This chapter focuses on gene interactions, whereby many different genes and their polypeptide products work together to carry out a biological process. Genetic approaches like suppression and synthetic enhancement find functionally related genes by examining changes to the mutant phenotypes that occur in double mutants. Genetic interactions find both direct and indirect interactions, so they are especially useful for obtaining a broad picture of the activities of the genes in the biological process; however, they usually do not provide many details about the specific roles. The chapter explains how information about the specific roles of the proteins is found using molecular approaches such as yeast two-hybrid screens or co-immunoprecipitation, the latter being considered the standard for proving that the proteins are part of the same complex in vivo.

Since its introduction as a laboratory organism 50 years ago, the nematode worm Caenorhabditis elegans has become one of the most widely used and versatile models for nearly all aspects of biological and genomic research. Many experiments in C. elegans begin with the generation and analysis of mutants that affect a specific biological process, so genetic techniques are the foundation of worm research. Many different aspects of biology are being studied in C. elegans , and three different recent Nobel Prizes have recognized six researchers working with worms. In addition, C. elegans was the first multicellular organism to have its genome sequenced, so many of the standard genomic methods have also been pioneered in C. elegans . In fact, many novel techniques and ideas are initially tested in C. elegans because of its versatility as a research organism. It is also appropriate for introducing undergraduate students to research, and some of its strengths and challenges for this purpose are discussed. © 2019 The Authors.

Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers that are associated with diarrhea. The genome of EAEC strain 042, a diarrheal pathogen validated in a human challenge study, encodes multiple colonization factors. Notable among them are aggregative adherence fimbriae (AAF/II) and a secreted antiaggregation protein (Aap). Deletion of aap is known to increase adherence, autoaggregation, and biofilm formation, so it was proposed that Aap counteracts AAF/II-mediated interactions. We hypothesized that Aap sterically masks heat-resistant agglutinin 1 (Hra1), an integral outer membrane protein recently identified as an accessory colonization factor. We propose that this masking accounts for reduced in vivo colonization upon hra1 deletion and yet no colonization-associated phenotypes when hra1 is deleted in vitro. Using single and double mutants of hra1, aap, and the AAF/II structural protein gene aafA, we demonstrated that increased adherence in aap mutants occurs even when AAF/II proteins are genetically or chemically removed. Deletion of hra1 together with aap abolishes the hyperadherence phenotype, demonstrating that Aap indeed masks Hra1. The presence of all three colonization factors, however, is necessary for optimal colonization and for rapidly building stacked-brick patterns on slides and cultured monolayers, the signature EAEC phenotype. Altogether, our data demonstrate that Aap serves to mask nonstructural adhesins such as Hra1 and that optimal colonization by EAEC is mediated through interactions among multiple surface factors. IMPORTANCE Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers of the human intestine and can cause diarrhea. Compared to other E. coli pathogens, little is known about the genes and pathogenic mechanisms that differentiate EAEC from harmless commensal E. coli. EAEC bacteria attach via multiple proteins and structures, including long appendages produced by assembling molecules of AafA and a short surface protein called Hra1. EAEC also secretes an antiadherence protein (Aap; also known as dispersin) which remains loosely attached to the cell surface. This report shows that dispersin covers Hra1 such that the adhesive properties of EAEC seen in the laboratory are largely produced by AafA structures. When the bacteria colonize worms, dispersin is sloughed off, or otherwise removed, such that Hra1-mediated adherence occurs. All three factors are required for optimal colonization, as well as to produce the signature EAEC stacked-brick adherence pattern. Interplay among multiple colonization factors may be an essential feature of exceptional colonizers.

The origin of beneficial mutations is fundamentally important in understanding the processes by which natural selection works. Using phage-resistant mutants in Escherichia coli as their model for identifying the origin of beneficial mutations, Luria and Delbrück distinguished between two different hypotheses. Under the first hypothesis, which they termed “acquired immunity,” the phages induced bacteria to mutate to immunity; this predicts that none of the resistant mutants were present before infection by the phages. Under the second hypothesis, termed “mutation to immunity,” resistant bacteria arose from random mutations independent of the presence of the phages; this predicts that resistant bacteria were present in the population before infection by the phages. These two hypotheses could be distinguished by calculating the frequencies at which resistant mutants arose in separate cultures infected at the same time and comparing these frequencies to the theoretical results under each model. The data clearly show that mutations arise at a frequency that is independent of the presence of the phages. By inference, natural selection reveals the genetic variation that is present in a population rather than inducing or causing this variation.

