Edward L. Organ

The Drosophila melanogaster stranded at second (sas) gene encodes a putative epidermal cell surface receptor required for larval development

Several lethal mutations were identified previously in the 84BD interval of the Drosophila melanogaster third chromosome (Lewis et al., 1980; Cavener et al., 1986b). We have examined the l(3)84Cd complementation group and found that mutants exhibit novel cuticular defects and die during larval development. The lethal phase occurs during the first larval molt or subsequently during the second instar larval stage; hence, we have named the gene stranded at second (sas). There are no apparent effects on the rate of development of embryos or first instar larvae. Second instar larvae which survive the molt exhibit a marked reduction in growth and eventually die as small second instar larvae. Incomplete penetrance in some weak sas alleles can yield fertile adults. In addition to the lethal phenotype, a segmentally repeated pattern of tanned spots is found within the ventral setal belts of mutant larvae. The position of the spots is always either between the fourth and fifth row of setae (cuticular projections) or between the first and second row of setae. The spots are adjacent to the muscle attachment sites in the setal belt region. Another common larval phenotype is the abnormal tanning of the ventral surface of the pharynx. The sas gene was cloned, and both the cuticular tanning and the larval lethal phenotypes were complemented by P-element-mediated transformation with a genomic DNA-cDNA construct. Three major sas transcripts are expressed throughout development in cuticle secreting epidermal tissues. The sas transcripts show stage- and tissue-specific patterns of expression with switches in transcript patterns occurring at the molts. The inferred 1348-amino-acid sequence suggests that sas encodes a cell surface protein which functions as a receptor. The putative extracellular region contains four tandem repeats of a cysteine-rich motif which is similar to a cysteine pattern present in procollagen and in thrombospondin. Following this region are at least three copies of a fibronectin type III class repeat. The short (35 amino acids) intracellular domain contains a sequence (NPXY) that has been implicated in endocytosis via coated pits.

Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection

BackgroundViruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.ResultsA gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor (Igf2r), a protease associated with insulin growth factor binding protein 5 (Prss11), and the CTCF transcriptional regulator (Ctcf). The disruption of Ctcf, which encodes a repressor of Igf2, was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension (E)-peptide sequence independently conferred high levels of cellular resistance to reovirus infection. Forced IGF-II expression results in a block in virus disassembly. In addition, Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo.ConclusionThese results indicate that IGF-II, and by inference other components of the IGF-II signalling pathway, can confer resistance to lytic reovirus infection. This report represents the first use of gene entrapment to identify host factors affecting virus infection. Concomitant transformation observed in some virus resistant cells illustrates a potential mechanism of carcinogenesis associated with chronic virus infection.

Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

Discovery of mammalian genes that participate in virus infection

BackgroundViruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.ResultsCandidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. One hundred fifty-one reovirus resistant clones were selected from cell libraries containing 2 × 105 independently disrupted genes, of which 111 contained mutations in previously characterized genes and functionally anonymous transcription units. Collectively, the genes associated with reovirus resistance differed from genes targeted by random gene entrapment in that known mutational hot spots were under represented, and a number of mutations appeared to cluster around specific cellular processes, including: IGF-II expression/signalling, vesicular transport/cytoskeletal trafficking and apoptosis. Notably, several of the genes have been directly implicated in the replication of reovirus and other viruses at different steps in the viral lifecycle.ConclusionsTagged sequence mutagenesis provides a rapid, genome-wide strategy to identify candidate cellular genes required for virus infection. The candidate genes provide a starting point for mechanistic studies of cellular processes that participate in the virus lifecycle and may provide targets for novel anti-viral therapies.

Tissue-specific regulatory elements of the Drosophila Gld gene

Putative cis-acting regulatory elements immediately upstream of the Gld promoter were identified by comparative analysis of three Drosophila species. A 509 bp region containing these elements and the Gld promoter region was shown to confer tissue-specific regulation to a reporter gene similar to the pattern observed for Gld mRNA and protein. A dispersed repeat with a core motif of TTAGA was also capable of directing the expression of a reporter gene to several epidermally derived tissues in which GLD is normally expressed. These tissues include male and female somatic reproductive organs. The TTAGA elements and a palindromic element act antagonistically to block expression of reporter gene in some tissues. Previously reported mutations of the heat shock response element resulted in the creation of three TTAGA elements. This mutated hsp70 promoter directs expression of a reporter gene to many of the same tissues as does the Gld TTAGA elements. We have found TTAGA elements near the promoter of two other genes which show an identical expression pattern in the male ejaculatory duct as Gld and the mutant hsp70.

Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication.

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.

Effects of Transforming Growth Factor-α (TGF-α) In Vitro and In Vivo on Reovirus Replication

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.

Effects of Transforming Growth Factor-α(TGF-α)In VitroandIn Vivoon Reovirus Replication

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.