From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes that encode the C. elegans orthologs of two splicing factors, the U2AF large subunit (UAF-1) and SF1/BBP (SFA-1). The uaf-1(n4588) mutation resulted in temperature-sensitive lethality and caused the unc-93 RNA transcript to be spliced using a cryptic 3′ splice site generated by the unc-93(e1500) missense mutation. The sfa-1(n4562) mutation did not cause the utilization of this cryptic 3′ splice site. We isolated four uaf-1(n4588) intragenic suppressors that restored the viability of uaf-1 mutants at 25°C. These suppressors differentially affected the recognition of the cryptic 3′ splice site and implicated a small region of UAF-1 between the U2AF small subunit-interaction domain and the first RNA recognition motif in affecting the choice of 3′ splice site. We constructed a reporter for unc-93 splicing and using site-directed mutagenesis found that the position of the cryptic splice site affects its recognition. We also identified nucleotides of the endogenous 3′ splice site important for recognition by wild-type UAF-1. Our genetic and molecular analyses suggested that the phenotypic suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588) and sfa-1(n4562) was likely caused by altered splicing of an unknown gene. Our observations provide in vivo evidence that UAF-1 can act in regulating 3′ splice-site choice and establish a system that can be used to investigate the in vivo regulation of RNA splicing in C. elegans.The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the  Caenorhabditis elegans, we found that mutations in two splicing factors, the U2AF large subunit and SF1/BBP suppress the rubberband Unc phenotype caused by a rare missense mutation in the gene unc-93. Mutations affecting the U2AF large subunit caused the recognition of a cryptic 3′ splice site generated by the unc-93 mutation, providing in vivo evidence that the U2AF large subunit can affect splice-site selection. By contrast, an SF1/BBP mutation that suppressed the rubberband Unc phenotype did not cause splicing using this cryptic 3′ splice site. Our genetic studies identified a region of the U2AF large subunit important for its effect on 3′ splice-site choice. Our mutagenesis analysis of in vivo transgene splicing identified a positional effect on weak 3′ splice site selection and nucleotides of the endogenous 3′ splice site important for recognition. The system we have defined should facilitate future in vivo analyses of pre–mRNA splicing.Eukaryotic genes contain intervening intronic sequences that must be removed from pre-mRNA transcripts by RNA splicing to generate functional messenger RNAs. While studying genes that encode and control a presumptive muscle potassium channel complex in the nematode  C. elegans, mRNA transcripts of at least 13% of predicted genes are alternatively spliced Drosophila gene Dscam (Down syndrome cell adhesion molecule), which through alternative splicing could potentially generate over 30,000 isoforms Eukaryotic genes contain intervening introns that are spliced from transcribed pre-mRNAs to generate functional coding mRNAs splicing factor one/branch-point binding protein) and the large and small subunits of U2AF (U2 auxiliary factor) Saccharomyces cerevisiae lacks a U2AF small subunit and a polypyrimidine sequence in its introns, and the recognition of a 3′ splice site is achieved by binding of SF1/BBP to a highly conserved consensus branch-point sequence Caenorhabditis elegans, there is no consensus branch-point sequence or long polypyrimidine sequence, and the recognition of a 3′ splice site is achieved by the binding of the U2AF large and small subunits to a consensus UUUUCAGR sequence in which “AG” is the 3′ splice site Pre-mRNA splicing involves five small nuclear ribonucleoprotein particles (snRNPs) and numerous associated factors rginine-Serine-rich RNA-binding SR proteins Splicing is also regulated by many AC. elegans is a genetically tractable organism and has been used to study a broad variety of biological problems. Our laboratory has analyzed a set of genes, unc-93, sup-9 and sup-10, that encode components of a presumptive C. elegans two-pore domain K+ channel complex and regulate muscle activity gain-of-function (gf) mutations in any of these three genes cause abnormal body-muscle contraction and are thought to activate the SUP-9 K+ channel. The gf mutant animals are defective in egg laying, sluggish and exhibit a rubberband phenotype: when prodded on the head, the animal contracts and relaxes along its entire body without moving backwards. Complete loss-of-function (lf) mutations of unc-93, sup-9 and sup-10 do not cause any obvious abnormalities Two-pore Acid Sensitive K+ channels TASK-1 and TASK-3 sup-10 encodes a novel single-transmembrane domain protein without identified mammalian homologs unc-93 encodes a multiple transmembrane-domain protein that defines a novel family of proteins conserved from C. elegans to mammals unc-93, sup-9 and sup-10 genes were not designed to identify genes essential for fertility or animal survival. To seek such essential genes, we performed a clonal genetic screen for suppressors of the locomotion defect caused by the unc-93 gf mutation e1500. In this paper, we describe our studies of two suppressors identified from this screen and the establishment of a reporter system for in vivo analysis of RNA splicing in C. elegans. We suggest that the U2AF large subunit affects 3′ splice site recognition and that some aspect of the function of the putative UNC-93/SUP-9/SUP-10 two-pore domain potassium channel complex depends on an unidentified gene the processing of which requires the functions of the U2AF large subunit and SF1/BBP.Previous genetic screens for genes that affect the activities of C. elegans genes unc-93, sup-9 and sup-10 cause a rubberband Unc phenotype, while lf mutations in these genes result in a phenotypically wild-type phenotype unc-93(e1500) mutation, with the goal of identifying mutations that also cause sterility or lethality  of P0 animals mutagenized with EMS (ethyl methanesulfonate) and isolated the suppressors n4588 and n4562. n4588 causes embryonic lethality at 25°C, and n4562 causes sterility at all temperatures. By mapping these mutations, we found that n4588 and n4562 are not alleles of any previously characterized suppressors of the rubberband Unc phenotype (see Rare gf mutations in the lity see . We scretype see .n4588 is a strong recessive suppressor of the locomotion defect and rubberband phenotype of unc-93(e1500) animals (n4588 is or is closely linked to a mutation that causes a recessive temperature-sensitive (ts) lethal phenotype and results in embryonic lethality at 25°C  C. elegans, together with the U2AF small subunit ortholog UAF-2, UAF-1 binds a consensus UUUUCAGR sequence, in which AG is the 3′ splice site RNA recognition motif) domains (U2AF homology motif) domain that binds the splicing factor SF1/BBP n4588 mutation lies between the U2AF small subunit-interacting domain and the first RRM domain of UAF-1a  unc-93(e1500) animals  in wild-type animals did not cause a rubberband Unc phenotype or any other visible abnormality (data not shown). Introducing stop codons or the n4588 T180I mutation into the full-length uaf-1a cDNA abrogated its rescuing activity (uaf-1a cDNA under control of a heat-shock promoter unc-93(e1500) by uaf-1(n4588) and the ts-lethality caused by uaf-1(n4588) (unc-93(e1500) animals with uaf-1 RNAi-expressing bacteria (uaf-1 is required for the rubberband Unc phenotype caused by unc-93(e1500).To test whether the point mutation found in the  animals . A predihenotype . Expressactivity . Heat-shbacteria  also parbacteria , suggestuaf-1 deletion mutation, n5222Δ, which removes the fourth exon (encoding part of the first RRM and part of the second RRM of UAF-1a) of the uaf-1a isoform (uaf-1a isoform are spliced together. uaf-1(n5222Δ)/+ animals grew and moved like the wild type, and uaf-1(n5222Δ)/+ did not suppress the rubberband Unc phenotype of unc-93(e1500) animals (data not shown). uaf-1(n5222Δ) homozygous mutants arrested and died at the late L1 to early L2 larval stages (based on body size), which precluded examination of the rubberband Unc behavior of n5222Δ homozygous animals (see uaf-1(n4588)/uaf-1(n5222Δ) suppressed the rubberband Unc phenotype of unc-93(e1500) animals as strongly as did homozygous uaf-1(n4588) (uaf-1(n4588) homozygotes, uaf-1(n4588)/uaf-1(n5222Δ) animals died embryonically at 25°C (data not shown). These results establish that n4588 is an allele of uaf-1 and that reducing the dosage of the uaf-1(n4588) allele by 50% does not affect the suppression of the rubberband Unc phenotype of unc-93(e1500) animals. These data suggest that uaf-1(n4588) causes either a reduction/loss of uaf-1 activity or an altered uaf-1 activity that is antagonized by the wild-type uaf-1 gene  . Similargene see .unc-93(e1500) are also caused by the unc-93(n200) mutation sup-9 and sup-10uaf-1(n4588) mutation could suppress the Unc phenotype caused by these other mutations (unc-93(n200) was suppressed by uaf-1(n4588) (sup-10(n983), which causes a rubberband Unc phenotype that is more severe than that of unc-93(n200) animals but less severe than that of unc-93(e1500) animals, was completely suppressed by uaf-1(n4588) (sup-9(n1550)uaf-1(n4588) (uaf-1(n4588) is an allele-specific suppressor of unc-93 but not a gene-specific suppressor of the rubberband Unc mutants and is distinct in its suppression pattern from other known suppressors of unc-93, sup-9 and sup-10 (see Locomotion defects similar to those caused by utations . Neither1(n4588) . sup-10(1(n4588) . The str1(n4588) . These dp-10 see .unc-93, sup-10 and sup-9 do not cause visible abnormalities in a wild-type background uaf-1, by generating double mutants containing uaf-1(n4588) and null mutations of unc-93, sup-10 or sup-9. We found that such double mutant animals grew and behaved indistinguishably from uaf-1(n4588) single mutant animals  can suppress gf mutations affecting other two-pore domain potassium channels, we generated double mutant animals containing the uaf-1(n4588) mutation and the unc-58(e665sd)egl-23(n601sd)twk-18(e1913sd)uaf-1(n4588)  .unc-93, sup-9, sup-10 or any of the other genes known to interact with these genes is reduced in uaf-1(n4588) animals, we examined unc-93, sup-10, sup-9, sup-18sup-11unc-93, sup-10 and sup-9, lf mutations in sup-18 and gf mutations in sup-11 can suppress the rubberband Unc phenotype caused by gf mutations in unc-93, sup-9 and sup-10. Using real-time qRT–PCR, we found no obvious reduction of the mRNA levels of these five genes (uaf-1(n4588) animals (data not shown), suggesting that the suppression of unc-93(e1500) by uaf-1(n4588) is not caused by a reduction of the level of the UAF-1 protein.To determine if the expression of ve genes . We alsounc-93 is altered by uaf-1(n4588). We examined the splicing of each exon of unc-93 in wild-type, uaf-1(n4588), unc-93(e1500), uaf-1(n4588) unc-93(e1500), unc-93(n200) and uaf-1(n4588) unc-93(n200) animals by RT–PCR  unc-93(e1500) animals (data not shown) using the PCR primer pairs at the 3′ end of exon 8 and the 5′ end of exon 9 (indicated in black in unc-93(e1500) animals the region between exon 8 and 9 corresponded to a weak but consistent RT–PCR product of a reduced length (unc-93(e1500) mutant animals (uaf-1(n4588) unc-93(e1500) animals, the RT–PCR product of reduced length was the most prominent product (unc-93(e1500) missense mutation, which has a G-to-A transition that changes amino acid 388 from Gly to Arg unc-93 exon 9 in unc-93(e1500) animals and 68% in uaf-1(n4588) unc-93(e1500) animals (uaf-1(n4588), unc-93(n200) or uaf-1(n4588) unc-93(n200) animals, all of which lack the cryptic 