SUMMARY A recent article by Maher et al. in GENETICS introduces an alternative approach to cell-type-specific gene knockdown in Caenorhabditis elegans, using nonsense-mediated decay. This strategy has the potential to be applicable to other organisms (this strategy requires that animals can survive without nonsense-mediated decay—not all can). This Primer article provides a guide and resource for educators and students by describing different gene knockdown methodologies, by assisting with the technically difficult portions of the Maher et al. article, and by providing conceptual questions relating to the article. Related article in GENETICS: Maher, K. N., A. Swaminathan, P. Patel, and D. L. Chase, 2013 A novel strategy for cell-autonomous gene knockdown in Caenorhabditis elegans defines a cell-specific function for the G-protein subunit GOA-1. Genetics 194: 363–373.

Mutations in the him-5 gene in Caenorhabditis elegans strongly reduce the frequency of crossovers on the X chromosome, with lesser effects on the autosomes. him-5 mutants also show a change in crossover distribution on both the X and autosomes. These phenotypes are accompanied by a delayed entry into pachytene and premature desynapsis of the X chromosome. The nondisjunction, progression defects and desynapsis can be rescued by an exogenous source of double strand breaks (DSBs), indicating that the role of HIM-5 is to promote the formation of meiotic DSBs. Molecular cloning of the gene shows that the inferred HIM-5 product is a highly basic protein of 252 amino acids with no clear orthologs in other species, including other Caenorhabditis species. Although him-5 mutants are defective in segregation of the X chromosome, HIM-5 protein localizes preferentially to the autosomes. The mutant phenotypes and localization of him-5 are similar but not identical to the results seen with xnd-1, although unlike xnd-1, him-5 has no apparent effect on the acetylation of histone H2A on lysine 5 (H2AacK5). The localization of HIM-5 to the autosomes depends on the activities of both xnd-1 and him-17 allowing us to begin to establish pathways for the control of crossover distribution and frequency.

ABSTRACT Enteroaggregative Escherichia coli (EAEC) strains are important diarrheal pathogens. EAEC strains are defined by their characteristic stacked-brick pattern of adherence to epithelial cells but show heterogeneous virulence and have different combinations of adhesin and toxin genes. Pathoadaptive deletions in the lysine decarboxylase ( cad ) genes have been noted among hypervirulent E. coli subtypes of Shigella and enterohemorrhagic E. coli . To test the hypothesis that cad deletions might account for heterogeneity in EAEC virulence, we developed a Caenorhabditis elegans pathogenesis model. Well-characterized EAEC strains were shown to colonize and kill C. elegans , and differences in virulence could be measured quantitatively. Of 49 EAEC strains screened for lysine decarboxylase activity, 3 tested negative. Most notable is isolate 101-1, which was recovered in Japan, from the largest documented EAEC outbreak. EAEC strain 101-1 was unable to decarboxylate lysine in vitro due to deletions in cadA and cadC , which, respectively, encode lysine decarboxylase and a transcriptional activator of the cadAB genes. Strain 101-1 was significantly more lethal to C. elegans than control strain OP50. Lethality was attenuated when the lysine decarboxylase defect was complemented from a multicopy plasmid and in single copy. In addition, restoring lysine decarboxylase function produced derivatives of 101-1 deficient in aggregative adherence to cultured human epithelial cells. Lysine decarboxylase inactivation is pathoadapative in an important EAEC outbreak strain, and deletion of cad genes could produce hypervirulent EAEC lineages in the future. These results suggest that loss, as well as gain, of genetic material can account for heterogeneous virulence among EAEC strains.

The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient.

Regulation of both the number and the location of crossovers during meiosis is important for normal chromosome segregation. We used sequence-tagged site polymorphisms to examine the distribution of all crossovers on the X chromosome during oogenesis and on one autosome during both oogenesis and spermatogenesis in Caenorhabditis elegans. The X chromosome has essentially one crossover during oogenesis, with only three possible double crossover exceptions among 220 recombinant X chromosomes. All three had one of the two crossovers in the same chromosomal interval, suggesting that crossovers in that interval do not cause interference. No other interval was associated with double crossovers. Very high interference was also found on an autosome during oogenesis, implying that each chromosome has only one crossover during oogenesis. During spermatogenesis, recombination on this autosome was reduced by approximately 30% compared to oogenesis, but the relative distribution of the residual crossovers was only slightly different. In contrast to previous results with other autosomes, no double crossover chromosomes were observed. Despite an increased frequency of nonrecombinant chromosomes, segregation of a nonrecombinant autosome during spermatogenesis appears to occur normally. This indicates that an achiasmate segregation system helps to ensure faithful disjunction of autosomes during spermatogenesis.

