 expression (Mital et al., 2005). AMA1 is a leading vaccine candidate in Plasmodium research, and is a secreted micronemal protein. A conditional expressor of TgAMA1 was generated, the expression of which was regulated with TATi-1. Studies with these mutants showed that tachyzoites not expressing TgAMA1 were no longer invasive and could not secrete rhoptries. No in vivo studies were reported with these mutants, but it is possible to envisage a vaccine application with this conditional TgAMA1 strain.

In summary, reverse genetic techniques have enabled the creation of mutant attenuated Toxoplasma strains with vaccine potential. The uracil auxotrophic mutant generated by disruption of CPSII is particularly promising. In addition, conditionally lethal mutant parasites have been generated that are infective and can induce a protective immune response without apparent detrimental effects to the host. It will be particularly important to demonstrate the safety of the mutants before such genetically modified organisms (GMOs) can be tested and used in the field.

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