3′ splice site caused by the unc-93(e1500) mutation.We tested whether the splicing of y RT–PCR . Every eexamined . However animals . Both no animals  and quan animals  analysesunc-93 transcript is predicted to encode a truncated protein lacking 12 amino acids in one of the predicted transmembrane domains unc-93 transcript in uaf-1(n4588) unc-93(e1500) animals encoded a functional UNC-93 protein, we expressed the cDNA in the body-wall muscles of sup-9(n1550); unc-93(lr12Δ) animals unc-93 cDNA in these animals restored the severe rubberband Unc phenotype. These results suggested that the alternatively spliced unc-93 transcript encoded a lf UNC-93 protein or possibly a dominant-negative UNC-93 protein. To test the latter possibility, we expressed either unc-93 wild-type cDNA or the alternatively spliced unc-93 cDNA in the body-wall muscles of unc-93(e1500) animals (unc-93(e1500)/+ animals have better locomotion than unc-93(e1500) animals unc-93 cDNA dramatically improved the locomotion of unc-93(e1500) animals (unc-93 transcript encoded an UNC-93 protein that could interfere with the endogenous UNC-93 function and cause the suppression of the rubberband Unc phenotype by uaf-1(n4588) , the transgene should also suppress the Unc phenotype of unc-93(e1500) animals. However, expression of the alternatively spliced unc-93 transcript in the body-wall muscles did not suppress the Unc phenotype of unc-93(e1500) animals  mutation, would alter the splicing of unc-93(e1500) exon 9, we fed animals with bacteria expressing dsRNA targeting uaf-1 and assessed unc-93 exon 9 splicing. As shown in unc-93(e1500) exon 9. The RNAi treatment did significantly reduce the level of UAF-1 protein  exon 9 similarly to that by the uaf-1(n4588) mutation is consistent with the hypothesis that uaf-1(n4588) does not reduce the function of UAF-1a but rather alters the function of UAF-1a, which leads to the recognition of the cryptic 3′ splice site of unc-93(e1500) exon 9 (see uaf-1(n4588) reduces uaf-1 function and that uaf-1(RNAi) does not reduce uaf-1 function as much.To examine whether reducing UAF-1 expression, like the  protein . That reon 9 see . Howeveruaf-1(n4588) suppressed the rubberband Unc phenotype of sup-10(n983) animals but did not suppress the rubberband Unc phenotype of unc-93(n200) and sup-9(n1550) animals (sup-10 mRNA in uaf-1(n4588) animals (sup-10(n983) transcript was alternatively spliced in uaf-1(n4588) mutants. uaf-1(n4588) did not cause the appearance of a sup-10 cDNA band different in size from the full-length sup-10 cDNA , uaf-1(n4588) and uaf-1(n4588); sup-10(n983) animals and failed to identify an alternatively spliced sup-10 transcript (data not shown). animals . Quantit animals . We exam-10 cDNA . We deteuaf-1(n4588) can affect the splicing of all genes known to be alternatively spliced, we tested for genetic interactions between uaf-1(n4588) and unc-52(e669). unc-52 encodes the C. elegans ortholog of human basement membrane-specific heparan sulfate proteoglycan core protein, and mutations affecting unc-52 cause adult paralysis unc-52(e669) can be suppressed by lf mutations of smu-1 and smu-2, genes that encode C. elegans homologs of mammalian splicing factors unc-52(e669) mutation causes a pre-mature stop in unc-52 exon 17 smu-1 and smu-2 lf mutations suppress unc-52(e669) by removing exon 17 and generating an alternatively spliced and functional transcript unc-52(e444) mutation causes a pre-mature stop in unc-52 exon 18, which is not removed in smu-1 and smu-2 mutant animals, leading to a transcript with a premature stop codon. Double mutants containing the unc-52(e444) mutation and the smu-1 or smu-2 mutations display an Unc phenotype unc-52(e669); uaf-1(n4588) and unc-52(e444); uaf-1(n4588) animals and found that uaf-1(n4588) did not suppress either unc-52(e669) or unc-52(e444) (uaf-1(n4588) mutation did not affect the alternative splicing of unc-52(e669) exon 17 and thus does not affect all cases of alternative splicing non-specifically.To test whether 52(e444) , implyinn4562 was also isolated from our clonal screen as a suppressor of the rubberband Unc phenotype of unc-93(e1500) animals. The suppressed phenotype was recessive, and n4562 caused a completely penetrant recessive sterility that was temperature independent and was tightly linked to its suppressor activity (see uaf-1(n4588), n4562 suppressed unc-93(e1500) and sup-10(n983) but did not suppress unc-93(n200) or sup-9(n1550)  . Therefon4562 to the right of LG IV (see unc-93(e1500) are located in this region. The genes uaf-2, encoding the C. elegans U2AF small subunit ortholog Y116A8C.32, encoding the SF1/BBP (splicing factor 1/branch-point binding protein) ortholog n4562. We determined the DNA sequences of coding regions of uaf-2 and Y116A8C.32 from n4562 animals and identified a nonsense mutation in Y116A8C.32, which we named sfa-1  (n4562 changed amino acid 458 from a Cys (TGT) to an opal stop (TGA) codon in a conserved C2HC-type zinc finger domain of the predicted SFA-1 protein (unc-93(e1500) by sfa-1(n4562) by expressing in body-wall muscles an SFA-1::GFP fusion protein driven by the myo-3 promoter unc-93(e1500) animals with bacteria expressing dsRNA targeting sfa-1 partially suppressed the rubberband Unc phenotype (We mapped G IV see . No know factor) . n4562 c protein . This muhenotype .sfa-1 deletion mutation, n5223Δ, which removes the third and fourth exons and a majority of the fifth exon  caused recessive embryonic lethality, and sfa-1(n5223Δ)/+ did not suppress the rubberband Unc phenotype of unc-93(e1500) animals (data not shown). sfa-1(n4562)/(n5223Δ) similarly caused embryonic lethalilty (data not shown), suggesting that the lethal phenotype of sfa-1(n5223Δ) homozygotes is caused by the sfa-1(n5223Δ) mutation. The embryonic lethality caused by sfa-1(n5223Δ) and sfa-1(n4562)/sfa-1(n5223Δ) precluded the use of n5223Δ for an analysis of the rubberband Unc phenotype, because our behavioral assay is performed with young adults (see We isolated an fth exon . Togetheults see .uaf-1(n4588), sfa-1(n4562) caused alternative splicing of unc-93(e1500) exon 9, we used RT–PCR to examine the splicing of unc-93(e1500) exon 9. As shown in sfa-1(n4562) did not cause increased alternative splicing of unc-93(e1500) exon 9. We tested the effect of sfa-1 on unc-93(e1500) exon 9 splicing by reducing sfa-1 expression using RNAi (sfa-1(RNAi) did not increase exon 9 alternative splicing  mutation causes a recessive sterile phenotype, which is less severe than the recessive embryonic lethality caused by sfa-1(n5223Δ)  or by sfa-1(n4562)/sfa-1(n5223Δ), (2) sfa-1(RNAi) phenocopies sfa-1(n4562) in the suppression of the rubberband Unc phenotype of unc-93(e1500) animals, and (3) sfa-1(RNAi) phenocopies sfa-1(n4562) in affecting the splicing of unc-93(e1500) exon 9, we propose that sfa-1(n4562) is a partial lf allele of sfa-1 and that the suppression of the rubberband Unc phenotype of unc-93(e1500) animals by sfa-1(n4562) is likely caused by reduced sfa-1 function.Because (1) the sfa-1(n4562) affects the alternative splicing of unc-93(e1500) exon 9 caused by the uaf-1(n4588) mutation, we generated uaf-1(n4588); sfa-1(n4562) animals with or without unc-93(e1500). Most uaf-1(n4588); sfa-1(n4562) animals (with or without unc-93(e1500)) died embryonically, and the few that hatched arrested at the L2 larval stage (based on body size) (To examine whether dy size) . We failsup-10 splicing in sfa-1(n4562) animals and failed to detect alternative splicing of the sup-10 transcript  offered a genetic approach to seek new regulators of RNA splicing by screening for suppressors of the ts-lethal phenotype. We performed a genetic screen for suppressors of uaf-1(n4588) ts-lethality at 25°C. From this screen, we isolated four intragenic suppressors, n5120, n5123, n5125 and n5127 (uaf-1(n5123) caused an I180F (ATT-to-TTT) change at the same residue mutated by n4588 (T180I) (ACT-to-ATT) (unc-93(e1500) (uaf-1(n4588 n5120) is predicted to cause a V179M (GTG-to-ATG) change in addition to the n4588 T180I mutation (uaf-1(n4588 n5120) weakly suppressed unc-93(e1500) (uaf-1(n4588 n5125) is predicted to cause a P177L (CCA-to-CTA) change in addition to the n4588 T180I mutation (uaf-1(n4588 n5125) also weakly suppressed unc-93(e1500) (uaf-1(n4588 n5127) is predicted to cause an M157I (ATG-to-ATA) change in addition to the n4588 T180I mutation (unc-93(e1500) (unc-93(e1500) suppressor activities of uaf-1(n4588 n5120), uaf-1(n4588 n5125) and uaf-1(n4588 n5127) were caused by uaf-1 mutations was confirmed by the observation that transgenes expressing uaf-1a (unc-93(e1500) suppressor phenotype of these mutants (unc-93(e1500) exon 9 in animals containing these uaf-1 mutations using Taqman RT–PCR. As shown in uaf-1 mutations exhibited differential effects on the splicing of unc-93(e1500) exon 9. uaf-1(n5123) had no apparent effect  alone). uaf-1(n4588 n5120) and uaf-1(n4588 n5125) weakly  and uaf-1(n4588 n5127) moderately (25%) increased the alternative splicing of unc-93(e1500) exon 9 (uaf-1(n4588) (68%)   and elim3(e1500) . uaf-1 . uaf-1 . uaf-1(nmutation  and was 3(e1500) . That thg uaf-1a  in the b mutants . We quan) exon 9 . None of8) (68%) .uaf-1(n4588) could suppress the Unc phenotype of unc-93(e1500) independently of unc-93 splicing, we generated transgenic animals that overexpressed in the body-wall muscles a full-length unc-93 cDNA containing the e1500 gf mutation. Since this cDNA generates a fully spliced form of unc-93 mRNA, if alternative splicing of unc-93(e1500) accounted for the suppression of the rubberband Unc phenotype, overexpressed unc-93(e1500) cDNA would not generate an alternatively spliced isoform and the animals would be as Unc in an uaf-1(n4588) background as in a wild-type background. As shown in unc-93(e1500) cDNA caused a strong rubberband Unc phenotype. The presence of the uaf-1(n4588) mutation reduced the severity of the rubberband Unc phenotype caused by the same transgenes. Similarly, overexpression of the unc-93(e1500) cDNA also caused a weaker rubberband Unc phenotype in sfa-1(n4562) animals than in wild-type animals (uaf-1(n4588) can suppress the unc-93(e1500) rubberband Unc phenotype through mechanism(s) other than by affecting the splicing of unc-93. This finding was consistent with our results showing that although sfa-1(n4562) suppressed the rubberband Unc phenotype of unc-93(e1500) animals, sfa-1(n4562) did not affect the alternative splicing of unc-93(e1500) exon 9, which also suggested that the suppression of unc-93(e1500) by sfa-1(n4562) was mediated by a mechanism other than by affecting the alternative splicing of unc-93  and the GFP reporter gene  and the exon 9 cryptic 3′ splice site (E9) in wild-type and ter gene  and placunc-93(e1500). In wild-type animals the splicing mimics that of the endogenous unc-93(e1500), with very little splicing at E9 (uaf-1(n4588) mutants occurred almost exclusively at E9 (>99%) (unc-93(e1500) was qualitatively but not quantitatively similar, occurring mostly at E9 (68%)  ; endogenE9 (68%) . These ruaf-1(n4588) animals, splicing occurred mostly at the new E9  (uaf-1(n4588) animals  (uaf-1(n4588) animals when two identical 3′ splice sites are present. We next switched the positions of I8 and E9 (uaf-1(n4588) animals, most splicing (80%) occurred at the new E9 (uaf-1(n4588) animals  . When I8ctively) , No. 3, ctively) . These r8 and E9 , No. 4. 