In Caenorhabditis elegans, sex is determined by the number of X chromosomes which, in turn, determines the expression of the X-linked gene xol-1. Recent work has shown that xol-1 expression is controlled by least two distinct regulatory mechanisms, one transcriptional and another post-transcriptional. The transcriptional regulator is a repressor acting in XX embryos; although the specific gene has not been identified, the chromosome region has been defined. A previously defined regulator of xol-1, known as fox-1, maps to a different region of the X chromosome and works post-transcriptionally, consistent with the identification of the fox-1 gene product as a putative RNA binding protein.

In Caenorhabditis elegans triploid animals with two X chromosomes (symbolized 3A;2X) are males. However, these triploid males can be feminized by making them mutant for recessive dosage compensation mutations, by adding X chromosome duplications or by microinjecting particular DNA sequences termed feminizing elements. None of these treatments affects diploid males. This study explores several aspects of these treatments in polyploids. The dosage compensation mutants exhibit a strong maternal effect, such that reduction of any of the dosage compensation gene functions in the mother leads to sex reversal of 3A;2X animals. Likewise, all X chromosome duplications tested cause both sex reversal and intersexual development of many 3A;2X animals. Microinjected feminizing element DNA does not cause extensive sex reversal, but does result in intersexual development in 3A;2X animals. Neither X chromosome duplications nor microinjected feminizing elements show that extreme maternal effect of the dosage compensation mutants, although there is indirect evidence for a maternal effect of the feminizing elements. In particular, very little feminizing element DNA needs to be microinjected in order to feminize triploid males, far less than what is needed for stable inheritance, implying that feminizing elements can work within the mother's gonad. However, even very high concentrations of microinjected feminizing elements do not affect sex determination in diploid males, suggesting that they are not part of the numerator of the X/A ratio. In addition, no pair of X chromosome duplications feminizes diploid males, suggesting that none of these duplications contains a numerator of the X/A ratio. Instead, I infer that an X-linked locus, as yet undefined, must be present in two copies for hermaphrodite development to ensue or that the two X chromosomes might interact.

Recessive mutations in three autosomal genes, him-1, him-5 and him-8, cause high levels of X chromosome nondisjunction in hermaphrodites of Caenorhabditis elegans, with no comparable effect on autosomal disjunction. Each of the mutants has reduced levels of X chromosome recombination, correlating with the increase in nondisjunction. However, normal or elevated levels of recombination occur at the end of the X chromosome hypothesized to contain the pairing region (the left end), with recombination levels decreasing in regions approaching the right end. Thus, both the number and the distribution of X chromosome exchange events are altered in these mutants. As a result, the genetic map of the X chromosome in the him mutants exhibits a clustering of genes due to reduced recombination, a feature characteristic of the genetic map of the autosomes in non-mutant animals. We hypothesize that these him genes are needed for some processive event that initiates near the left end of the X chromosome.

Annals of the New York Academy of SciencesVolume 625, Issue 1 p. 524-531 The Genetics of Response to Volatile Anesthetics in Caenorhabditis elegansa PHILIP G. MORGAN, PHILIP G. MORGAN Department of Anesthesiology University Hospitals of Cleveland and Case Western Reserve University 2078 Abington Road Cleveland, Ohio 44106Search for more papers by this authorMARGARET M. SEDENSKY, MARGARET M. SEDENSKY Department of Anesthesiology University Hospitals of Cleveland and Case Western Reserve University 2078 Abington Road Cleveland, Ohio 44106Search for more papers by this authorPHILIP M. MENEELY, PHILIP M. MENEELY Department of Genetics Fred Hutchinson Cancer Research Center Seattle, Washington 98104Search for more papers by this author PHILIP G. MORGAN, PHILIP G. MORGAN Department of Anesthesiology University Hospitals of Cleveland and Case Western Reserve University 2078 Abington Road Cleveland, Ohio 44106Search for more papers by this authorMARGARET M. SEDENSKY, MARGARET M. SEDENSKY Department of Anesthesiology University Hospitals of Cleveland and Case Western Reserve University 2078 Abington Road Cleveland, Ohio 44106Search for more papers by this authorPHILIP M. MENEELY, PHILIP M. MENEELY Department of Genetics Fred Hutchinson Cancer Research Center Seattle, Washington 98104Search for more papers by this author First published: June 1991 https://doi.org/10.1111/j.1749-6632.1991.tb33883.xCitations: 8 a PGM was supported in part by an American Society of Anesthesiologists Starter Grant 6426412. MMS was supported in part by a National Institutes of Health Grant 1R29GM41385-01A1. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume625, Issue1Molecular and Cellular Mechanisms of Alcohol and AnestheticsJune 1991Pages 524-531 RelatedInformation

