Boule JB et al. (2005) Nature "The yeast Pif1p helicase removes telomerase from telomeric DNA."

Boule JB, Zakian VA, Vega LR

[Nature, 2005]

Telomeres are the physical ends of eukaryotic chromosomes. Genetic studies have established that the baker''s yeast Pif1p DNA helicase is a negative regulator of telomerase, the specialized reverse transcriptase that maintains telomeric DNA, but the biochemical basis for this inhibition was unknown. Here we show that in vitro, Pif1p reduces the processivity of telomerase and releases telomerase from telomeric oligonucleotides. The released telomerase is enzymatically active because it is able to lengthen a challenger oligonucleotide. In vivo, overexpression of Pif1p reduces telomerase association with telomeres, whereas depleting cells of Pif1p increases the levels of telomere-bound Est1p, a telomerase subunit that is present on the telomere when telomerase is active. We propose that Pif1p helicase activity limits telomerase action both in vivo and in vitro by displacing active telomerase from DNA ends.

Karashima T et al. (1997) Worm Breeder's Gazette "A C. elegans homologue of DAZ/boule is involved in progression through meiosis during oogenesis"

Karashima T, Sugimoto A, Yamamoto M

[Worm Breeder's Gazette, 1997]

Human DAZ (Deleted in Azoospermia), mouse Dazla, and Drosophila boule encode a family of proteins with a single RNP-type RNA-binding motif and are thought to be essential for meiosis during spermatogenesis (Nature, 381: 783-785, 1996). The C.elegans cDNA project identified a putative homologue of DAZ/boule. This cDNA (yk62b4, kindly supplied by Yuji Kohara) appears to contain the complete open reading frame for the gene and its predicted amino acid sequence contains a putative RNA-binding domain most similar to Boule, as well as a motif found in all DAZ family members (see figure). We tentatively call this gene Ce-boule . Ce-boule maps close to clr-1 on Chromosome II. To examine whether Ce-boule is involved in meiosis in C. elegans, antisense Ce-boule RNA was injected into gonads of wildtype L4 hermaphrodites. F1 hermaphrodite progeny derived from the injected P0s were sterile at high penetrance. The sterile F1s had normal somatic gonads and normal-looking sperm (by both Nomarski and DAPI staining), but oogenesis was severely affected: with Nomarski microscopy, gonad arms were filled with mostly small nuclei, and none of the cells displayed the characteristic morphology of developing or mature oocytes. By DAPI staining, majority of the nuclei looked similar to mitotic cells. Possible pachytene nuclei were occasionally observed in the proximal arms, but no diplotene-diakinesis nuclei were found. Although the morphology of the nuclei appears mitotic, the phenotype is not tumorous (unlike gld-1(null) or glp-1(gf)), judging from the density of the nuclei in the gonad. The injected P0s themselves showed a similar phenotype after they laid approximately 30 fertilized eggs. Thus, antisense Ce-boule injection seems to affect either the transition from mitosis to meiosis or the early stage of meiosis during oogenesis. The expression pattern of Ce-boule in the adult hermaphrodite germ line was studied by in situ hybridization and it was found that Ce-boule mRNA shows a graded pattern in the gonad. The signal was detected starting at the mitosis/meiosis transition zone and increased in intensity toward the pachytene region.The strong signal was maintained through the proximal arm except in mature oocytes, where the signal was distinctly weaker. No signal was seen in mature sperm. This expression pattern is consistent with the hypothesis that Ce-boule is required during oogenesis, for the meiotic entry and/or the prophase of meiosis I. It is surprising that DAZ/boule's possible counterpart in C.elegans appears to be essential for oogenesis and dispensable for spermatogenesis, as both DAZ and boule seem to be mainly associated with male sterility. For further characterization, we are currently screening for mutants of Ce-boule by a PCR-based method and by looking for sterile mutants in the corresponding chromosomal region. We are grateful to Tim Schedl and Lisa Kadyk for helpful suggestions and guidance.

Ramos CG et al. (2014) J Bacteriol "Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia."

Feliciano JR, da Costa PJ, Ramos CG, Grilo AM, Leitao JH

[J Bacteriol, 2014]

Volume 195, no. 16, p. 35143523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitao, J. Bacteriol. 193:15151526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitao, Microbiology 156:896908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitao, Microb. Pathog. 48:168177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

Karashima T et al. (2000) Development "Caenorhabditis elegans homologue of the human azoospermia factor DAZ is required for oogenesis but not for spermatogenesis."