8 and E9 . Similare new E9 , No.4. H animals , No. 4 sunc-93 is expressed in body-wall muscles myo-3 promoter myo-3 promoter and the unc-93 promoter (unc-93(e1500) involves a mechanism that is not promoter-specific.The pattern of alternative splicing in cell culture can depend on the promoter used C. elegans consensus 3′ splice site TTTTcag uaf-1(n4588) animals (uaf-1(n4588) animals is similar to that of transgene No.1  animals, splicing occurred exclusively at the new E9 . We next changed each of the non-T bases to T from −7 to −4 of E9 (uaf-1(n4588) animals occurred exclusively at E9   animals, splicing at the new E9 was reduced  mutation by uaf-1(n4588) and sfa-1(n4562) remains to be determined. Four observations indicate that although uaf-1(n4588) causes alternative splicing of unc-93(e1500) exon 9, this alternative splicing is not the basis of the suppression. First, unc-93(e1500)/unc-93(lf) heterozygous animals are as Unc as unc-93(e1500) homozygous animals unc-93 expression by 50% does not reduce the rubberband Unc phenotype. By contrast, uaf-1(n4588 n5127) reduced unc-93(e1500) expression by 25% , and these animals were strongly suppressed. Also, in uaf-1(n4588) unc-93(e1500) animals, the unc-93(e1500) transcript was reduced by 68% , and these animals might have been expected to be slightly less Unc than unc-93(e1500)/unc-93(lf) animals but instead were strongly suppressed. Thus, the level of reduction of the unc-93(e1500) transcript does not correlate with the level of the suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588 n5127) and uaf-1(n4588). Second, the strong rubberband Unc phenotype caused by overexpression of the unc-93(e1500)-specific cDNA in body-wall muscles was partially suppressed by uaf-1(n4588), suggesting that unc-93(e1500) splicing is not needed for uaf-1(n4588)-mediated suppression. Third, sfa-1(n4562) suppressed unc-93(e1500) without affecting the splicing of unc-93(e1500) exon 9, and sfa-1(n4562) partially suppressed the rubberband Unc phenotype caused by overexpression of the unc-93(e1500) cDNA in the body-wall muscles. Again, suppression can occur without affecting unc-93(e1500) mRNA splicing. Similarly, we did not identify an alternatively spliced sup-10 transcript in either uaf-1(n4588); sup-10(n983) or sfa-1(n4562); sup-10(n983) animals, suggesting that sup-10(n983) was suppressed by uaf-1(n4588) and sfa-1(n4562) by a mechanism other than alternative splicing of the sup-10 transcript. Fourth, reducing the expression of uaf-1 and sfa-1 by RNAi suppressed the rubberband Unc phenotype of unc-93(e1500) but did not cause altered splicing of unc-93(e1500) exon 9. Based on these arguments, we propose that uaf-1 and sfa-1 mutations suppress unc-93(e1500) and sup-10(n983) by affecting the splicing of one or more unidentified genes required for the expression of the unc-93(e1500) and sup-10(n983) rubberband Unc phenotype. We cannot exclude the possibility that the alternative splicing of unc-93(e1500) contributed to the suppression of unc-93(e1500) by uaf-1 mutations.The mechanism(s) of the suppression of the Unc phenotype caused by the unc-93, sup-9 and sup-10 are of three classes. First, lf mutations in any of these three genes are recessive suppressors of the rubberband Unc phenotypes caused by gf mutations in any of these three genes, because the functions of all three genes are necessary for expression of the Unc phenotype sup-11 are strong dominant suppressors of unc-93(e1500) and unc-93(n200) and partial recessive suppressors of sup-9(n1550) and sup-10(n983)sup-11 suppression is unknown. Third, lf mutations of sup-18 are strong recessive suppressors of sup-10(n983) and weak recessive suppressors of unc-93(e1500), unc-93(n200) and sup-9(n1550)sup-18 suppression is also unknown. uaf-1(n4588) and sfa-1(n4562) define a new class of suppressors: they are recessive and allele-specific for unc-93 gf mutations (unc-93(e1500) was suppressed, but unc-93(n200) was not) but not gene-specific (sup-10(n983) was also suppressed). Previous genetic and molecular studies from our laboratory led to the hypothesis that UNC-93, SUP-9 and SUP-10 form a protein complex in the body-wall muscles in vivo functions regulated in different ways. As mentioned above, we propose that mutations in uaf-1 and sfa-1 affect the splicing of one or more unknown genes required for unc-93 and sup-10 activity. This unknown gene might be required specifically for the expression of the rubberband Unc phenotype caused by unc-93(e1500) or sup-10(n983) but have a negligible role in the expression of the rubberband Unc phenotypes caused by unc-93(n200) or sup-9(n1550). Similarly, sup-11 and sup-18 could affect functions of the UNC-93/SUP-9/SUP-10 complex distinct from that affected by uaf-1 and sfa-1.The known suppressors of gf mutations of Saccharomyces cerevisiae and Schizosaccharomyces pombe and the fruit fly Drosophila melanogaster have significantly facilitated the understanding of the in vivo function and regulation of these splicing factors S. cerevisiae identified genetic and biochemical interactions between the U2AF large subunit and SF1/BBP S. pombe provided in vivo evidence that the U2AF subunits are required for splicing Drosophila null mutations of the U2AF large or small subunits cause lethality C. elegans, reducing the expression of uaf-1 or sfa-1 by RNAi causes lethality uaf-1 and sfa-1 and allow the survival of animals in permissive conditions, such as at lower temperatures or when derived from heterozygous mothers. These mutations provide a valuable resource for analyzing the function and regulation of the U2AF large subunit and SF1/BBP genes in vivo in animals.The SF1/BBP and U2AF proteins are critical splicing factors that regulate splicing by binding the branch-point sequence and the 3′ splice sites in vivo. Distinguishing different 3′ splice sites is a critical aspect of alternative splicing.The recognition of 3′ splice sites is achieved by interactions between SF1/BBP and the U2AF large and small subunits, which together bind specific intronic sequences unc-93(e1500) missense mutation generates a new cryptic 3′ splice site (AG) within exon 9 (ACTGcag). This site differs from the consensus 3′ splice site for C. elegans (TTTTcag) C. elegans than is TTTTcag or the intron 8 endogenous 3′ splice site (AATTcag)  intron 8 and the cryptic non-consensus site in unc-93(e1500) exon 9 followed this prediction, as only 1.3% of the splicing events utilized this cryptic 3′ splice site. Strikingly, however, the n4588 missense mutation in uaf-1 shifted this specificity, causing splicing to occur mostly at the cryptic site, generating 68% of aberrantly spliced transcripts. This result suggests that UAF-1 might play an important role in determining the choice among alternative 3′ splice sites in vivo (see discussion below).The AATTcag) . Based on4588 mutation did not cause an apparent change of UAF-1 protein level, and reducing UAF-1 using RNAi did not increase the relative amount of alternatively spliced exon 9, suggesting that uaf-1(n4588) might alter UAF-1 function. However, RNAi-treatment did not abolish the expression of UAF-1 (unc-93(e1500) exon 9 because of residual UAF-1 protein in RNAi-treated animals. Thus, the altered splicing of unc-93(e1500) exon 9 in uaf-1(n4588) unc-93(e1500) mutants might reflect the consequence of the absence of UAF-1 activity. It is also possible that uaf-1(n4588) causes both a loss of function and an altered function of UAF-1, which cause the suppression of the rubberband Unc phenotype of the unc-93(e1500) animals and the altered splicing of unc-93(e1500) transcript, respectively.The of UAF-1 , and we unc-93 transcript appeared to be spliced similarly in wild-type and uaf-1(n4588) animals, suggesting that uaf-1(n4588) did not alter the recognition of most wild-type 3′ splice sites. We also found that uaf-1(n4588) did not suppress the Unc phenotype caused by the unc-52(e669) mutation, which can be suppressed by mutations in the splicing factor genes smu-1 and smu-2. We conclude that the uaf-1(n4588) mutation does not affect all cases in which alternative splicing is possible.The other 14 introns of the uaf-1(n4588). Three of the suppressors  carried both the original n4588 mutation and a second site mutation in uaf-1. These three new uaf-1 mutations reduced the alternative splicing of unc-93(e1500) exon 9 to levels intermediate between those of uaf-1(n4588) and wild-type animals. This finding supports the hypothesis that UAF-1 is important in 3′ splice-site choice. The fourth intragenic suppressor, n5123, affected the same site as the original n4588 mutation by generating a phenylalanine codon different from both the wild-type codon (threonine) and the codon generated by the n4588 mutation (isoleucine). The uaf-1(n5123) allele behaves like the uaf-1(+) allele, suggesting that this mutation restored the normal specificity of UAF-1. The amino acids affected by these uaf-1 mutations  are confined to a region between the U2AF small subunit-interacting domain We isolated four intragenic suppressors of the temperature-sensitive lethality caused by C. elegans, the first two nucleotides (−2 to −1) of 3′ splice sites are more highly conserved than nucleotides −7 to −3 in vivo.In C. elegans introns than there are copies of the strong 3′ splice site sequence TTTTcag (uaf-1(n4588) mutation does not appear to affect the splicing of most other introns of unc-93  was morf unc-93 , which hSecond, we conclude that the nucleotides at −7, −5 and −4 were more important than the nucleotide at −6 for wild-type UAF-1 to recognize the sequence of I8 , which iC. elegans  and E9 (ACTGcag) are both rare 3′ splice sites compared to TTTTcag, which is found in about 26% of the approximate 40000 introns analyzed, and is the most commonly used 3′ splice site in  elegans  [29],  was used to balance the uaf-1(n5222Δ) locus.unc-93(e1500) animals (P0) were mutagenized with EMS (ethyl methanesulfonate) as described 1 progeny from these animals were picked to single wells of 24-well culture plates with OP50 bacteria grown on NGM agar. F2 progeny were observed using a dissecting microscope to identify animals with improved locomotion. From ∼10,000 F1 clones screened, 100 independent suppressed strains were isolated. 97 of the isolates, including two weak recessive sterile suppressors and 95 recessive fertile suppressors, were kept as frozen stocks for possible later study. Three stronger suppressors that caused or were closely linked to mutations that caused sterility (n4562) or ts-lethality (n4588 and n4564) were chosen for further analysis. The analysis of n4564 is ongoing.Synchronized L4 n4588 to the left of dpy-1 on LGIII based on the suppression of unc-93(e1500) using standard methods. As the suppressor activity and ts-lethality were very closely linked, e.g., more than 500 n4588 unc-93(e1500)/+ unc-93(e1500) individuals failed to segregate Sup non-Let progeny, we then followed the phenotype of ts-lethality to further map n4588. We mapped n4588 to the right of nucleotide 186577 on BE0003N10 (cosmid BE0003N10 sequences refer to nucleotides of accession no. AC092690) using 10 Vab recombinants recovered after crossing vab-6(e697) n4588 hemaphrodites with males of the Hawaiian strain CB4856 n4588 dpy-1(e1) hemaphrodites with males from the Hawaiian strain CB4856. We determined the coding sequences of four genes in this interval, uaf-1, rab-18, kbp-4 and par-2, and identified a missense mutation in the third exon of the uaf-1a isoform.We mapped n4562 were very closely linked, e.g., over 500 unc-93(e1500); n4562/+ individuals failed to segregate Sup non-Ste progeny, we followed the sterility phenotype to map n4562 to the right of dpy-4 on LG IV using standard methods. We next mapped n4562 to the right of nucleotide 37163 on Y43D4A (accession no. AL132846) using 234 Dpy recombinants recovered after crossing dpy-4(e1166) n4562 with males of the Hawaiian strain CB4856. The sequences of coding exons of sfa-1 and uaf-2, both located in this region, were determined, and a Cys458Opal (TGT-to-TGA) mutation was identified in sfa-1.As the suppressor activity and