A clonal genetic screen for mutants causing defects in larval tracheal morphogenesis in Drosophila

The initial establishment of the tracheal network in the Drosophila embryo is beginning to be understood in great detail, both in its genetic control cascades and in its cell biological events. By contrast, the vast expansion of the system during larval growth, with its extensive ramification of preexisting tracheal branches, has been analyzed less well. The mutant phenotypes of many genes involved in this process are probably not easy to reveal, as these genes may be required for other functions at earlier developmental stages. We therefore conducted a screen for defects in individual clonal homozygous mutant cells in the tracheal network of heterozygous larvae using the mosaic analysis with a repressible cell marker (MARCM) system to generate marked, recombinant mitotic clones. We describe the identification of a set of mutants with distinct phenotypic effects. In particular we found a range of defects in terminal cells, including failure in lumen formation and reduced or extensive branching. Other mutations affect cell growth, cell shape, and cell migration.     

Temperature-sensitive plant mutants isolated in vitro

Summary Conditional-lethal, temperature-sensitive plant mutants have been isolated using a simple protoplast cloning method. The leaf protoplasts used were obtained from sterile, haploid shoot cultures of Nicotiana plumbaginifolia. Recessive mutations are described at three loci: ts1, ts2 and ts3. The mutations are lethal when either tissue cultures or plants are incubated at 33°C but not at 26°C.     

Temperature-sensitive mutations in Drosophila melanogaster. XI. Male sterile mutants of the Y chromosome

Eight temperature-sensitive (ts) male sterile mutations have been induced by ethyl methanesulfonate treatment of Y chromosomes derived from a selected temperature-resistant Amherst wild-type stock of Drosophila melanogaster. Males carrying such mutated Y chromosomes (Y^ts) are sterile when raised at 29°C but fertile when reared at 22°C. Complementation tests of the mutants with Y chromosome fragments, deletions, and inter se localized all eight to the long arm of the chromosome in four different complementation groups.When Y^ts-bearing males, reared to adulthood at 22°C, were subjected to a 48-hr regimen at 29°C and mated to fresh virgin females daily, a significant reduction in fertility resulted 5 days after initiation of 29°C treatments. This period of sterility was transient (48–72-hr duration) and corresponded to a temperature-sensitive period (TSP) of spermatogenesis during the primary spermatocyte stage. A more precise definition of the TSP utilized exposure of subadult males to 29°C at selected developmental periods during which only certain germ cell stages are present. Upon eclosion adult males were subjected to a similar schedule of consecutive matings of 12-hr duration in order to detect any delay in the appearance of fertility. Different ts males could be distinguished by the resultant pattern of sterility, and the TSP of different mutations thus localized to either primary spermatocyte or immediately post-meiotic stage.Associated with Y^ts-mediated sterility, spermiogenesis is defective at restrictive temperature as evidenced by the production of nonmotile sperm and a failure to transfer such sperm to the female during copulation. In addition, electron microscopy detected a variety of ultrastructural abnormalities, including defects of axoneme formation, irregularities of Nebenkern derivative development, and failures of separation from the syncitial state or mature cyst with subsequent degeneration.     

Remembering Obaid Siddiqi,a pioneer in the study of temperature-sensitive paralytic mutants in Drosophila

Although Obaid Siddiqi’s major research focus in neurogenetics was on chemosensation and olfaction in Drosophila, he made seminal contributions to the study of temperature-sensitive paralytic mutants that paved the way for research that we and many other investigators have continued to pursue. Here we recount Siddiqi’s investigation and the impact it had on our own studies especially at a formative stage of our careers. We acknowledge our debt to Obaid Siddiqi and remember him fondly as an inspired and inspiring scientist, mentor, role model and human being.     

Perturbations of model membranes induced by pathogenic dynorphin A mutants causing neurodegeneration in human brain

Several effects of the endogenous opioid peptide dynorphin A (Dyn A) are not mediated through the opioid receptors. These effects are generally excitatory, and result in cell loss and induction of chronic pain and paralysis. The mechanism(s) is not well defined but may involve formation of pores in cellular membranes. In the 17-amino acid peptide Dyn A we have recently identified L5S, R6W, and R9C mutations that cause the dominantly inherited neurodegenerative disorder Spinocerebellar ataxia type 23. To gain further insight into non-opioid neurodegenerative mechanism(s), we studied the perturbation effects on lipid bilayers of wild type Dyn A and its mutants in large unilamellar phospholipid vesicles encapsulating the fluorescent dye calcein. The peptides were found to induce calcein leakage from uncharged and negatively charged vesicles to different degrees, thus reflecting different membrane perturbation effects. The mutant Dyn A R6W was the most potent in producing leakage with negatively charged vesicles whereas Dyn A L5S was virtually inactive. The overall correlation between membrane perturbation and neurotoxic response [3] suggests that pathogenic Dyn A actions may be mediated through transient pore formation in lipid domains of the plasma membrane.     

