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[
Methods Cell Biol,
1995]
Geneticists like to point out that the ultimate test of a proposed function for a gene and its encoded product (or products) in a living organism involves making a mutant and analyzing its phenotype. This is the goal of reverse genetics: a gene is cloned and sequenced, its transcripts and protein coding sequence are analyzed, and a function may be proposed; one must then introduce a mutation in the gene in a living organism to see what the functional consequences are. The analysis of genetic mosaics takes this philosophy a step further. In mosaics, some cells of an individual are genotypically mutant and other cells are genotypically wild type. One then asks what the phenotypic consequences are for the living organism. This is not the same as asking what cells transcribe the gene or in what cells the protein product of the gene is to be found, but rather it is asking in what cells the wild-type gene is needed for a given function...
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[
1977]
Native myosin purified from the wild-type, N2, and a body-wall defective mutant, E675, of the nematode contains two myosins, each homogeneous for different heavy chains. These myosins can be resolved from one another on hydroxyapatite and, when cleavaged with CNBr, they yield different peptide-fragments. In E190, one of the homogeneous myosins is absent.
e190 and
e675 are alleles of the same gene,
unc-54. The myosin lacking in E190 is the same one affected in E675. This suggests that
unc-54 is the structural gene for a myosin heavy chain. In order to determine the role of these different myosins, we plan to use antibodies to locate the myosins on thick filaments from body-wall muscle. Additionally, we are studying the patterns of synthesis and degradation of the two myosins in the wild-type and muscle-defective mutants in order to discover how the observed stoichiometry is maintained.
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[
WormBook,
2005]
Mutations in many genes can result in a similar phenotype. Finding a number of mutants with the same phenotype tells you little about how many genes you are dealing with, and how mutable those genes are until you can assign those mutations to genetic loci. The genetic assay for gene assignment is called the complementation test. The simplicity and robustness of this test makes it a fundamental genetic tool for gene assignment. However, there are occasional unexpected outcomes from this test that bear explanation. This chapter reviews the complementation test and its various outcomes, highlighting relatively rare but nonetheless interesting exceptions such as intragenic complementation and non-allelic non-complementation.
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[
WormBook,
2005]
Genetic suppression has provided a very powerful tool for analyzing C. elegans. Suppression experiments are facilitated by the ability to handle very large numbers of individuals and to apply powerful selections. Because the animal grows as a self-fertilizing diploid, both dominant and recessive suppressors can be recovered. Many different kinds of suppression have been reported. These are discussed by category, with examples, together with discussion of how suppressors can be used to interpret the underlying biology, and to enable further experimentation. Suppression phenomena can be divided into intragenic and extragenic classes, depending on whether the suppressor lies in the same gene as the starting mutation, or in a different gene. Intragenic types include same-site replacement, compensatory mutation, alteration in splicing, and reversion of dominant mutations by cis- knockout. Extragenic suppression can occur by a variety of informational mechanisms, such as alterations in splicing, translation or nonsense-mediated decay. In addition, extragenic suppression can occur by bypass, dosage effects, product interaction, or removal of toxic products. Within signaling pathways, suppression can occur by modulating the strength of signal transmission, or by epistatic interactions that can reveal the underlying regulatory hierarchies. In C. elegans biology, the processes of muscle development, vulva formation and sex determination have provided remarkably rich arenas for the investigation and exploitation of suppression.
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[
1987]
Nematode sperm are crawling cells that exhibit a type of locomotion characteristic of an entire class of protozoa as well as numerous embryonic, differentiated, and transformed metazoan cells. Despite considerable variation in morphology and speed of locomotion expressed by these various types of crawling, or amoeboid, cells, there is general agreement that in all cases locomotion is propelled by cytoplasmic contraction involving myosin-induced sliding of actin filaments and regulated, in ways that are not fully understood, by a spectrum of actin-binding proteins. We began to study the motility of sperm of Caenorhabditis elegans hoping to exploit the mutability of this cell in order to analyze the molecular basis of amoeboid movement genetically. Much to our surprise, we discovered that sperm motility is not driven by an actin-based mechanism. Subsequent work, however, has shown that nematode sperm do share many fundamental properties with other amoeboid cells. As a consequence, sperm continue to serve as a profitable model for understanding how cells crawl and, at the same time, have allowed us to examine a new type of cellular motor.
