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Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing

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      Abstract

      The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing.

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      Most cited references 36

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      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

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      The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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        The genetics of Caenorhabditis elegans.

        Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
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          The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.

          The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.
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            Author and article information

            Affiliations
            [* ]Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55454
            []Department of Biology, Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112
            Author notes
            [1]

            Present address: Division of Biological Sciences, Section of Neurobiology, University of California, San Diego, La Jolla, CA 92093.

            [2 ]Corresponding author: University of Minnesota, 6-160 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455. E-mail: rougvie@ 123456umn.edu
            Journal
            G3 (Bethesda)
            Genetics
            G3: Genes, Genomes, Genetics
            G3: Genes, Genomes, Genetics
            G3: Genes, Genomes, Genetics
            G3: Genes|Genomes|Genetics
            Genetics Society of America
            2160-1836
            10 October 2016
            December 2016
            : 6
            : 12
            : 4077-4086
            27729432
            5144976
            GGG_034165
            10.1534/g3.116.034165
            Copyright © 2016 Edelman et al.

            This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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            Figures: 4, Tables: 2, Equations: 0, References: 38, Pages: 10
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            Investigations

            Genetics

            caenorhabditis elegans, lin-42, heterochrony, molting

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