sterility of uaf-1(n5222Δ) removes nucleotides 9786 to 11082 of YAC Y92C3B. sfa-1(n5223Δ) removes nucleotides 207818 to 208925 of YAC Y116A8C.Genomic DNA pools from EMS-mutagenized animals were screened for deletions using PCR as described uaf-1(n4588) L1 animals were prepared, and levels of control genes  and tested genes  were quantified from each biological replicate. For the Taqman probe-based assay , the probes , one sample for each assay was quantified. PCR primers and Taqman probes are listed in Total RNA was prepared using Trizol according to the manufacture's instructions (Invitrogen), treated with RNase-Free DNase I (New England Biolabs) and followed by incubation at 75°C for 10 minutes to inactivate DNase I. First-strand cDNA was synthesized with random hexamer primers (New England Biolabs) using the Superscript II or III First-Strand Synthesis Kit (Invitrogen). Quantitative RT–PCR was performed using either a DNA Engine Opticon System (MJ Research) or a Mastercycler realplex system (Eppendorf). For the SYBR green-based assay (DNA Engine Opticon System), each 30 µl PCR reaction contained 1 to 10 ng RT template, 0.5 mM PCR primers and 15 µl 2× SYBR Green PCR Master Mix (Applied Biosystems). Three independent samples of synchronized wild-type (N2) and e probes  were labuaf-1(n4588) animals (P0) at the L4 larval stage grown at 15°C were mutagenized with EMS as described 0 animals were allowed to grow to young adults at 15°C in a mixed population and bleached, and F1 progeny were synchronized at the early L1 stage by starvation in S medium 1 animals were placed on 50 Petri plates  with NGM agar seeded with OP50 and permitted to grow to young adults at 15°C and then moved to 25°C. After six days at 25°C, the animals were grown at 20°C for six days and examined each day for the presence of living F2 animals. About 50,000 F1 progeny from a mixture of more than 10,000 P0 animals were screened. From the screen, we recovered 13 surviving F2 animals from 13 different F1 plates. Six of the 13 suppressors were intragenic suppressors representing four different mutations: one was n5120, one was n5123, three were identical to n5125 and one was n5127. It is possible that the three isolates containing the n5125 mutation were derived from the same P0 animal, because all of the P0 animals were in a mixed population when bleached to release eggs. The other seven suppressors were extragenic mutations, i.e., n4588/+; sup/+ animals segregated n4588-like progeny. Some or all of the extragenic isolates could have been derived from the same P0 animal.Synchronized unc-93(e1500)) were fed HT115(DE3) bacteria containing plasmids directing the expression of dsRNAs targeting either uaf-1 or sfa-1 on NGM plates with 1 mM IPTG and 0.1 mg/ml Ampicillin 1 progeny of the unc-93(e1500) animals (escapers) were examined for suppression of locomotion defects. Animals were washed from plates, rinsed three times with H2O, and resuspended in Trizol (Invitrogen) for preparation of total RNA or in 2× protein loading buffer  for SDS-PAGE analysis. We generated the DNA construct expressing dsRNA targeting uaf-1 (see below). The bacterial strain expressing dsRNA targeting sfa-1 was obtained from a whole-genome RNAi library sfa-1 coding sequences.Young adult animals , 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol), boiled for 5 minutes, and samples were then loaded onto 8% polyacrylamide gels containing SDS. Protein samples were transferred from polyacrylamide gels to Immobilon-P Transfer Membranes (Millipore). Primary and secondary antibody incubations were performed with 5% non-fat milk in TBST  at room temperature for one hour each. Signals were visualized using Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) and X-ray film . Primary antibody was rabbit anti-UAF-1(1∶20000) Animals were washed from plates with Hunc-93(e1500) by uaf-1(n4588) or sfa-1(n4562), uaf-1 and sfa-1 cDNAs were subcloned to vector pPD93.97 using BamHI and AgeI restrictions sites. uaf-1b cDNA was amplified with PCR using uaf-1a cDNA as template and subcloned to pPD93.97 using BamHI and AgeI restrictions sites. uaf-1a cDNA was subcloned to pPD49.83 (for heat-shock induced expression of uaf-1a cDNA) using BamHI and SacI restriction sites. An XhoI/SpeI fragment of uaf-1a cDNA subcloned in a pGEM-TA easy vector (Promega) was subcloned to pPD129.36 (for the uaf-1 RNAi construct) using XhoI and NheI sites. To test whether the truncated unc-93(Δ) cDNA caused by altered splicing of the unc-93(e1500) transcript in uaf-1(n4588) mutants encodes a functional UNC-93 protein, unc-93 cDNA and unc-93(Δ) cDNA were subcloned to pPD93.97 using BamHI(blunt) and AgeI sites. To test whether uaf-1(n4588) or sfa-1(n4562) mutations could suppress the Unc phenotype caused by ectopic expression of the unc-93(e1500) cDNA, unc-93(e1500) cDNA was subcloned to pPD93.97 using BamHI(blunt) and AgeI sites. To examine the effect of nucleotide substitutions on the recognition of the intron 8 endogenous 3′ splice site and the exon 9 cryptic 3′ splice site, we fused the genomic sequence between exon 8 and exon 10 of unc-93(e1500) in-frame with the GFP gene of pPD93.97 using BamHI and AgeI sites. We replaced the myo-3 promoter of pPD93.97 with a 2 kb promoter of unc-93 using PmlI and BamHI sites. Point mutations in uaf-1(stop codons), the unc-93 (e1500 mutation) or mutated transgenes were introduced using QuickChange II or III Site-Directed Mutagenesis Kit (Stratagene) with primers containing corresponding mutations. PCR was performed using Eppendorf Cyclers, and DNA products were resolved using agarose gels. DNA sequence determination was performed with an ABI Prism 3100 Genetic Analyzer. PCR primers are listed in To rescue the suppression of the Unc phenotype of Arabidopsis genomic DNA and 10 µg/ml of the transgene of interest. When the transgene did not cause the expression of a GFP fusion protein, 10 µg/ml pPD95.86-GFP plasmid  or 5 µg/ml pmyo-2dsRED (expressing RFP in pharynx) was added to the injection mixture as a visible fluorescence marker to identify animals carrying the transgene.Germline transgene experiments were performed as described We downloaded approximate 40,000 unique intronic sequences from WormMart (WormBase Release 195) and processed the sequences using BBEdit and MS Excel softwares. Identical 3′ splice sites (positions −7 to −1) were grouped and counted.Figure S1uaf-1(n4588) does not reduce the mRNA levels of genes that genetically interact with unc-93. Real-time RT-PCR analyses of mRNA levels of constitutively expressed genes , a body wall muscle specific gene (myo-3) and genes involved in the rubberband Unc phenotype . The levels of endogenous rpl-26 mRNA of each sample were quantified in parallel real-time RT-PCR experiments and used as loading controls. Each data set represents the average of duplicate experiments of three biological replicates of synchronized L1 animals. Error bars, standard errors.(0.32 MB TIF)Click here for additional data file.Figure S2uaf-1 RNAi reduces UAF-1 protein level. (A) Animals were fed bacteria expressing control empty vector or dsRNA targeting uaf-1. A western blot was prepared using a UAF-1 polyclonal antibody uaf-1(RNAi) dramatically reduced the protein level of UAF-1. (B) Coomassie blue staining indicated that the loading of total protein was similar among the samples. (C) Quantification using NIH ImageJ software of UAF-1 levels in wild-type or unc-93(e1500) animals treated with either control empty vector or uaf-1(RNAi). uaf-1(RNAi) treatment reduced UAF-1 levels in both wild-type and unc-93(e1500) animals .(0.30 MB TIF)Click here for additional data file.Figure S3uaf-1(n4588) and sfa-1(n4562) do not cause obvious alternative splicing of the sup-10 transcript. (A) Total RNAs from animals of the indicated genotypes were prepared and RT-PCR experiments were performed to amplify the full-length sup-10 cDNA using PCR primers covering the 5′ start codon and the 3′ stop codon. No sup-10 transcript with an abnormal size was detected for wild-type, sup-10(n983), uaf-1(n4588) and uaf-1(n4588); sup-10(n983) animals. DNA sequences of the sup-10 RT-PCR bands from all the four genotypes were determined. No sup-10 transcript with altered splicing was observed. (B) No alternatively spliced sup-10 transcript was identified from wild-type, sup-10(n983), sfa-1(n4562) and sfa-1(n4562); sup-10(n983) animals using RT-PCR experiments and DNA sequence determination. Genotypes are indicated at the top.(0.37 MB TIF)Click here for additional data file.Figure S4uaf-1(n4588) and sfa-1(n4562) cause synthetic lethality. Anesthesized animals of the indicated genotypes were observed with Nomarski optics. For wild-type, uaf-1(n4588) and sfa-1(n4562), animals were photographed 24 hours after the mid-L4 stage . For uaf-1(n4588); sfa-1(n4562) double mutants, animals were observed each day for up to ten days after hatching. No animals grew beyond the size of a wild-type animal of the L2 larval stage. Two escapers of the uaf-1(n4588); sfa-1(n4562) genotype that survived embryonic lethality are shown. Scale bar: 200 µm.  sqv mutants of Caenorhabditis elegans are defective in vulval epithelial invagination. Proc Natl Acad Sci U S A 96: 968–973.)(0.79 MB TIF)Click here for additional data file.Table S1uaf-1(n4588) does not cause synthetic defects with loss-of-function mutations of unc-93, sup-9 and sup-10 and does not suppress other Unc mutants. To examine whether uaf-1 functions redundantly with unc-93, sup-9 or sup-10, we compared double mutants containing uaf-1(n4588) and lf mutations of unc-93, sup-9 or sup-10 to uaf-1(n4588) single mutants for locomotion, growth, and gross morphology. No differences were observed. Double mutants with uaf-1(n4588) and gf mutations of unc-58, egl-23 and twk-18 were compared to single mutants carrying these gf mutations, and no visible suppression of the Unc phenotypes was observed. Double mutants containing uaf-1(n4588) and unc-52(e444) or unc-52(e669) mutations were compared to single mutants of either unc-52(e444) or unc-52(e669) mutations, and no visible suppression of the Unc phenotypes was observed. Pvl: protruding vulva. Ste: sterile. Dpy: dumpy. Egl: egg-laying defective.(0.04 MB DOC)Click here for additional data file.Table S2unc-93 transcript does not encode a functional UNC-93 protein product. Transgenes driving the expression of the alternatively spliced unc-93 cDNA (unc-93 cDNA(Δ)) did not rescue the suppression of sup-9(n1550) by the unc-93(lr12Δ) mutation, while transgenes expressing a wild-type unc-93 cDNA rescued the suppression.The alternatively spliced (0.02 MB DOC)Click here for additional data file.Table S3unc-93 transcript likely does not encode a dominant-negative UNC-93 protein product. Tansgenes driving the expression of the unc-93 cDNA(Δ) did not suppress the rubberband Unc phenotype of unc-93(e1500) animals, while transgenes expressing a wild type unc-93 cDNA suppressed the Unc phenotype. As loss of function of unc-93 results in phenotypically wild-type animals, the lack of suppression of unc-93(e1500) by the unc-93(Δ) transgenes suggests that the function of unc-93 was not antagonized by unc-93(Δ), indicating that the unc-93(Δ) cDNA does not encode a dominant-negative UNC-93 protein.The alternatively spliced (0.02 MB DOC)Click here for additional data file.Table S4Sequences and distributions of the three 3′ splice sites we analyzed. Approximate 40,000 unique introns were analyzed, and the numbers and ratios of all types of 3′ splice sites were calculated. The list here includes the three sites we analyzed in our mutagenesis experiments shown in (0.03 MB DOC)Click here for additional data file.Table S5List of PCR primers and Taqman probes.(0.10 MB DOC)Click here for additional data file.