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[
Methods Cell Biol,
1995]
ACeDB (A Caenorhabditis elegans Data Base) is a data management and display system that contains a wide range of genomic and other information about C. elegans. This chapter provides an overview of ACeDB for the C. elegans user, focusing in particular on the Macintosh version Macace. Previous reviews of AceDB include those of Thierry-Mieg and Durbin (1992) and Durbin and Thierry-Mieg (1994), which describe the general properties of the whole system, and that by Dunham et al. (1994), which discussed the use of AceDB for physical map data collection and assembly. ACeDB was developed by Jean Thierry-Mieg and Richard Durbin primarily for the C. elegans project, when the genomic sequencing project was just beginning in 1990. The original aim was to create a single database that integrated the genetic and physical maps with both genomic sequence data and the literature references. The forerunner of ACeDB was the program CONTIG9 (Sulston et al., 1988), which was developed to maintain and edit the physical map. CONTIG9 served researchers around the world by providing critical on-line access to the current physical map as it was being constructed (Coulson et al., 1986). This policy of immediate access allowed members of the worm community to see the same data as the people making the map, and proved very successful in maximizing use of the map. The same approach was adopted as a template for ACeDB. These two principles, developing a comprehensive database for all types of genomic and related data and providing public access to the data in the same form as used by the data-collecting laboratories, have continued to underlie developments of ACeDB. Over the last 5 years, a wide range of genome projects relating to other organisms have taken the ACeDB program and used it to develop databases for their own data. ACeDB has been used both in public projects designed to redistribute public data in a coordinated fashion and laboratory-based projects for collecting new data. Others, such as the C. elegans ACeDB, have used the database for both purposes. The reason it has been possible to adapt ACeDB so widely is that its flexible data structure allows new types of objects and new types of information about these objects to be added easily. This chapter describes (1) how to obtain ACeDB and documentation for it, (2) how to access and use the information in ACeDB, and (3) how to use ACeDB as a laboratory-based data managing system. Some of what we discuss is specific to the nematode database, but other information applies to the basic computer software program and, hence, to any database using the ACeDB program.
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Cell proliferation and cell death are two opposing sides of the same coin. The development and homeostasis of multicellular organisms requires the accurate control of both processes. The death that occurs normally as a process of development is called 'programmed cell death' or 'apoptosis'. The phenomenon of developmentally programmed cell death has been known for more than 100 years, but it is only in the last decade that its molecular mechanisms have begun to be uncovered. The nematode Caenorhabditis elegans is one of the few experimental systems in which a genetic approach is available to dissect the processes of programmed cell death, and in fact, findings from studies using C. elegans have already played a very important role in elucidating these mechanisms. Here, we summarise the progress to date in understanding how cell death is controlled in C. elegans, and review the main machinery of programmed cell death/apoptosis with respect to its evolutionary conservation between C. elegans and other species.
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[
Methods Cell Biol,
1995]
Sequence analysis of cosmids from C. elegans and other organisms currently is best done using the random or "shotgun" strategy (Wilson et al., 1994). After shearing by sonication, DNA is used to prepare M13 subclone libraries which provide good coverage and high-quality sequence data. The subclones are assembled and the data edited using software tools developed especially for C. elegans genomic sequencing. These same tools facilitate much of the subsequent work to complete both strands of the sequence and resolve any remaining ambiguities. Analysis of the finished sequence is then accomplished using several additional computer tools including Genefinder and ACeDB. Taken together, these methods and tools provide a powerful means for genome analysis in the nematode.
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[
Modern Cell Biology,
1994]
During the development of any multicellular organism, the behavior of any given cell can be influenced in two ways: by its ancestry, i.e., by the particular pattern of determinants it inherits (lineal programming); or by its environment, i.e., the signals it receives from other cells. In C. elegans, the relative importance of these two factors for the development of any given cell can be examined with an unusually high degree of precision. There are a number of reasons for this, but perhaps the most important is that the cell lineage, the particular pattern of cell divisions and differentiations that occur in development, is known, and is largely the same from animal to animal. Alterations in the lineage, therefore, can be understood in terms of altered developmental decisions of
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[
1960]
For the purpose of the present chapter the noun 'cultivation' is to be taken as the maintenance, in the laboratory, of a population of organisms belonging to a desired species through successive generations and subcultures over a prolonged period of time (weeks, months, or years). This is a deliberate restriction of the term. The noun 'culture' is most aptly used for a population within a circumscribed vessel or container (test-tube, Petri dish, U.S. Bureau of Plant Industry watch glass, etc.); it is also used in a looser, more general way (as "in culture") to cover conditions of substantial growth whether or not leading to cultivation in the strict sense