mRNAs that contain premature stop codons are unstable in most eukaryotes, but the mechanism of their degradation is largely unknown. We demonstrate that functions of the six C. elegans stag genes are necessary for rapid turnover of nonsense mutant mRNAs of the unc-54 myosin heavy chain gene. Nonsense aUeles of uric-54 express mRNAs that are unstable in stag(+) genetic backgrounds but have normal or near normal stability in stag(-) backgrounds, stag mutations also stabilize mRNA of unc-54(r293), a small deletion that removes the unc-54 polyadenylation site and expresses an aberrant mRNA. Most uric-54 nonsense mutations are recessive in both stag(+) and stag(-) genetic backgrounds. However, four specific alleles are recessive when stag (+) and dominant when snag (-). These stag-dependent dominant alleles express nonsense mutant polypeptides that disrupt thick filament and/or sarcomere assembly. All four alleles are predicted to express nonsense fragment polypeptides that contain most of the myosin globular head domain without an attached rod segment. By degrading messages that contain premature stop codons, the stag genes eliminate mRNAs that encode potentially toxic protein fragments. We propose that this system of mRNA turnover protects cells from their own errors of transcription, mRNA processing, or mRNA transport.[Key Words: mRNA turnover; Caenorhabditis elegans; smg genes; mRNA surveillance] Received July 9, 1993; revised version accepted August 6, 1993.The steady-state level of a eukaryotic mRNA is established by its relative rates of synthesis and degradation. It is increasingly apparent that mRNA degradation is an important aspect of gene expression and its regulation (for reviews, see Atwater et al. 1990;Peltz et al. 1991). The half-lives of different mRNAs can vary from a few minutes to a few weeks. For example, the half-lives of c-myc and c-fos can be as short as 30 mins (Kruijer et al. 1984;Muller et al. 1984;Kindy and Sonnenshein 1986), the half-life of B-globin mRNA is >24 hr (Ross and Pizarro 1983), and the half-life of Xenopus vitellogenin mRNA in the presence of estrogen is -3 weeks (Brock and Shapiro 1983). The stability of many mRNAs is regulated by cellular and environmental stimuli. For example, the half-lives of certain histone mRNAs change during the cell cycle (Hereford et al. 1981), tubulin mRNA tumover is regulated by the concentration of unpolymerized tubulin (Cleveland 1988), estrogen increases the half-life of vitellogenin mRNA (Brock and Shapiro 1983), and heat shock stabilizes HSP70 mRNA (DiDomenico et al. 1982). Regulated mRNA stability is widespread, but we know very little about the molecular mechanisms involved.mRNA degradation presumably involves both cis-acting sequences that identify a mRNA for degradation and trans-acting factors that degrade (or regulate degradation of) the message. A number of cis-acting sequences that Corresponding author.are required for regulated or constitutive mRNA turnover have been defined. The iron-responsive element regulates stability of transfe...

The COPAS Biosorter is a flow cytometer designed to accommodate large objects the size of Caenorhabditis elegans. This instrumentation brings high-speed automated analysis and sorting to this small model organism. The Biosort system optically analyzes and sorts living multicellular organisms on the basis of fluorescent protein expression patterns and other optical signatures, at rates up to about 100 organisms per second. The Biosort is capable of fluorescently analyzing and sorting multicellular organisms that are many-fold larger than single cells. Animals pass through a laser beam focused to the center of the flow cell. This beam is narrower than the animal so that multiple measurements are made per animal, which means that the organism is optically scanned along its long axis as it flows. Stable laminar flow in the flow cell acts to orientate the animal with the flow stream. Fluorescent locations along the axis of the animal are sequentially excited as the organism flows through the line of focus. The fluorescent properties of commonly used reagents in the research field allow the user to detect fluorescent protein expression, lectin and antibody binding, and autofluorescence. The ability to dispense organisms as they emerge from the flow cell allows for the collection of those organisms that have certain optical properties defined by the researcher. Also, dispensing allows for the precise distribution of specific numbers of animals for analysis that can vary with organism numbers.

Independent reversions of mutations affecting three different Caenorhabditis elegans genes have each yielded representatives of the same set of extragenic suppressors. Mutations at any one of six loci act as allele-specific recessive suppressors of certain allels of unc-54 (a myosin heavy chain gene), lin-29 (a heterochronic gene), and tra-2 (a sex determination gene). The same mutations also suppress certain alleles of another sex determination gene, tra-1, and of a morphogenetic gene, dpy-5. In addition to their suppression phenotype, the suppressor mutations cause abnormal morphogenesis of the male bursa and the hermaphrodite vulva. We name these genes smg-1 through smg-6 (suppressor with morphogenetic effect on genitalia), in order to distinguish them from mab (male abnormal) genes that can mutate to produce abnormal genitalia but which do not act as suppressors (smg-1 and smg-2 are new names for two previously described genes, mab-1 and mab-11). The patterns of suppression, and the interactions between the different smg genes, are described and discussed. In general, suppression is recessive and incomplete, and at least some of the suppressed mutations are hypomorphic in nature. A suppressible allele of unc-54 contains a deletion in the 3' noncoding region of the gene; the protein coding region of the gene is apparently unaffected. This suggests that the smg suppressors affect a process other than translation, for example mRNA processing, transport, or stability.

The VAST BioImager system is a set of tools developed for zebrafish researchers who require the collection of images from a large number of 2-7 dpf zebrafish larvae. The VAST BioImager automates larval handling, positioning and orientation tasks. Color images at about 10 μm resolution are collected from the on-board camera of the system. If images of greater resolution and detail are required, this system is mounted on an upright microscope, such as a confocal or fluorescence microscope, to utilize their capabilities. The system loads a larvae, positions it in view of the camera, determines orientation using pattern recognition analysis, and then more precisely positions to user-defined orientation for optimal imaging of any desired tissue or organ system. Multiple images of the same larva can be collected. The specific part of each larva and the desired orientation and position is identified by the researcher and an experiment defining the settings and a series of steps can be saved and repeated for imaging of subsequent larvae. The system captures images, then ejects and loads another larva from either a bulk reservoir, a well of a 96 well plate using the LP Sampler, or individually targeted larvae from a Petri dish or other container using the VAST Pipettor. Alternative manual protocols for handling larvae for image collection are tedious and time consuming. The VAST BioImager automates these steps to allow for greater throughput of assays and screens requiring high-content image collection of zebrafish larvae such as might be used in drug discovery and toxicology studies.

We have investigated the structural features of spontaneous deletions in Caenorhabditis elegans. We cloned and sequenced the junctions of 16 spontaneous deletions affecting the unc-54 myosin heavy-chain gene and compared their sequences with those of the wild type. We analyzed these sequences in an attempt to identify structural features of the gene that are consistently involved in the spontaneous deletion process. Most deletions (15 of 16) removed a single contiguous region of DNA, with no nucleotides inserted or rearranged at the deletion junctions; one deletion was more complex. unc-54 deletions were small, averaging 600 base pairs in length, and were randomly distributed throughout the gene. Unlike deletions that occur in Escherichia coli, spontaneous unc-54 deletions did not contain statistically significant direct or inverted repeats at or near their termini. Except for their small average size, we have not identified any distinguishing features of their sequence or structure. We discuss these results with regard to the mechanisms for spontaneous deletion in eucaryotic and procaryotic cells.