Caenorhabditis elegans genome exists, however the application of these valuable biological tools has been limited due to the incomplete correlation between their genetic and physical characterization.A collection of genetic deficiencies covering over 70% of the We have applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) to the high resolution, molecular characterization of several genetic deficiency and duplication strains in a 5 Mb region of Chromosome III. We incorporate this data into a physical deficiency map which is subsequently used to direct the positional cloning of essential genes within the region. From this analysis we are able to quickly determine the molecular identity of several previously unidentified mutations.Caenorhabditis elegans. Consequently we have generated a valuable physical mapping resource, which we have demonstrated can aid in the rapid molecular identification of mutations of interest.We have applied accurate, high resolution molecular analysis to the characterization of genetic mapping tools in  Caenorhabditis elegans genome has been generated by various research groups over the past three decades ), BC4638 (sDf127(s2428) [sDp3]) and BC4690 (sDf128(s2786) [sDp3])  known to cover the region under investigation in this study [To implement this strategy all of the is study . For thiis study .sDp3 from the background the ability to accurately resolve breakpoint positions in the ambiguously mapped deficiency strains improved significantly. A comparison of oaCGH data for sDf127 balanced by sDp3 and this same deficiency out-crossed to hT2 (qIs48) highlights this improvement  dpy-5(e61) I/hT2 [bli-4(e937) let-?(q782) qIs48] , BC4697 – sDf121(s2098) unc-32(e189) III; sDp3 , BC7162 – dpy-17(e164) sDf125(s2424) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC4638 – dpy-17(e164) sDf127(s2428) unc-32(e189) III; sDp3 , BC7163 – dpy-17(e164) sDf127(s2428) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC4690 – dpy-17(e164) sDf128(s2786) unc-32(e189) III; sDp3 , BC7164 – dpy-17(e164) sDf128(s2786) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC7244 – sDf130(s2427) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC7181 – dpy-17(e164) sDf135(s2767) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC3737 – sDp8; eT1, BC4158 sDp3; dpy-17(e164) let-722(s2448) unc-32(e189) III, BC4160 – sDp3; dpy-17(e164) let-722(s2450) unc-32(e189) III, BC4228 – sDp3; dpy-17(e164) let-722(s2588) unc-32(e189), BC4262 – sDp3; dpy-17(e164) let-754(s2622) unc-32(e189) III, BC4173 – dpy-17(e164) let-766(s2463) unc-32(e189) III; sDp3, BC4167 – sDp3; dpy-17(e164) let-716(s2457) unc-32(e189) III, BC4266 – sDp3; dpy-17(e164) let-716(s2626) unc-32(e189) III, BC4192 – sDp3; dpy-17(e164) let-768(s2482) unc-32(e189) III, BC4232 – sDp3; dpy-17(e164) let-768(s2592) unc-32(e189) III, BC4268 – sDp3; dpy-17(e164) let-768(s2628) unc-32(e189) III, BC2842 – dpy-18(e364)/eT1 III; unc-46(e177) let-444(s1418)/eT1 [let-500(s2165)]V, MT3022 – nDf20/sma-2(e502) unc-32(e189)III.Nematodes were grown as previously described  on 150 msDp3 balanced strains into 5 ul of lysis buffer  and freeze cracked in Liquid N2 followed by digestion (1 hour at 60°C followed by 15 min at 95°C). 1 ul of this DNA preparation was subsequently used in a 25 ul PCR reaction.DNA preparation for oaCGH samples was done as previously described . For mutsDp3 balanced strains a C. elegans whole genome array was used, based on Wormbase release WS120, and available from NimbleGen Systems Inc. [hT2 (qIs48) balanced strains was performed with a whole genome C. elegans array designed with overlapping 50-mer probes targeting primarily annotated exons and micro-RNAs [For the analysis of cro-RNAs . oaCGH scro-RNAs . Copy nucro-RNAs  and are PCR amplification across the region of the breakpoint was performed with nested primers (Primer sequences and amplification conditions available upon request) and sequenced using standard molecular methods.MRJ carried out the experiments, data analysis and drafted the manuscript. JSM assisted in sample preparation. SF designed the oaCGH array. DGM helped to draft the manuscript. DLB conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Identified mutants of essential genes and their associated RNAi phenotype.Click here for file


Hox genes play a central role in axial patterning during animal development. They are clustered in the genome and specify cell fate in sequential domains along the anteroposterior (A-P) body axis in a conserved order that is co-linear with their relative genomic position. In the soil worm Caenorhabditis elegans, this striking rule of co-linearity is broken by the anterior Hox gene ceh-13, which is located between the two middle Hox paralogs, lin-39 and mab-5, within the loosely organized nematode Hox cluster. Despite its evolutionary and developmental significance, the functional consequence of this unusual genomic organization remains unresolved.ceh-13 in different developmental processes, and found that its expression and function are not restricted to the anterior body part. We show that ceh-13 affects cell migration and fusion as well as tissue patterning in the middle and posterior body regions too. These data reveal novel roles for ceh-13 in developmental processes known to be under the control of middle Hox paralogs. Consistently, enhanced activity of lin-39 and mab-5 can suppress developmental arrest and morphologic malformation in ceh-13 deficient animals.In this study we have investigated the role of Hox genes in C. elegans which display region-specific accumulation and function along the A-P axis, the expression and functional domain of the anterior Hox paralog ceh-13 extends beyond the anterior region of the worm. Furthermore, ceh-13 and the middle Hox paralogs share several developmental functions. Together, these results suggest the emergence of the middle-group Hox genes from a ceh-13-like primordial Hox ancestor.Our findings presented here show that, unlike other  Hox genes lin-lin-Hox gd manner ,12. The l Figure  11,12].,12.Hox g mutants . This suceh-13 lf mutant background. To visualize these cells, we used an integrated green fluorescent protein- (GFP) labeled mec-7 reporter  called vulval precursor cells (VPCs), which lie ventrally along the anterior-posterior axis  and some of the most anterior and posterior Pn.p cells remain unfused VzhIs1 IV; unc-119(e2498)IIIswIs1 IImuIs16 ?; dpy-20(e1282)IVbxls12 [egl-5::gfp]jcIs1 IV; unc-29(e193)ImuIs62 ?mig-13(mu225)X; lin-15(n765ts)X; muIs62 ceh-13(sw1)IIIceh-13(ok737)IIImab-5(e1751gf)IIImab-5(e1751gf)III; ceh-13(sw1)IIImab-5(e1751gf)III; muIs35 Vmab-5(e1751gf)III; ceh-13(sw1)III; muIs35 Vmig-13(mu294)X; muIs35 Vceh-13(sw1)III; mig-13(mu294)X; muIs35 Vceh-13(sw1)III; muIs62 ceh-13(sw1)III; muIs35 Vceh-13(ok737)III; muIs35 Vlet-60 (n1064gf)IVlin-8(n111)II; lin-15B(n765ts)Xlin-53(n833)I; lin-15B(n765ts)Xceh-13(sw1)III; let-60 (n1064gf)IVceh-13(sw1)III; lin-8(n111)II; lin-15B(n765ts)Xceh-13(ok737)III; lin-8(n111)II; lin-15B(n765ts)Xceh-13(sw1)III; lin-53(n833)I; lin-15B(n765ts)Xok737 allele is a deletional derivative of ceh-13. It removes the end of the first exon and more than half of the first intron, resulting in a frame shift and the generation of a premature stop codon 26 base pairs after the downstream deletional breakpoint. Based on phenotypic characterization (see below), ok737 is likely to represent another null allele of ceh-13. Homozygous ceh-13(ok737) mutant animals exhibit a variable abnormal morphology phenotype accompanied with a highly penetrant embryonic or larval lethality: 96% (621/647) of ok737 mutants die at various stages of embryogenesis or at early larval stages. The arrested embryos and larvae display serious body malformations, especially at the anterior and central body parts. The ceh-13(ok737) escapers, which are able to develop into fertile adults, show less severe morphological defects than the arrested mutants, and have a decreased brood size (data not shown), a short body length and delayed developmental rate, as compared with the wild type. Homozygous ceh-13(ok737) mutants were isolated from the VC509 balanced strain of genotype ceh-13(ok737) III/hT2[bli-4(e937) let-?(q782) qIs48] . For sequencing the mutant allele, genomic PCR was performed with the following forward and reverse primers: 5'-tga gct cca ctg aat gtt atg g-3' and 5'-tat gac gaa ccg gtc ttt cc-3'.mec-7::gfp reporter .For clustering the ceh-13 and synMuv mutant animals. EA performed the phylogenetic analysis of the nematode Hox genes. FM designed experiments, wrote the manuscript. KTV supervised the work, designed experiments, performed cloning experiments, wrote the manuscript. All authors read and approved the final manuscript.BT made the majority of the experiments, wrote the manuscript, performed statistical analysis. T. V designed experiments, performed the expression analysis of the Hox genes, wrote the manuscript. Á R performed vulval analysis in


The association of the human herpesvirus-8/Kaposi's sarcoma (KS)-associated herpesvirus (HHV-8/KSHV) serology with various malignancies in Tanzania is not currently well established while previous studies were based on either PCR or immunofluorescence assays [IFA] but not with a sensitive enzyme-linked immunosorbent assay (ELISA). Selected archival diagnostic biopsies (n = 184) and sera from indigenous patients with KS (n = 120), non-KS tumors (n = 24) and non-neoplastic lesions (n = 40) at Muhimbili National Hospital (MNH), Tanzania, were evaluated by diagnostic histopathology, immunohistology [anti-HHV-8 latency-associated nuclear antigen (LANA)] and serology for HIV (ELISA) and HHV-8 (IFA and ELISA).About 66.3% (n = 122) cases including AIDS-associated Kaposi's sarcoma (AKS) (n = 93), reactive conditions (n = 28) and only one non-KS tumour were HIV positive. Endemic KS (EKS) patients were mostly males  who were less  predominant in AIDS-associated (AKS). A high (89%) percentage of patients with anti-HHV-8 antibodies was found in the cohort including the HIV positive (92%) cases, males (81.2%), KS patients (93%), non-KS tumors (92%), and reactive conditions (75%). All HHV-8 seronegative KS cases were nodular stage whereas both sera and corresponding biopsies from early stage KS were HHV-8+. Assay sensitivity, positive predictive value (PPV) and specificity were 98.6%, 93.5% and 16.7% for IFA and 93.5%, 98.6% and 50.0% for ELISA respectively.HHV-8 seroprevalence at MNH appears high as expected among AKS cases and males but also in non-KS patients. ELISA showed a combination of high HHV-8 sensitivity as well as higher PPV and specificity than IFA which however, showed higher sensitivity. The apparent stage-dependent, inverted serum HHV-8 immunoreactivity supports a notion of viral immune-segregation during KS development. Routine HHV-8 screening should be considered particularly in patients at risk of KS and for selection of blood/organ donations. The HIV and AIDS epidemic has dramatically increased the frequency of different malignancies particularly Kaposi's sarcoma (KS) and certain malignant lymphoma (ML) which are associated with the novel human herpesvirus type 8 (HHV-8)/Kaposi's sarcoma-associated herpes virus (KSHV) and have become a major health concern in sub-Saharan Africa including Tanzania -3. The pA total of 184 selected biopsies and corresponding sera available at MNH/MUHAS between 1990 and 2001 from indigenous African patients were included in the study. Of these patients, 120(65%) had KS, 24(13%) non-KS tumors and 40(22%) non-malignant (reactive) conditions. The male:female ratio was 2.75:1 for KS, 3:1 for non-KS tumors and almost 1:1 for reactive, non-malignant conditions Figure  and  were nodular stage Figure  with cut2 = 7.96, p = 0.0048)  and at the Immunopathology Lab . Furthermore, available biopsies and corresponding sera from non-KS tumors and reactive conditions from the same period were also evaluated. The biopsies of reactive conditions included all histologically non-malignant/inflammatory cases whose sera were available for analysis. None of the patients had received any antiretroviral (ARV) or anti-tumour therapy before the biopsy was taken.Routine hematoxylin and eosin (H & E) staining was done as previously described ,26 and mAvailable sera from patients with histologically diagnosed KS, non-KS malignancies as well as reactive conditions were comparatively evaluated for HIV and HHV-8 serology. Serology for HIV-1 evaluation (ELISA) was performed at Microbiology/Immunology MUHAS as previously described ,28.HHV-8 serology by IFA was performed [Swedish Institute for Infectious Diseases Control (SMI)] on cytospins of BCBL-1* cells  using pa2. To induce lytic gene transcription, cells were cultured with 20 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA)  as previously described, [® slides . The slides were fixed for 10 minutes in 4% paraformaldehyde (PFA) and washed before immunostaining as previously described [The HHV-8 infected body cavity-based lymphoma (BCBL-1) cells (kindly provided by G. Gaidano) were derived from a primary effusion lymphoma (PEL) ,31, and scribed, ,28,32 anescribed ,8.For HHV-8 antibody evaluation an IgG enzyme immunoassay (EIA) Kit  Biotrin International  was used. The Biotrin HHV-8 ELISA is a direct EIA based on the binding of HHV-8-specific antibodies to lytic peptide antigens coupled to microtitre test strips. Bound antibodies are detected by an anti-human IgG peroxidase conjugate and a 3,3',5,5'-tetramethylbenzidine (TMB) dark blue substrate reaction . The use of lytic peptide epitopes derived from various HHV-8 viral proteins ensures both a high sensitivity and specificity. The Kit has no detectable cross-reactivity with HIV/EBV antibodies. Control sera included those from known HHV-8+ KS patients while the HHV-8 infected BCBL-1 cells provided an internal positive control for IFA studies. The negative controls for both IFA and ELISA included sera from known HIV and HHV-8 negative non-KS patients and a buffer (PBS).Immunostaining with the avidin-biotin complex (ABC) immunoperoxidase technique was used on KS sections (5 μm) for detection of HHV-8 latency-associated nuclear antigen (LANA) to compliment histopathology and serology, as previously described ,26,33. NFor microscopy and microphotography an Olympus BX60, microscope with a digital camera (Sony DKC-5000) was used as previously described ,19,33.The EPI INFO 6 statistical software programme  was used. P-values of ≤0.05 were considered statistically significant. The Fisher exact test was used to test the significance level where the sample size was small.The authors declare that they have no competing interests.


Signaling downstream of Ras is mediated by three major pathways, Raf/ERK, phosphatidylinositol 3 kinase (PI3K), and Ral guanine nucleotide exchange factor . Ras signal transduction pathways play an important role in breast cancer progression, as evidenced by the frequent over-expression of the Ras-activating epidermal growth factor receptors EGFR and ErbB2. Here we investigated which signal transduction pathways downstream of Ras contribute to EGFR-dependent transformation of telomerase-immortalized mammary epithelial cells HME16C. Furthermore, we examined whether a highly transcriptionally regulated ERK pathway target, PHLDA1 (TDAG51), suggested to be a tumor suppressor in breast cancer and melanoma, might modulate the transformation process.Cellular transformation of human mammary epithelial cells by downstream Ras signal transduction pathways was examined using anchorage-independent growth assays in the presence and absence of EGFR inhibition. TDAG51 protein expression was down-regulated by interfering small hairpin RNA (shRNA), and the effects on cell proliferation and death were examined in Ras pathway-transformed breast epithelial cells.Activation of both the ERK and PI3K signaling pathways was sufficient to induce cellular transformation, which was accompanied by up-regulation of EGFR ligands, suggesting autocrine EGFR stimulation during the transformation process. Only activation of the ERK pathway was sufficient to transform cells in the presence of EGFR inhibition and was sufficient for tumorigenesis in xenografts. Up-regulation of the PHLDA1 gene product, TDAG51, was found to correlate with persistent ERK activation and anchorage-independent growth in the absence or presence of EGFR inhibition. Knockdown of this putative breast cancer tumor-suppressor gene resulted in increased ERK pathway activation and enhanced matrix-detached cellular proliferation of Ras/Raf transformed cells.Our results suggest that multiple Ras signal transduction pathways contribute to mammary epithelial cell transformation, but that the ERK signaling pathway may be a crucial component downstream of EGFR activation during tumorigenesis. Furthermore, persistent activation of ERK signaling up-regulates TDAG51. This event serves as a negative regulator of both Erk activation as well as matrix-detached cellular proliferation and suggests that TDAG51 opposes ERK-mediated transformation in breast epithelial cells. The development and progression of breast cancer is the result of multiple genetic changes, which lead to complex alterations in signal transduction networks in breast cancer cells relative to their normal epithelial counterparts. Signaling differences between tumor and normal cells are reflected in altered gene expression patterns, a finding that has been widely investigated using various molecular techniques. Patterns of differential gene expression have been used for classification and prognostication of certain cancers and may be valuable for prospectively predicting responsiveness to therapeutic treatments . In addiRas activation is a common intermediary in signaling pathways initiated by a variety of cell surface receptors, and signaling pathways downstream of Ras have been implicated repeatedly in oncogenesis. Ras proteins are frequently mutated to an activated form in human cancers, particularly in tumors of the pancreas, colon, thyroid, and lung . Ras mutThe Ras oncogene binds numerous effectors that in turn activate a variety of signaling pathways. The most highly characterized of these are the Raf proteins, PI3K, and the Ral guanidine nucleotide exchange factors , although Ras does recruit and activate other potentially transforming effectors . The disin vitro transformation. Studies using immortalized mouse EpH4 mammary epithelial cells have implicated Raf as well as PI3K pathways in supporting transformation and tumorigenesis [human immortalized mammary epithelial cells, Raf and PI3K clearly contribute to transformation, although each is usually not sufficient for tumor formation in animal models [In mammary epithelial cells, both species- as well as cell line-dependent differences in Ras downstream signaling pathways have been found to induce tumorigenesis and/or anchorage-independent growth, a measure of igenesis . For huml models ,15. In fl models .Dissecting the physiological consequences of individual Ras-mediated signaling pathways with respect to mammary epithelial transformation is of obvious interest. The ability of activated Ras and Raf to induce autocrine expression of epidermal-like growth factors has been implicated in the protection of MCF10A mammary epithelial cells from anoikis . Using HWe performed a functional analysis of one gene in particular, TDAG51, whose expression is regulated by ERK through EGFR dependent and independent mechanisms. The loss of TDAG51 mRNA and protein has been correlated with breast adenocarcinoma and melanoma progression in clinical samples ,18. The 2. For induction of proteins from the tetracycline-inducible retroviral expression vector pLRT, 250 ng/mL of doxycycline (Sigma-Aldrich) was added to culture media.HME16C human mammary cells  were culV12), H-RasG12V, E37G (RasV12G37), H-RasG12V, T35S (RasV12S35), H-RasG12V, Y40C (RasV12C40), and Rlf-CAAX [Constructs for the inducible expression of H-Ras, H-Ras effector domain mutants, and Rlf-CAAX were created by PCR subcloning the sequences of HA-tagged H-RasG12V (RasRlf-CAAX  into theRlf-CAAX . The genRlf-CAAX . PolycloRlf-CAAX ). After Western blots were performed using Chemilluminescence (Pierce). Antibodies used for western blots included anti-Ras mAb , rat anti-HA mAb , anti-phospho Erk mAb , goat anti- Erk2 , rabbit anti-phospho-Akt Ser473 , goat anti-Akt , or an anti-alpha tubulin mAb , followed by detection using appropriate anti-mouse, rabbit, or goat HRP-conjugated secondary antibodies (Jackson Labs) and ECL detection. For Ral activation assays, the Ral Activation Assay Kit  was used according to the manufacturer's protocol.4 cells were plated per well in 0.36% bacto-agar (BD Biosciences), w/v in growth media, on a 0.6% bacto-agar support in 6-well culture plates. Prior to cell plating, 250 ng/mL doxycycline, DMSO vehicle, or the anti-EGFR compound PD153035 , was added to the agar, as appropriate. Appropriately-supplememented fresh media was added to wells every 4 days. Colonies greater than 100 μm in diameter were counted after 18 days using a 2× lens equipped with a graded grid to determine colony size.Cells were trypsinized, neutralized, and 1.2 × 10Cells were trypsinized and plated in 500 μL appropriate growth media into 24-well ultra-low attachment plates (Corning) at 25,000 cells per well. At the times indicated, 50 μL of alamar blue  was added to each well and incubated for 6 hours under standard culture conditions. The amount of alamar blue reduction was quantified by measuring the fluorescence of each sample at 530 nm excitation/590 nm emission, corrected for the background fluorescence in wells containing media but no cells. The number of cells for initial plating and the alamar blue incubation times were determined by generating a standard curve of fluorescence versus cell number incubated with alamar blue for various times. Plating cells initially at 25,000/well insures that fluorescence measurements will occur on a linear part of the curve with a 6-hour incubation time over the course of an experiment. It was determined that TDAG51-specific shRNA did not affect the redox potential of HME16C at various cell numbers. Statistical analysis was performed using 2-way analysis of variance (ANOVA) with Bonferroni post-tests using the GraphPad Prism software program .Tumorigenicity assays were done essentially as described . To induTotal RNA was extracted using Trizol reagent (Invitrogen) after treatment of cells with 250 ng/mL doxycycline for 72 hours to induce gene expression, or with doxycycline and 0.25 μM EGFR receptor kinase inhibitor PD153035 as indicated. Twenty micrograms of RNA were used for cDNA generation, and cDNA labeled with Cy3 or Cy5 monofunctional reactive dye to amino allyl-modified dUTP incorporated into cDNA using the FairPlay Microarray labeling kit (Stratagene). Labeled cDNA was hybridized to long-oligo (70-mer) cDNA microarrays from the NCI/CCR Microarray Center, NCI, Frederick, MD, according to standard protocols .