Sugimoto A, Yamamoto M, Karashima T

[Development, 2000]

DAZ (Deleted in Azoospermia), the putative azoospermia factor gene in human, encodes a ribonucleoprotein-type RNA-binding protein required for spermatogenesis. A Drosophila homologue of DAZ, called boule, is also essential for spermatogenesis. A mouse homologue, Dazla, is implicated in both spermatogenesis and oogenesis. Here, we report the identification and characterization of daz-1, the single DAZ homologue in the nematode Caenorhabditis elegans. Loss of daz-1 function caused sterility in hermaphrodites, by blocking oogenesis at the pachytene stage of meiosis I. Epistasis analysis suggested that this gene executes its function succeeding gld-1, which governs the early pachytene stage in the oogenic pathway. Spermatogenesis did not appear to be affected in daz-1 hermaphrodites. Males defective in daz-1 produced sperm fully competent in fertilization. Analysis employing sex-determination mutants indicated that the daz-1 function was required for meiosis of female germline regardless of the sex of the soma. Transcription of daz-1 was restricted to the germline, starting prior to the onset of meiosis and was most conspicuous in cells undergoing oogenesis. Thus, daz-1 in C. elegans is an essential factor for female meiosis but, unlike other DAZ family members so far reported, it is dispensable for male meiosis.

Bryer J et al. (1997) International C. elegans Meeting "Current Perspectives of the hsp70 Gene Family in CAENORHABDITIS ELEGANS"

Baillie DL, Bryer J

[International C. elegans Meeting, 1997]

Our lab has previously identified six members of the hsp70 gene family in Caenorhabditis elegans (hsp-1, hsp-2, hsp-4, hsp-6 and the two pseudogenes hsp-2 and hsp-5). We present here, a current computational examination of the hsp70 gene family. To date, ten putative members of the hsp70 family in C. elegans have been identified. In Saccharomyces cerevisiae, for which the sequence of the entire genome is known, there are fourteen members of the hsp70 gene family. In yeast, the hsp70 family members have been shown to comprise two subfamilies. Utilizing phylogenetic methods, the nine C. elegans members are shown here, to likewise, fall into distinct subfamilies. Also shown is a comparison between the hsp70 gene family in C. elegans, S. cerevisiae and with the more ancestral Esherichia coli family members. It is interesting to note that mitochondrial members from the worm and yeast are more closely related to the bacterial members than to other eukaryotic members. This contributes to the mitochondrial origin theory of a bacterial- eukaryotic cell symbiotic relationship. The genomic sequence of the four recently identified members all contain introns. It was known that three of the other hsp70 gene members contain introns. The presence of introns within hsp70 genes is uncommon, thought to be partially due to the evidence for splicing mechanism inhibition under heat stress conditions. Within multicellular eukaryotes, nematodes are proving to be the execption to the lack of introns in hsp70 genes (see the Brugia malayi sequence). This work is supported by an NSERC scholarship to JB and an NSERC operating grant to DLB.

Davide Faggionato et al. (2006) European Worm Meeting "Learning Lessons from dys-1 and snf-6 - Having Acetylcholine as a Teacher"

Davide Faggionato, Ralf Baumeister, Oyunbileg Nyamsuren

[European Worm Meeting, 2006]

We study the function/dysfunction of mutations of genes that in humans have been associated with degenerative disorders. Recently, we identified components of an ubiquitylation complex as regulators of muscle assembly and turnover. Specifically, mutational inactivation of ubiquitylation suppressed paralysis of unc-45 mutant animals that show a severe myofibril disorganization 1.. In order to get an idea whether this is a generally applicable way to reduce the phenotypic consequences of mutations in muscle genes, we focused on other genes affecting muscle assembly and function.. dys-1 encodes the C. elegans orthologue of human dystrophin, mutations of the latter being responsible for Duchenne muscular dystrophy. One of the available mutants in dys-1, cx18, displays a surprisingly mild impairment of movement, that, however, is strongly enhanced in the background of hlh-1/MyoD, generating a synthetic paralysis. However, dys-1(cx18) animals also display subtle phenotypes like increased sensitivity to aldicarb, suggesting an additional neuronal function of this gene 2.. In a previous screen for ethanol resistance, mutations in the muscular sodium-acetylcholine cotransporter gene snf-6 were identified. Surprisingly, snf-6 mutations strongly resemble the dys-1 mutant phenotype in an hlh-1 background, suggesting a link between cholinergic signalling and dystrophin function.. In order to understand dys-1 dysfunction, we performed a detailed comparative analysis of both mutants and will present the data in our poster.. 1Hoppe T, Cassata G, Barral JM, Springer W, Hutagalung AH, Epstein HF, Baumeister R. Regulation of the myosin-directed chaperone UNC-45 by a novel E3/E4-multiubiquitylation complex in C. elegans. Cell. (2004) 118(3), 337-49. 2Bessou C, Giugia JB, Franks CJ, Holden-Dye L, Segalat L. Mutations in the Caenorhabditis elegans dystrophin-like gene dys-1 lead to hyperactivity and suggest a link with cholinergic transmission. Neurogenetics. 1998 Dec;2(1):61-72