Drosophila eiger mutants are sensitive to extracellular pathogens

We showed previously that eiger, the Drosophila tumor necrosis factor homolog, contributes to the pathology induced by infection with Salmonella typhimurium. We were curious whether eiger is always detrimental in the context of infection or if it plays a role in fighting some types of microbes. We challenged wild-type and eiger mutant flies with a collection of facultative intracellular and extracellular pathogens, including a fungus and Gram-positive and Gram-negative bacteria. The response of eiger mutants divided these microbes into two groups: eiger mutants are immunocompromised with respect to extracellular pathogens but show no change or reduced sensitivity to facultative intracellular pathogens. Hence, eiger helps fight infections but also can cause pathology. We propose that eiger activates the cellular immune response of the fly to aid clearance of extracellular pathogens. Intracellular pathogens, which can already defeat professional phagocytes, are unaffected by eiger.     

Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant loss-of-function mutations in SERCA indicates novel intra- and intermolecular interactions for SERCA in vivo, overlooked by current structural models.     

Temperature-sensitive mutations causing reversible paralysis in Caenorhabditis elegans

A method has been developed for the isolation of temperature-dependent paralytic mutants of the nematode Caenorhabditis elegans, based on a screening procedure using short-time exposure to 30 degrees C. Of ten mutants isolated, eight lose their motilities between 30 degrees C and 33 degrees C without prominent changes in body forms. The other two strains that are mainly described in this report are accompanied by alterations in body forms. One mutation, cn101, is recessive and an allele of cha-1. The cn101 mutant shows reversible paralysis at 30 degrees, accompanied by a hypercontracted and coiled body form. At the restrictive temperature, the strain is resistant to all tested inhibitors of acetylcholinesterase (AChE). Another mutation, designated mah-2 (cn110), is a sex-linked semidominant that is mapped as 0.6 map units left of dpy-6. The cn110 mutant is rapidly paralyzed at the restrictive temperature and has a straight and rigid body form; the mutant rapidly recovers when the temperature is lowered. No disorganization of the muscle structure was detected by polarized light and electron microscopic inspection.     

Temperature-sensitive mutants of RNase E in Salmonella enterica

RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys, Ile207→Ser, Ile207→Asn, and Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.     

Koilocytes are enriched for alkaline-labile sites

This study investigated possible variations in the chromatin structure of koilocytes resulting from human papillomavirus (HPV) infection. Alkaline-labile sites (ALS) were detected with the DNA breakage detection–fluorescence in situ hybridization (DBD-FISH) technique using a whole human genome DNA probe obtained from individuals without koilocytosis. The variable levels of ALS present were measured quantitatively using image analysis after whole-genome DNA hybridization. A significant increase in the number of ALS was observed in koilocytes compared with normal cells. We demonstrated that the presence of ALS could be an indicator of chromatin change in koilocytes caused by HPV infection.     

Connecting cell-cycle activation to neurodegeneration in Drosophila

Studies in cell-culture systems and in postmortem tissue from human disease have suggested a connection between cell-cycle activation and neurodegeneration. The fruit fly Drosophila melanogaster has recently emerged as a powerful model system in which to model neurodegenerative diseases. Here we review work in the fly that has begun to address some of the important questions regarding the relationship between cell-cycle activation and neurodegeneration in vivo, including recent data implicating cell-cycle activation as a downstream effector of tau-induced neurodegeneration. We suggest how powerful research tools in Drosophila might be utilized to approach fundamental questions that remain.     

Temperature-sensitive mutants of vesicular stomatitis virus are conditionally defective particles that interfere with and are rescued by wild-type virus.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to complementation groups I, II and IV inhibited the replication of wild-type vesicular stomatitis virus when mixed infections were carried out in BHK21 cells at 32, 37, and 39.5 C. The group IV mutant (ts G 41) was most effective in this regard; wild-type virus yields were inhibited almost 1,000-fold in mixed infections with this mutant at 32 C. In the case of group I and II mutants, inhibition of wild-type virus replication at 37 and 39.5 C was accompanied by an enhancement (up to 15,000-fold) of the yields of the coinfecting ts mutant. The yields of the group IV mutant (ts G 41) were not enhanced by mixed infections with wild-type virus at any temperature, although this mutant inhibited wild-type virus replication at all temperatures. The dominance of the replication of ts mutants at 37 C provides a rationale for the selection and maintenance of ts virus in persistently infected cells.