® SYBR® Green qPCR Supermix UDG real-time RT-PCR kit (Invitrogen) and a Rotor-Gene3000™ thermocycler with Rotor-Gene 5.0.37 software (Corbett Research) that calculates relative PCR synthesis rates by comparative quantification. The specificity of product synthesis was verified by melting curve analysis by the Rotor-Gene 5.0.37 software, and by running of real-time PCR products on 2% agarose gels to verify product size and rule out primer-dimer contribution to calculated values. The sequence-specific primers used in real-time RT-PCR can be found in Additional file cDNA was synthesized from RNA obtained for microarray analysis using the SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen). Quantification of relative cDNA levels for each gene was accomplished using the PlatinumCells grown under anchorage-independent conditions were spun down, washed once in sterile PBS, and suspended in a 50% mixture of PBS and ACCUMAX cell detachment solution  for 10 minutes at 25°C to dissociate cell clumps. After dissociation, cells were washed once in cold PBS and then fixed with 70% ethanol. Fixed cells were treated with RNAse A for 20 minutes at 37°C, and nuclear DNA was stained with 50 μg/mL propidium iodide at 4°C. Cell cycle profiles were generated using a FACSCalibur flow cytometer (BD Biosciences) and modeling cell cycle phases with the cell cycle option of FlowJo flow cytometry software  after gating for viable, single cells. Statistical analysis was performed using 2-way ANOVA with Bonferroni post tests using the GraphPad Prism software program .6 cells per well in 6-well ultra-low attachment plates or 5 × 105 cells per dish in 60 mm tissue culture dishes for attached control cells. Attached cells were treated with either DMSO or 10 μM EdU for 4 hours, and cells grown in low attachment plates were treated with DMSO or 10 μM EdU for 24 hours. Using FlowJo flow cytometry software , DMSO-treated control cells were used to determine the threshold above which cells could be considered EdU positive, and the fraction of cells incorporating EdU was then determined for experimental samples.Measurement of cell proliferation by 5-ethynyl-2'-deoxyuridine (EdU) incorporation was measured using the Click-iT EdU cell proliferation Assay Kit for Flow Cytometry (Invitrogen) according to the manufacturer's protocol. Briefly, cells were plated at 1 × 10The release of lactose dehydrogenase (LDH) into cell culture supernatant was measured using the cytotoxicity detection kit LDH  according to the manufacturer's instructions.V12, the H-RasV12 effector domain mutants (EDM) H-RasV12G37, H-RasV12S35, and H-RasV12C40, and the constitutively activated version of a RalGEF, Rlf-CAAX, were used to infect telomerase immortalized HME16C human mammary epithelial cells. Anti-Ras and anti-HA western blotting demonstrated approximately equal levels of ectopic Ras expression among Ras-infected cells, with slightly lower levels in HME16C RasV12-infected cells relative to EDM-infected cells  western blotting was performed to detect activated, phosphorylated Akt. In cells that were serum starved for 24 hours and matrix detached for 6 hours, elevated levels of phosphorylated Akt were observed in RasV12-, RasV12C40-, and RasV12G37-infected HME16C, with highest levels present in RasV12-infected cells , identical to previously reported results [To compare our results to others, we verified the function of pLRT vector driven Ras EDM mutants and Rlf-CAAX in HEK-HT cells, which previously have been reported to form colonies in soft agar upon expression of H-RasRlf-CAAX . In our  results . Express results .V12-infected HME16C cells formed rapidly growing, fluid-filled tumors with an average latency of 4 weeks and a mean tumor volume of 808.8 mm3 at 6 weeks . Empty vector-, RasV12G37-, RasV12C40-, and Rlf-CAAX-infected cells failed to form palpable tumors four months after injection. The metastatic potential of RasV12- and RasV12G37-expressing cell lines was tested by tail-vein injection in nude mice, but no metastatic lesions were observed by histological analysis in lungs, liver, spleen, or kidneys at 16 weeks post injection (not shown).Anchorage-independent growth often predicts the ability of cells to grow as xenografted tumors in immuno-compromised mice. Tumor formation was assessed following subcutaneous inoculation. The RasV12- and Ras EDM-infected cells . Therefore, we sought to determine if autocrine signaling by the EGFR was required for the anchorage-independent growth of Ras- or Ras EDM-infected cells. RasV12- and Ras EDM-infected HME16C cells were grown in soft agar in the presence or absence of the EGFR-specific inhibitors, PD153035 and PD168393. Complete inhibition of colony formation for RasV12G37-infected and RasV12C40-infected cells was observed at 0.25 μM PD153035, a concentration that specifically inhibits EGFR [V12- and RasV12S35-infected cells formed colonies efficiently at this same concentration of inhibitor  data analysis was performed for the datasets, and a delta value of 0.4 was chosen for each dataset, which maintains the estimated false discovery rate (FDR) below 1% for each. A summary of the genes up- or down-regulated greater than 2-fold in the H-RasV12- and Ras effector domain mutant-infected HME16C cell lines is presented in Additional file Activation of the Ras oncogene is accompanied by the stimulation of multiple signal transduction pathways leading to the activation or repression of numerous transcription factors as well as changes in mRNA translation and stability, and thus, the modulation of gene expression. To determine which gene expression changes accompany the transformation of HME16C human epithelial cells by activated Ras, we examined our transformed HME16C cells by cDNA microarray analysis. To do this, RNA was isolated from H-RasV12-, RasV12S35-, and RasV12G37-infected cells were induced with doxycycline and subsequently incubated either in the presence or absence of 0.25 μM PD153035, and microarray analysis comparisons were made to vehicle-treated pLRT-infected cells. Almost all Ras and Ras EDM-induced upregulated transcriptional responses were blocked by pharmacological inhibition of EGFR, consistent with previous reports for inhibition of Raf-regulated transcription [V12 and RasV12S35 cells, although the relative fold increase was reduced following EGFR inhibition. By comparison, PHLDA1 was down-regulated in PD153035-treated RasV12G37 relative to vehicle-treated cells (not shown). Therefore, PHLDA1 represents a Raf/ERK-responsive gene whose expression parallels EGFR-independent HME16C mammary epithelial cell transformation.To evaluate the effect of EGFR inhibition on gene expression, RasV12- and Ras EDM-infected cells to levels that correlated with the level of ERK activation and the extent of anchorage-independent growth , and the expression of the encoded TDAG51 protein approximately paralleled PHLDA1 RNA expression  in cell proliferation assays, indicating a higher rate of DNA synthesis in cells with reduced TDAG51 protein . Sequences of primers used for real-time RT-PCR analysis are as follows: amphiregulin Forward 5'-CGCCGGTGGTGCTGTCGCTCTT-3' Reverse 5'-TCACTCACAGGGGAAATCTCACTC-3', anterior gradient 2 Forward 5'-GGGGTGACCAACTCATCTGGACTCAG-3' Reverse 5'-GACATACTGGCCATCAGGAGAAAGGTGT-3', CD24 Forward 5'-GCTCCTACCCACGCAGATTTATT-3' Reverse 5'-CACGAAGAGACTGGCTGTTGACT-3', c-met Forward 5'-AAATGGCCACGGGACAACACAA-3' Reverse 5'-TGGGCTGGGGTATAACATTCAAGA-3', CTGF Forward 5'-CTGCCCGGGAAATGCTGCGAGGAGT-3' Reverse 5'-CTGCAGGAGGCGTTGTCATTGGTAA-3', CYR61 Forward 5'-CGGCCCAAGTACTGCGGTTCCT-3' Reverse 5'-ATTGGCATGCGGGCAGTTGTAGTT-3', Decorin Forward 5'-ACTTCTGCCCACCTGGACACAACA-3' Reverse 5'-ATGGCAGAGCGCACGTAGACACA-3', DICER-1 Forward 5'-CAGGAAATACCCGTGCAACCAACTA-3' Reverse 5'-GCATTACGGCCATCACAGGACTTC-3', E-cadherin Forward 5'-GGTATCTTCCCCGCCCTGCCAATCC-3' Reverse 5'-AACCGCTTCCTTCATAGTCAAACACGAG-3', EphA2 Forward 5'-CCCCTTCCGCCCCACACTACCTCACAGC-3' Reverse 5'-ACACGGCCCGCATTCCCCAGACTCG-3', Epiregulin Forward 5'-TTGTATTTTTAGTAGAGGCGGGGTTTCA-3' Reverse 5'-TCGGGCACAGATGTTCAAGTCAC-3', ETS-1 Forward 5'-ACTCGGGGGCCAGGACTCTTTTGAA-3' Reverse 5'-CACGGTCCCGCACATAGTCCTTGAA-3', ETS variant gene 5 Forward 5'-TCGGGGACGTCTACGGTTTCTACT-3' Reverse 5'-AAGACTGTAAACGGCTACCATTGA-3', FAT-2 Forward 5'-GCTGGACATCAAACGGGCTAACAT-3' Reverse 5'-ACCGCATCTGAACCCCCACTGAAT-3', HMGA-1 Forward 5'-GCTCACCCTGCCCGCTCCCAACC-3' Reverse 5'-GCCCCAGCCCCTCTTCCCCACAAA-3', HMGA-2 Forward 5'-CTGATAAGCAAGAGTGGGCGGGTGAGAA-3' Reverse 5'-ACAGGGAGTGGGTTGGGGTGGTATTTGA-3', Secreted frizzled-related protein 1 Forward 5'-CCCGCTCCCTTTCCCTCCATA-3' Reverse 5'-GCTCTCACTTTCCGCCCAATCC-3', Tenascin C Forward 5'-GCGGCCCAGAGCGAGGAA-3' Reverse 5'-TATTGCGATGTTGTCACTGGGAGATTTT-3', TGF-a Forward 5'-CCGCCTTGGTGGTGGTCTCC-3' Reverse 5'-AGGGCGCTGGGCTTCTCGTG-3', TGF-β2 Forward 5'-CCCGCCCACTTTCTACAGACCCTACT-3' Reverse 5'-CATTCGCCTTCTGCTCTTGTTTTCAC-3', TGF-β receptor 2 Forward 5'-ACAGTGGCAGTCAAGATCTTTCCCTATG-3' Reverse 5'-CAACTCCGTCTTCCGCTCCTCAG-3', TRIM29 Forward 5'-ATGCATGCGCCACGTTGAGAAGAT-3' Reverse 5'-GGTGAAGCGGCCAGGAGACGAG-3'.Click here for file