Muneyoshi Otori et al. (2002) Mid-west Worm Meeting "Identification and Comparison of C. briggsae and C. remanei homologs of C. elegans daz-1"

Takeshi Karashima, Masayuki Yamamoto, Muneyoshi Otori

[Mid-west Worm Meeting, 2002]

DAZ family proteins play important roles in meiosis and gametogenesis, and all family members carry of a well-conserved RRM (RNA Recognition Motif) in their N-terminus followed by a highly diverged C-terminal tract. We have previously characterized the C. elegans daz-1 gene, which belongs to the Boule subtype of this family. The daz-1 gene is necessary for oogenesis but not for spermatogenesis. Immunohistochemical analysis has shown that the DAZ-1 protein is mainly localized in the mitotic and early meiotic regions in hermaphrodite gonads. Here we report an initial characterization of the daz-1 homologs in two nematode species closely related to C. elegans, namely C. briggsae and C. remanei. We isolated a full-length C. briggsae daz-1 (Cb-daz-1) cDNA, based on the EST sequence reported by P. Kuwabara, using the RACE PCR technique. A full-length C. remanei daz-1 (Cr-daz-1) cDNA was also isolated by RACE, using degenerate PCR primers prepared according to a sequence alignment of Ce-daz-1 and Cb-daz-1. The deduced amino acid sequences indicated that the N-terminal RRM region was highly conserved among the three species (79% identity), implicating a possible conservation of their target RNA molecules. However, the C-terminal region following the RRM was diverged among them. Ce-DAZ-1 (499 a.a.) had a long C-terminus (349 a.a.), which was unique compared to the DAZ family proteins in other species. Cr-DAZ-1 (452 a.a.) also had a long C-terminus (297 a.a.), but fewer amino acid residue matched in this region than in the RRM region. Cb-DAZ-1 was the smallest among the three (224 a.a.), with only 61 amino acid residues in the C-terminal region.

Maruyama R et al. (2000) Japanese Worm Meeting "DAZ-1, a C. elegans homolog of DAZ (Deleted in Azoospermia), localizes to cytoplasm of the mitotic germ cells"

Maruyama R, Yamamoto M, Karashima T, Sugimoto A

[Japanese Worm Meeting, 2000]

The DAZ family genes encode RNA-binding proteins and are involved in germ cell development in a variety of organisms. Human DAZ ( Deleted in Azoospermia ) is present on the Y chromosome and is essential for spermatogenesis. Drosophila boule is required for the entry to meiosis during spermatogenesis, and is expressed only in males. Mice deficient in Dazla , a mouse homolog of DAZ , are sterile in both sexes, exhibiting defects in germ cell development and survival. In contrast, Xdazl , a Xenopus homolog of DAZ , is essential for early differentiation of primordial germ cells (PGCs) and their migration through endoderm. To understand the function of DAZ family proteins, we characterized the C. elegans homolog of DAZ , daz-1 . The hermaphrodite daz-1 null mutant was completely sterile due to arrest of oogenesis at the late pachytene stage of meiosis I. However, spermatogenesis was normal in both daz-1 males and hermaphrodites. We produced affinity-purified polyclonal antibodies against DAZ-1 protein. In immunoblot analysis, the antibodies recognized a single band of the expected size in the N2 adult extract, but no band in the daz-1 adult extract. Using these antibodies, we examined expression and localization of DAZ-1 protein. DAZ-1 was observed only in the cytoplasm of germ line cells, starting from L2 through adulthood. The DAZ-1 signal was intense in the distal region of a gonadal arm, where germ cells undergo mitosis. The entry to meiosis was accompanied by a decrease of DAZ-1 expression. This mitotic expression of DAZ-1 is puzzling, because phenotypes of the daz-1 mutant have suggested that DAZ-1 is required for the progression through the pachytene stage in oogenesis. One possible explanation is that, DAZ-1 may regulate an essential function for meiosis pre-meiotically.