The mechanism and site(s) of action of volatile anesthetics are unknown. In all organisms studied, volatile anesthetics adhere to the Meyer-Overton relationship--that is, a ln-ln plot of the oil-gas partition coefficients versus the potencies yields a straight line with a slope of -1. This relationship has led to two conclusions about the site of action of volatile anesthetics. (i) It has properties similar to the lipid used to determine the oil-gas partition coefficients. (ii) All volatile anesthetics cause anesthesia by affecting a single site. In Caenorhabditis elegans, we have identified two mutants with altered sensitivities to only some volatile anesthetics. These two mutants, unc-79 and unc-80, confer large increases in sensitivity to very lipid soluble agents but have little or no increases to other agents. In addition, a class of extragenic suppressor mutations exists that suppresses some altered sensitivities but specifically does not suppress the altered sensitivity to diethyl ether. There is much debate concerning the molecular nature of the site(s) of anesthetic action. One point of discussion is whether the site(s) consists of a purely lipid binding site or if protein is involved. The simplest explanation of our observations is that volatile anesthetics cause immobility in C. elegans by specifically interacting with multiple sites. This model is in turn more consistent with involvement of protein at the site(s) of action.

The signal for sex determination in the nematode Caenorhabditis elegans is the ratio between the number of × chromosomes and the number of sets of autosomes (the X/A ratio). Animals with an X/A ratio of 0.67 (a triploid with two × chromosomes) or less are males. Animals with an X/A ratio of 0.75 or more are hermaphrodites. Thus, diploid males have one × chromosome and diploid hermaphrodites have two × chromosomes. However, the difference in X‐chromosome number between the sexes is not reflected in general levels of X‐linked gene expression because of the phenomenon of dosage compensation. In dosage compensation, X‐linked gene expression appears to be ‘turned down’ in 2X animals to the 1X level of expression. An intriguing and unexplained finding is that mutations and X‐chromosome duplications that elevate X‐linked gene expression also feminize triploid males. One way that this relationship between sex determination and X‐linked gene expression may be operating is discussed.

The authors studied the wild type strain, N2, and three mutant strains of the nematode, Caenorhabditis elegans, in order to measure genetically produced changes in responses to nine volatile anesthetics. They determined the anesthetic ED50s of N2 for thiomethoxyflurane, methoxyflurane, chloroform, halothane, enflurane, isoflurane, fluroxene, flurothyl, and diethylether. The log-log relationship of the oil-gas partition coefficients (O/G) and the ED50s of these agents for N2 yields a straight line with a slope of -.997 with a R2 of .98 over a range of O/G (at 37 degrees C) from 48 to 7230. When the O/Gs are corrected to 22 degrees C, the slope is -.964 with an R2 of .98. This relationship is similar to that found in other animals. Two mutant strains, unc-79 and unc-80, show altered responses to these anesthetics. These strains are two to three times more sensitive than N2 to anesthetics with an O/G greater than that of halothane (220 at 37 degrees C), yet they differ little from N2 in response to anesthetics with lower O/Gs. unc-79 and unc-80 are about 30% more sensitive than N2 to diethylether. The double mutant unc-79; unc-80 is more sensitive to halothane, isoflurane, and fluroxene than is either mutant alone. The authors believe these data indicate an alteration at the site of action of volatile anesthetics in unc-79 and unc-80. They also postulate that the interaction of unc-79 and unc-80 indicate these genes code for enzymes in a common pathway, and that unc-79 precedes unc-80 in this pathway.

X chromosome duplications have been used previously to vary the dose of specific regions of the X chromosome to study dosage compensation and sex determination in Caenorhabditis elegans. We show here that duplications suppress and X-linked hypomorphic mutation and elevate the level of activity of an X-linked enzyme, although these two genes are located in a region of the X chromosome that is not duplicated. The effects do not depend on the region of the X chromosome duplicated and is stronger in strains with two doses of a duplication than in strains with one dose. This is evidence for a general elevation of X-linked gene expression in strains carrying X-chromosome duplications, consistent with the hypothesis that the duplications titrate a repressor acting on many X-linked genes.