C. elegans, attenuation of the insulin-like signal transduction pathway increases adult lifespan of the nematode. We demonstrate that the O-GlcNAc cycling enzymes OGT and OGA, which add and remove O-GlcNAc respectively, modulate lifespan in C. elegans. Median adult lifespan is increased in an oga-1 deletion strain while median adult life span is decreased upon ogt-1 deletion. The O-GlcNAc-mediated effect on nematode lifespan is dependent on the FoxO transcription factor DAF-16. DAF-16 is a key factor in the insulin-like signal transduction pathway to regulate reproductive development, lifespan, stress tolerance, and dauer formation in C. elegans. Our data indicates that O-GlcNAc cycling selectively influences only a subset of DAF-16 mediated phenotypes, including lifespan and oxidative stress resistance. We performed an affinity purification of O-GlcNAc-modified proteins and observed that a high percentage of these proteins are regulated by insulin signaling and/or impact insulin pathway functional outcomes, suggesting that the O-GlcNAc modification may control downstream effectors to modulate insulin pathway mediated cellular processes.O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a regulatory, nuclear and cytoplasmic post-translational glycosylation of proteins associated with age-related diseases such as Alzheimer's, Parkinson's, and type II diabetes. Global elevation of O-GlcNAc levels on intracellular proteins can induce insulin resistance, the hallmark of type II diabetes, in mammalian systems. In  Insulin resistance precedes and is a hallmark of type II diabetes . DespiteC. elegans, the conserved insulin-like signaling pathway regulates lifespan as well as reproductive development, stress response, and dauer formation , CF1553 muIs84[pAD76 (sod-3::GFP)], CL2070 dvls70[integrat-edhsp-16-2::GFP+pRF4], ET434 oga-1(ok1207), ET435 ogt-1(ok1474), ET436 oga-1(ok1207); ogt-1(ok1474), ET439 oga-1(ok1207); daf-2(e1370), ET438 ogt-1(ok1474); daf-2(e1370), ET437 oga-1(ok1207); daf-16(mu86), ET468 ogt-1(ok1474); daf-16(mu86), ET469 oga-1(ok1207); age-1(hx546), ET393 ogt-1(ok1474); age-1(hx546), ET471 oga-1(ok1207); sgk-1(ok538), ET470 ogt-1(ok1474); sgk-1(ok538), ET472 oga-1(ok1207); pdk-1(mg142), and ET445 oga-1(ok1207); akt-1(mg144). The oga-1(ok1207) and ogt-1(ok1474) mutants in strains ET434 and ET435 were outcrossed 6 times against N2 in our laboratory with the mutations followed by PCR. The mutation in ogt-1(ok1474) and oga-1(ok1207) alleles were verified using PCR primers provided by the C. elegans Gene Knockout Project at OMRF, which is part of the International C. elegans Gene Knockout Consortium. The mutations in double mutant strains were followed by PCR and restriction enzyme analysis or by DNA sequencing spanning the region corresponding to the mutations, where appropriate.The following nematode strains were used in this study: N2 wild type Bristol, RB1342 All lifespan assays were performed using 85-125 animals for each genotype per assay at 20°C on NGM plates seeded with OP50 bacteria following established procedures [Survival curves were analyzed by the Log-rank (Mantel-Cox) and Wilcoxon tests using GraphPad Prism (version 5.0) to determine the significance between mutant and wild-type controls as indicated in Table Oxidative stress resistance assays were performed in multi-well plates by immersing adults in M9 media containing 100 mM paraquat  using 10-20 animals for each genotype per assay at 20°C ,40. A coPostembryonic developmental timing was assayed by collecting comma-stage embryos from animals maintained at 20°C. The exact time of hatching was recorded, and then animals were transferred onto separate OP50-seeded NGM plates. After the third molt, animals were inspected more frequently (every 15 mins) to record the exact time of the fourth molt. The assay was repeated several times using at least 10 animals for each genotype per assay, and multiple experimental data were pooled together for final analysis. For fecundity analysis, individual larval stage animals were placed on OP50-seeded NGM plates at 20°C. Offspring were counted by moving them onto a fresh plate one-by-one. The assay was repeated multiple times with at least 10 animals per genotype in each assay, and data were pooled together for final analysis.Adult animals were flash frozen in liquid nitrogen in lysis buffer (50mM HEPES pH 7.8 and 300mM NaCl). Frozen worms were crushed in a pestle pre-chilled with liquid nitrogen. The frozen ground worms were re-suspended in cold lysis buffer (with DNaseI and protease inhibitor cocktail from Roche) and subjected to sonication for 6-8 seconds followed by centrifugation at 16,000 rpm for 1 hour at 4°C. The middle portion (not including the pellet or floating lipids) of each tube was collected for analysis. An equal amount of whole animal lysate was separated on a 10% NuPAGE Bis-Tris gel (Invitrogen) followed by transfer onto a PVDF membrane (Millipore). The membrane was probed with anti-O-GlcNAc antibody from Affinity Bioreagents (1:2000 in 3% BSA dissolved in 1XTBS + 0.05% Tween20). The immunoblot was visualized using a chemiluminescent substrate . The same membrane was re-probed with anti-α-tubulin antibody (Sigma) to normalize protein loading.E. coli strain HT115 transformed with pPD129.36 clones from an RNAi library provided by Julie Ahringer for gene inactivation experiments, as previously described [All RNAi experiments were performed at 20°C unless otherwise mentioned using escribed ,43.sod-3::gfp and hsp-16-2::gfp reporters were assayed in 10-day-old adults.Experimental animals for analysis were grown on feeding RNAi for more than one generation. All animals were imaged at single shutter setting, and equal nuclear and cytoplasmic areas were chosen for light intensity measurement using OpenLab 5.5 software. The average of three mean light intensity measurement values per cell (in arbitrary units) for both nuclear and cytoplasmic regions were recorded for multiple cells in each treated and untreated animals. The assay was repeated several times with at least 10 animals per genotype in each assay at 20°C. Data from multiple experiments were pooled for final analysis. BothC. elegans protein database from NCBI with filtering for a false-discovery rate of less than 1%, as previously described [Adult animals were collected and flash frozen in liquid nitrogen in 50mM HEPES pH 7.8 and 50mM NaCl buffer. Frozen worms were crushed with a mortar and pestle pre-chilled with liquid nitrogen. The frozen ground worms were re-suspended in the above buffer (with DNaseI and protease inhibitor cocktail from Roche), and disrupted by sonication followed by centrifugation at 16,000 rpm for 1 hour at 4°C. Clear supernatant was collected and O-GlcNAc-modified proteins were identified as previously described . Brieflyescribed . The numSupplemental Movie 1.oga-1(ok1207), wild type (N2), and ogt-1(ok1474) animals at day 12 of adulthood. Note that, while oga-1 mutant and wild-type animals are highly active, the ogt-1 mutant animal is considerably slower, a phenotype similar to that of older (day 18) wild-type animals (data not shown). At an earlier time point (day 6) all three genotypes had indistinguishable movement (data not shown).Three 10-second movie recordings, from left to right,


CRC xenografts differed in their p53 status (wt or mut) and in their microsatellite instability phenotype . Five CRC xenografts were established from clinical samples. All five had a functional p53, two were MSI+and three were MSI–. Tumour-bearing nude mice were treated intraperitonealy (i.p.) with CPT-11. At 10 mg kg–1of CPT-11, four injections at 4-day intervals, four of the five xenografts responded to CPT-11 (growth delay of up to 10 days); the non-responder tumour was MSI−. At 40 mg kg−1of CPT-11, six injections at 4-day intervals, the five CRCs displayed variable but marked responses with complete regressions. In order to assess the role of p53 status in CPT-11 response, four CRC lines were used. HT29 cell line was MSI−/ Ala273-mutp53, its subclone HT29A3 being transfected by wtp53. LoVo cell line was MSI+/ wtp53, its subclone X17LoVo dominantly expressed Ala273-mutp53 after transfection. LoVo tumours (MSI+/ mutp53) were more sensitive than X17LoVo (MSI+/ mutp53. HT 29 tumours (MSI−Imutp53), were refractory to CPT-11 while HT29A3 tumours (MSI−/ wtp53) were sensitive, showing that wtp53 improves the drug-response in these MSI−tumours. Levels of mRNA expression of top1, fasR, TP53 and mdr1 were semi-quantified by reverse transcription polymerase chain reaction. None of these parameters correlated with CPT-11 response. Taken together, these observations indicate that MSI and p53 alterations could be associated with different CPT-11 sensitivities; MSI phenotype moderately influences the CPT-11 sensitivity, MSI+being more sensitive than MSI−CRC freshly obtained from patients, mutp53 status being associated with a poor response to CPT-11. © 2000 Cancer Research CampaignBiological parameters influencing the response of human colorectal cancers (CRCs) to CPT-11, a topoisomerase 1 (top1) inhibitor, were investigated using a panel of nine CRCs xenografted into


In the structure, the CdII atom is six-coordinated by two azide anions and three pyrazine-2-carboxylate ligands. Each pyrazine-2-carboxylate ligand bridges three CdII atoms, whereas the azide ligand bridges two CdII atoms, resulting in the formation of a two-dimensional metal–organic polymer developing parallel to the (100) plane.The title compound, [Cd(C DOI: 10.1107/S1600536809012227/dn2435Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Silicon and germanium containing heteroaromatic sulfides have been prepared usingphase transfer catalytic (PTC) system thiol / Si or Ge containing alkyl halide / solid KOH / 18-crown-6 / toluene. The target sulfides were isolated in yields up to 92 %. It has been found that 2-{[dimethyl (β-triethylgermylethyl)-silylmethyl]thio}-1-methylimidazole and 2-{[dimethyl(β-triphenylsilylethyl)silyl-methyl]thio}benzothiazole are the most active cholesterol level lowering andvasodilating agents.


In the Materials and Methods section two optimal primer sequences were switched by mistake with sub-optimal primer sequences. The correct sentences should be:"We designed a universal-forward primer with an M13-tail added at the 5' end (M13BC-FW 5'-TGT AAA ACG ACG GCC AGT-HAA YCA YAA RGA YAT YGG -3'), similar to other primers previously used in other barcode approaches [e.g. 13]. Then, we searched for a reverse primer that allowed the specific amplification of vertebrate COI sequences while avoiding the co-amplification of invertebrate DNA (BCV-RV1 59-GCY CAN ACY ATN CCY ATR (T)(A)-3')."


Interleukin-6 (IL-6) is a multifunctional cytokine that regulates the immune response, acute phase anaphylactic reaction, and haematopoiesis. Lipopolysaccharide (6–24 μg/ml) significantly induced IL-6 release from murine spleen cells. In cultured rabbit synovial cells interleukin-1  induced IL-6 production in a concentration-dependent manner. Triazolodiazepine (Tri) is a hetrazepine platelet-activating factor antagonist. In this study we found that Tri (0.1–10 μmol/l) exerted strong inhibitory effects on LPS stimulated IL-6 production in murine spleen cells. Kinetic studies showed that the inhibition of IL-6 release was time-independent. In rabbit synovial cells Tri also reduced IL-6 release induced by IL-1 and tumour necrosis factor. Inhibition of cytokine production by Tri may partially explain its wide and strong anti-inflammatory effects.