T Karashima et al. (1999) International C. elegans Meeting "daz-1, a C. elegans homologue of DAZ (Deleted in Azoospermia), is required for the progression of meiosis in oogenesis"

T Karashima, A Sugimoto, M Yamamoto

[International C. elegans Meeting, 1999]

The DAZ family is composed of proteins with a conserved RNP-type RNA-binding motif. Members of this family appear to be involved in meiosis in various organisms. For example, deletion of the DAZ gene cluster on the human Y chromosome correlates with azoospermia. A mutation in the Drosophila homologue of DAZ , boule , results in complete male sterility due to a defect in spermatogenesis. From the genome sequence data, we identified a single C. elegans homologue of DAZ , and named it daz-1 . To elucidate the function of this gene, we isolated a deletion allele of daz-1 , termed tj3 , by the PCR-oriented screen. The daz-1(tj3) homozygous hermaphrodite was sterile with complete penetrance. The gonad of the daz-1 adult hermaphrodite was full of small nuclei, with no cells displaying the characteristic morphology of developing or mature oocytes. However, apparently normal sperm were seen in the spermatheca. By DAPI staining, the majority of the germ nuclei showed morphology similar to those of the pachytene stage, and no diplotene-diakinesis nuclei were found. On the other hand, daz-1 males produced fully-functional sperm. Thus, unlike other DAZ genes, C. elegans daz-1 appears to be despensable for spermatogenesis but essential for meiosis during oogenesis. The effect of daz-1(tj3) was examined in several germ-cell sex determination mutants. In feminized XX animals of fem-3(lf); daz-1 and fog-3(lf); daz-1 , defective oogenesis was observed. X0 animals of fog-3(lf); daz-1 , which had male soma and female germline, showed similar pachytene arrest of the presumptive oocytes. In contrast, the XX animals of fem-3(gf); daz-1 , which had female soma and male germline, produced apparently normal sperm. Taken together, the consequence of the daz-1(tj3) mutation depends on the sexual identity of the germline, regardless of the somatic sex. Northern analysis and in situ hybridization showed that, in hermaphrodite, daz-1 mRNA is present exclusively in germline, starting to emerge at L2 stage. A much weaker signal was detected in adult male gonad. From these observations, we speculate that daz-1 function is essential during oogenesis, especially for the progression of prophase of meiosis I.

Muneyoshi Otori et al. (2002) Japanese Worm Meeting "Identification and Comparison of C. briggsae and C. remanei homologs of C. elegans daz-1"

Takeshi Karashima, Muneyoshi Otori, Masayuki Yamamoto

[Japanese Worm Meeting, 2002]

DAZ family proteins play important roles in meiosis and gametogenesis, and all family members carry of a well-conserved RRM (RNA Recognition Motif) in their N-terminus followed by a highly diverged C-terminal tract. We have previously characterized the C. elegans daz-1 gene, which belongs to the Boule subtype of this family. The daz-1 gene is necessary for oogenesis but not for spermatogenesis. Immunohistochemical analysis has shown that the DAZ-1 protein is mainly localized in the mitotic and early meiotic regions in hermaphrodite gonads. Here we report an initial characterization of the daz-1 homologs in two nematode species closely related to C. elegans , namely C. briggsae and C. remanei . We isolated a full-length C. briggsae daz-1 ( Cb-daz-1 ) cDNA, based on the EST sequence reported by P. Kuwabara, using the RACE PCR technique. A full-length C. remanei daz-1 ( Cr-daz-1 ) cDNA was also isolated by RACE, using degenerate PCR primers prepared according to a sequence alignment of Ce-daz-1 and Cb-daz-1 . The deduced amino acid sequences indicated that the N-terminal RRM region was highly conserved among the three species (79% identity), implicating a possible conservation of their target RNA molecules. However, the C-terminal region following the RRM was diverged among them. Ce-DAZ-1 (499 a.a.) had a long C-terminus (349 a.a.), which was unique compared to the DAZ family proteins in other species. Cr-DAZ-1 (452 a.a.) also had a long C-terminus (297 a.a.), but fewer amino acid residue matched in this region than in the RRM region. Cb-DAZ-1 was the smallest among the three (224 a.a.), with only 61 amino acid residues in the C-terminal region. Possible function of Cb-daz-1 and Cr-daz-1 was investigated by RNAi. Interestingly, RNAi of Cb-daz-1 in C. briggsae caused a Mog phenotype in F1 hermaphrodites. In mature P0 animals, Cb-daz-1 RNAi 'switched off' oogenesis that had already started, and induced re-initiation of spermatogenesis. In contrast, trials to block Cr-daz-1 function by RNAi in C. remanei have generated no obvious phenotype so far, the reason for which is under investigation. Comparison of the structure and function of the three nematode DAZ-1 proteins will be important in elucidation of the molecular function of the DAZ family proteins.