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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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[
Methods Cell Biol,
1995]
In studying embryos of many species, methods of fragmenting and culturing embryonic tissues or cells have been useful for addressing questions of blastomere autonomy in early and later embryogenesis, for exposure to drugs or other agents that perturb specific processes, and for direct labeling of DNA or RNA. For Caenorhabditis elegans workers, the small size of the embryo and the impermeability of the chitinous eggshell and inner vitelline membrane have made such experiments difficult. A method of permeabilization and blastomere isolation, a culture system that will support further cellular development and differentiation, and assay methods for assaying the degree of development and its relative normality after experimental manipulation are minimal requirements for a satisfactory C. elegans embryonic culture system. Methods of isolating early blastomeres have included crushing of the eggshell and extrusion, laser ablation of neighboring blastomeres within an itact eggshell, laser puncturing of the eggshell producing extrusion, and digestion of the eggshell followed by shearing or manual stripping of the vitelline membrane. This last method is described in detail below. Permeabilization of complete embryos can be achieved by the same methods; in addition, one-cell embryos within the shell can be permeabilized to certain drugs such as cytochalasin D by gentle pressure on an overlying
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[
1994]
In order to contribute to the understanding of the organization and function of genes in the genome of Caenorhabditis elegans, we have undertaken a genetic approach. This type of approach relies upon the availability of mutant strains. Although there are many specific applications, in general genetic deduction depends upon the removal of a single component, and subsequent inference from phenotypic alterations as to the function of that component. The biology of C. elegans makes it very amenable to genetic manipulation...
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[
1990]
The biological processes collectively called aging are being dissected in our laboratory using classic genetic analyses akin to those used in the dissection of other fundamental biological processes, e.g., development or metabolism. Many pitfalls are inherent in the genetic analysis of components of fitness; many result from effects of inbreeding. These inbreeding effects have been avoided by the use of the small free-living nematode Caenorhabditis elegans. The hermaphroditic life-style of this animal facilitates the analysis of life span and senescence by permitting the direct isolation and genetic analysis of long-lived mutants and recombinant inbred (RI) lines without complications resulting from inbreeding problems. Both approaches to obtaining long-lived genotypes have been used effectively in the analysis of the aging processes of C. elegans and the reader will find a brief summary of
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[
1984]
Current knowledge concerning the protein components of muscle is based largely on biochemical analysis of myofibrillar preparations. Such in vitro studies are limited because direct evidence for the in vivo function of isolated proteins is difficult to obtain. In vitro techniques, futhermore, are restricted often to the study of abundant proteins. Very little is known about minor sarcomeric components or how the sarcomere is assembled overall. Certainly, the assembly and function of a structure as complex as the sarcomere require many more than the dozen or so proteins commonly studied. Genetic techniques provide an alternative approach to the study of muscle. Mutations that cause muscle disfunction define genes required to construct a normal muscle. cell. The nature of mutant defects provides insights into the functions of the wild-type gene products. Genetic analysis is not restricted to the study of abundant proteins. The genes for even low-abundance proteins are subject to mutation, and if a gene product is required for muscle assembly or function, such mutants will be muscle-defective. Macromolecular complexes provide special opportunities for genetic intervention, because gene products that directly interact in the structure can often be identified. Mutational defects that affect one member of an interacting pair of proteins can be compensated by mutations affecting its interacting partner. Not all genes, hwoever, are directly accessible to genetic analysis. For example, genes whose products are essential for cell or organism viability and genes having more than one functional copy present special problems.....
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[
1977]
The soil nematode Caenorhabditis elegans was selected 11 years ago by Sydney Brenner as an experimental organism suitable for the isolation of many behavioral mutants and small enough for anatomical analysis of such mutants with the electron microscope. Two distinct goals motivated the initial studies of this organism: first, the hope that some of the mutants would have simple anatomical alterations that could be directly correlated with their behavioral defects, allowing the assignment of specific functions to specific neurons, and second, the hope that the detailed analysis of the kinds of alterations induced by individual mutations and the classes of cells affected by given mutations would reveal general features of the genetic program that specifies the development of the organism. Over the past 11 years the number of investigators working on C. elegans has increased to about 75 and is still growing. Nearly 3,000 different mutants have been isolated and different investigators are pursuing their effects on different cells. My own research is in the development of the nervous system. In particular, I would like to learn something about the workings of the complex black box that connects individual genes to the determination of the morphology of developing neurons. Are there gene products whose specific function is to determine the morphology of cells? If so, what are these gene products and how do they act in the developing cell? One would anticipate that mutations in such hypothetical genes would cause specific morphological alterations in cells. Because the morphology of a neuron determines its function, by selecting behavioral mutants altered in the function of the nervous system one might commonly find mutants that alter the morphology of neurons, and some of these might be in specific morphological genes. It is my hope that it will be possible to compare such mutants to the wild type in order to identify the defective gene products and thereby learn something about the role of normal gene products in determining the development of neurons. In this paper I will first summarize the results of several years' work on one specific class of mutants in the nematode, sensory mutants, work performed both in my laboratory and that of my colleagues Jim Lewis and Jonathan Hodgkin. Second, I will discuss frankly some of the difficulties and frustrations we have experienced in trying to interpret the effects of these specific mutants. Some of these difficulties illustrate problems endemic to genetic studies of development. Third, I will describe the more recent work performed in my labortory that is being directed toward genetic analysis of the structure and function of a
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[
1983]
In the preparation of this review, I have made the basic assumption that the desire of the reader is to understand the biological basis of organismic aging. Given this premise, the organism of choice should be one that offers the most immediate hope of arriving at such an understanding. An ideal organism should have a short lifespan; be inexpensive to maintain; be experimentally malleable by a variety of techniques including molecular, morphological, genetic, and biological approaches; and be the object of study in a sufficient number of different laboratories to assure the accumulation of a critical mass of data. The nematode, Caenorhabditis elegans, admirably fulfills all of these basic requirememts. Researchers in the field of aging are faced with a large number of different theories which purport to explain the molecular basis of organismic aging. There are two major reasons for this proliferation of theoretical views. First, aging is an extremely complex phenomenon involving changes in a number of different physiological systems; these physiological changes are often detected, but proof that any one of the changes is responsible for aging is lacking. Second, the focus of a great deal of the research in the field has not been so much on understanding the biological basis of the entire aging process as on understanding one or another of the consequences of this process, particularly in humans and other mammals. The mammalian model systems may often be quite inappropriate for addressing the more basic, long-term questions about the